TY - JOUR AB - The extracellular fibrinogen-binding protein (Efb) is one of nearly a dozen proteins secreted by Staphylococcus aureus to inhibit complement activation or amplification. The C-terminal domain of Efb (Efb-C) forms a high-affinity interaction with the thioester-containing domain of C3b (TED/C3d), thereby blocking formation of the C3 proconvertase complex through an allosteric mechanism. However, further functional consequences of Efb-C binding to C3b remain unexplored. Here, we identified a previously unknown interaction between Efb-C, C3b, and the complement regulatory molecule FHR2 (factor H-related protein 2). Since the FHR2/C3b interaction is centered upon the 2 C-terminal-most domains of FHR2 (FHR2[3-4]) and the TED/C3d domain of C3b, we tested whether Efb-C could influence the FHR2(3-4)/C3d interaction. We observed a significant enhancement of FHR2(3-4)/C3d binding in the presence of Efb-C. We studied the FHR2(3-4)/C3d/Efb-C complex by X-ray crystallography and found that Efb-C forms few direct interactions with FHR2(3-4). Yet, the presence of Efb-C also enhanced binding of FHR2(3-4) and full-length FHR2 to C3b, suggesting that the effect of Efb-C on the FHR2/C3b interaction arises from increased accessibility of the FHR2-binding site. We found that enhanced FHR2 binding did not impact the rate of C3 convertase formation more than Efb-C alone, nor did it impart decay acceleration or cofactor activity. However, we observed potent, synergistic inhibition of complement downstream of C5 activation by Efb-C and FHR2 but not by Efb-C and FHR2(3-4). Our results show that Efb-C binding to C3b exerts additional inhibitory effects on the central complement components beyond blocking formation of the C3 proconvertase alone. AU - Duan, H.* AU - Körtvely, E.* AU - Mary, J.L.* AU - Hauck, S.M. AU - Geisbrecht, B.V.* C1 - 76106 C2 - 58426 TI - The C-terminal domain of Staphylococcus aureus Efb recruits FHR-2 to C3b, synergistically inhibiting the terminal complement pathway. JO - J. Immunol. PY - 2025 SN - 0022-1767 ER - TY - JOUR AB - The BAFF/APRIL-system with the two cytokines BAFF and APRIL and their three receptors, transmembrane activator and CAML interactor (TACI), BAFF receptor, and B-cell maturation Ag, is important for B cell maintenance. The BAFF/APRIL system is a therapeutic target in B cell-derived malignancies and autoimmune diseases. However, unexpected outcomes of clinical trials with atacicept (TACI-Fc) underline our incomplete understanding of this system. Shedding of the three receptors is one important regulatory element. In humans, TACI exists in two isoforms generated through alternative splicing in their extracellular portion: TACI-long (l) has two cysteine-rich domains, whereas TACI-short (s) lacks the first low-affinity one. In this study, we discriminated soluble (s) forms of TACI-l and TACI-s with newly generated mAbs and found that both were spontaneously released from activated human B cells, with a predominance of sTACI-l. Furthermore, sTACI-l was also the dominant isoform in human serum. Vaccination with the mRNA vaccine from BioNTech does not significantly affect the serum levels of sTACI-l. Both TACI-s and TACI-l were shed by a disintegrin and metalloproteinase domain-containing protein 10. TACI-l and TACI-s formed homo- and hetero-oligomers in soluble and membrane-bound forms. Both sTACI-l and sTACI-s acted as decoy receptors for BAFF, but only sTACI-l also efficiently inhibited APRIL. Dimerization of sTACI-l enhanced its decoy functions only slightly. Together, we extend our knowledge of the complexity of the BAFF/APRIL system by identifying and characterizing the two soluble isoforms of TACI. AU - Fichtner, M.L.* AU - Rübsamen, H.* AU - Smolle, M.* AU - Schaller, J.* AU - Feederle, R. AU - Bültmann, A.* AU - Kümpfel, T.* AU - Schneider, P.* AU - Thaler, F.S.* AU - Meinl, E.* C1 - 67852 C2 - 54330 CY - 9650 Rockville Pike, Bethesda, Md 20814 Usa SP - 199-208 TI - Features of isoforms of human soluble TACI. JO - J. Immunol. VL - 211 IS - 2 PB - Amer Assoc Immunologists PY - 2023 SN - 0022-1767 ER - TY - JOUR AB - CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and β-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine. AU - Abdulhaqq, S.* AU - Ventura, A.B.* AU - Reed, J.S.* AU - Bashirova, A.A.* AU - Bateman, K.B.* AU - McDonald, E.* AU - Wu, H.L.* AU - Greene, J.M.* AU - Schell, J.B.* AU - Morrow, D.* AU - Wisskirchen, K. AU - Martin, J.N.* AU - Deeks, S.G.* AU - Carrington, M.N.* AU - Protzer, U. AU - Früh, K.* AU - Hansen, S.G.* AU - Picker, L.J.* AU - Sacha, J.B.* AU - Bimber, B.N.* C1 - 63634 C2 - 51639 CY - 9650 Rockville Pike, Bethesda, Md 20814 Usa SP - 2913-2921 TI - Identification and characterization of antigen-specific CD8+ T cells using surface-trapped TNF-α and single-cell sequencing. JO - J. Immunol. VL - 207 IS - 12 PB - Amer Assoc Immunologists PY - 2021 SN - 0022-1767 ER - TY - JOUR AB - IL-4 receptor signaling is supposed to play a major role in anti-inflammatory polarization and proliferation of adipose tissue macrophages. In this study, we examined the metabolic and inflammatory phenotype of C57BL/6J mice (IIl4ra) with LysM-dependent knockout (IIl4ra Δmyel) of the IL-4 receptor α-chain (IL-4Rα), the mandatory signaling component of IL-4 and IL-13, on chow and high-fat diet. Lean IIl4ra Δmyel mice showed decreased insulin sensitivity, no divergent adipose tissue macrophage polarization, but an increased percentage of CD8+ T cells in visceral adipose tissue. After 20 wk of a high-fat diet, IIl4ra Δmyel mice exhibited higher glucose tolerance, no changes in the lymphocyte compartment and fewer M1 macrophages in visceral adipose tissue. In vivo adipose tissue macrophage proliferation measured by BrdU incorporation was unaffected by Il4ra knockout. Interestingly, we show that IL-4Rα signaling directly augmented Itgax (Cd11c) gene expression in bone marrow-derived macrophages and increased the amount of CD11c+ macrophages in adipose tissue explants. Myeloid cell-specific knockout of Il4ra deteriorated insulin sensitivity in lean mice but improved parameters of glucose homeostasis and partially protected from adipose tissue inflammation in obese mice. Hence, IL-4Rα signaling probably plays a minor role in maintaining the macrophage M2 population and proliferation rates in vivo. Moreover, our data indicate that IL-4 signaling plays a proinflammatory role in adipose tissue inflammation by directly upregulating CD11c on adipose tissue macrophages. AU - Ackermann, J.* AU - Arndt, L.* AU - Kirstein, M.* AU - Hobusch, C.* AU - Brinker, G.* AU - Klöting, N. AU - Braune, J.* AU - Gericke, M.* C1 - 63641 C2 - 51687 CY - 9650 Rockville Pike, Bethesda, Md 20814 Usa SP - 3081-3089 TI - Myeloid cell-specific IL-4 receptor knockout partially protects from adipose tissue inflammation. JO - J. Immunol. VL - 207 IS - 12 PB - Amer Assoc Immunologists PY - 2021 SN - 0022-1767 ER - TY - JOUR AB - Foxp(3+) regulatory T cells are well-known immune suppressor cells in various settings. In this study, we provide evidence that knockout of the relB gene in dendritic cells (DCs) of C57BL/6 mice results in a spontaneous and systemic accumulation of Foxp(3+) T regulatory T cells (Tregs) partially at the expense of microbiota-reactive Tregs. Deletion of nfkb2 does not fully recapitulate this phenotype, indicating that alternative NF-kappa B activation via the RelB/p52 complex is not solely responsible for Treg accumulation. Deletion of Re1B in DCs further results in an impaired oral tolerance induction and a marked type 2 immune bias among accumulated Foxp(3+) Tregs reminiscent of a tissue Treg signature. Tissue Tregs were fully functional, expanded independently of IL-33, and led to an almost complete Treg-dependent protection from experimental autoimmune encephalomyelitis. Thus, we provide clear evidence that RelB-dependent pathways regulate the capacity of DCs to quantitatively and qualitatively impact on Treg biology and constitute an attractive target for treatment of autoimmune diseases but may come at risk for reduced immune tolerance in the intestinal tract. AU - Andreas, N.* AU - Potthast, M. AU - Geiselhöringer, A.-L. AU - Garg, G.* AU - de Jong, R.J. AU - Riewaldt, J.* AU - Russkamp, D. AU - Riemann, M.* AU - Girard, J.-P.* AU - Blank, S. AU - Kretschmer, K.* AU - Schmidt-Weber, C.B. AU - Korn, T.* AU - Weih, F.* AU - Ohnmacht, C. C1 - 57013 C2 - 47433 CY - 9650 Rockville Pike, Bethesda, Md 20814 Usa SP - 2602-2613 TI - RelB deficiency in dendritic cells protects from autoimmune inflammation due to spontaneous accumulation of tissue T regulatory cells. JO - J. Immunol. VL - 203 IS - 7 PB - Amer Assoc Immunologists PY - 2019 SN - 0022-1767 ER - TY - JOUR AB - Mice deficient for ADP-ribosyltransferase diphteria toxin-like 1 (ARTD1) are protected against microbially induced inflammation. To address the contribution of ARTD1 to inflammation specifically in myeloid cells, we generated an Artd1(Delta Myel) mouse strain with conditional ARTD1 deficiency in myeloid lineages and examined the strain in three disease models. We found that ARTD1, but not its enzymatic activity, enhanced the transcriptional activation of distinct LPS-induced genes that included IL-12, TNF-alpha, and IL-6 in primary bone marrow derived macrophages and LPS-induced IL-12/18-IFN-gamma signaling in Artd1(Delta Myel) mice. The loss of Artd1 in myeloid cells also reduced the T(H)1 response to Helicobacter pylori and impaired immune control of the bacteria. Furthermore, Artd1(Delta Myel) mice failed to control tumor growth in a s.c. MC-38 model of colon cancer, which could be attributed to reduced T(H)1 and CD8 responses. Together, these data provide strong evidence for a cell-intrinsic role of ARTD1 in myeloid cells that is independent of its enzymatic activity and promotes type I immunity by promoting IL-12/18 expression. AU - Kunze, F.A.* AU - Bauer, M.* AU - Komuczki, J.* AU - Lanzinger, M.* AU - Gunasekera, K.* AU - Hopp, A.K.* AU - Lehmann, M. AU - Becher, B.* AU - Müller, A.* AU - Hottiger, M.O.* C1 - 55576 C2 - 46424 CY - 9650 Rockville Pike, Bethesda, Md 20814 Usa SP - 1406-1416 TI - ARTD1 in myeloid cells controls the IL-12/18-IFN-γ axis in a model of sterile sepsis, chronic bacterial infection, and cancer. JO - J. Immunol. VL - 202 IS - 5 PB - Amer Assoc Immunologists PY - 2019 SN - 0022-1767 ER - TY - JOUR AB - Homing of pathogenic CD4+ T cells to the CNS is dependent on α4 integrins. However, it is uncertain whether α4 integrins are also required for the migration of dendritic cell (DC) subsets, which sample Ags from nonlymphoid tissues to present it to T cells. In this study, after genetic ablation of Itga4 in DCs and monocytes in mice via the promoters of Cd11c and Lyz2 (also known as LysM), respectively, the recruitment of α4 integrin-deficient conventional and plasmacytoid DCs to the CNS was unaffected, whereas α4 integrin-deficient, monocyte-derived DCs accumulated less efficiently in the CNS during experimental autoimmune encephalomyelitis in a competitive setting than their wild-type counterparts. In a noncompetitive setting, α4 integrin deficiency on monocyte-derived DCs was fully compensated. In contrast, in small intestine and colon, the fraction of α4 integrin-deficient CD11b+CD103+ DCs was selectively reduced in steady-state. Yet, T cell-mediated inflammation and host defense against Citrobacter rodentium were not impaired in the absence of α4 integrins on DCs. Thus, inflammatory conditions can promote an environment that is indifferent to α4 integrin expression by DCs. AU - Sie, C.* AU - Perez, L.G.* AU - Kreutzfeldt, M.* AU - Potthast, M. AU - Ohnmacht, C. AU - Merkler, D.* AU - Huber, S.* AU - Krug, A.* AU - Korn, T.* C1 - 56894 C2 - 47418 SP - 1417-1427 TI - Dendritic cell accumulation in the gut and central nervous system Is differentially dependent on α4 integrins. JO - J. Immunol. VL - 203 IS - 6 PY - 2019 SN - 0022-1767 ER - TY - JOUR AB - CMV is a prevalent human pathogen. The virus cannot be eliminated from the body, but is kept in check by CMV-specific T cells. Patients with an insufficient T cell response, such as transplant recipients, are at high risk of developing CMV disease. However, the CMV-specific T cell repertoire is complex, and it is not yet clear which T cells protect best against virus reactivation and disease. In this study, we present a highly resolved characterization of CMV-specific human CD8(+) T cells based on enrichment by specific peptide stimulation and mRNA sequencing of their TCR beta-chains (TCR beta). Our analysis included recently identified T cell epitopes restricted through HLA-C, whose presentation is resistant to viral immunomodulation, and well-studied HLA-B-restricted epitopes. In eight healthy virus carriers, we identified a total of 1052 CMV-specific TCR beta sequences. HLA-C-restricted, CMV-specific TCR beta clonotypes dominated the ex vivo T cell response and contributed the highest-frequency clonotype of the entire repertoire in two of eight donors. We analyzed sharing and similarity of CMV-specific TCR beta sequences and identified 63 public or related sequences belonging to 17 public TCR beta families. In our cohort, and in an independent cohort of 352 donors, the cumulative frequency of these public TCR beta family members was a highly discriminatory indicator of carrying both CMV infection and the relevant HLA type. Based on these findings, we propose CMV-specific TCR beta signatures as a biomarker for an antiviral T cell response to identify patients in need of treatment and to guide future development of immunotherapy. AU - Huth, A. AU - Liang, X. AU - Krebs, S.* AU - Blum, H.* AU - Moosmann, A. C1 - 55065 C2 - 46021 CY - 9650 Rockville Pike, Bethesda, Md 20814 Usa SP - 979-990 TI - Antigen-specific TCR signatures of cytomegalovirus infection. JO - J. Immunol. VL - 202 IS - 3 PB - Amer Assoc Immunologists PY - 2018 SN - 0022-1767 ER - TY - JOUR AB - Copyright © 2018 by The American Association of Immunologists, Inc. IL-7 therapy has been evaluated in patients who do not regain normal CD4 T cell counts after virologically successful antiretroviral therapy. IL-7 increases total circulating CD4 and CD8 T cell counts; however, its effect on HIV-specific CD8 T cells has not been fully examined. TRAF1, a prosurvival signaling adaptor required for 4-1BB-mediated costimulation, is lost from chronically stimulated virus-specific CD8 T cells with progression of HIV infection in humans and during chronic lymphocytic choriomeningitis infection in mice. Previous results showed that IL-7 can restore TRAF1 expression in virus-specific CD8 T cells in mice, rendering them sensitive to anti-4-1BB agonist therapy. In this article, we show that IL-7 therapy in humans increases the number of circulating HIV-specific CD8 T cells. For a subset of patients, we also observed an increased frequency of TRAF1+HIV-specific CD8 T cells 10 wk after completion of IL-7 treatment. IL-7 treatment increased levels of phospho-ribosomal protein S6 in HIV-specific CD8 T cells, suggesting increased activation of the metabolic checkpoint kinase mTORC1. Thus, IL-7 therapy in antiretroviral therapy-treated patients induces sustained changes in the number and phenotype of HIV-specific T cells. AU - Wang, C.* AU - Edilova, M.I.* AU - Wagar, L.E.* AU - Mujib, S.* AU - Singer, M.* AU - Bernard, N.F.* AU - Croughs, T.* AU - Lederman, M.M.* AU - Sereti, I.* AU - Fischl, M.A.* AU - Kremmer, E. AU - Ostrowski, M.A.* AU - Routy, J.P.* AU - Watts, T.H.* C1 - 52512 C2 - 44047 CY - Bethesda SP - 558-564 TI - Effect of IL-7 Therapy on Phospho-Ribosomal Protein S6 and TRAF1 Expression in HIV-Specific CD8 T Cells in Patients Receiving Antiretroviral Therapy. JO - J. Immunol. VL - 200 IS - 2 PB - Amer Assoc Immunologists PY - 2018 SN - 0022-1767 ER - TY - JOUR AB - Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation. However, the necessity of MASP-3 for pro-FD cleavage has been questioned, because AP activity is still observed in sera from MASP-1/3-deficient Malpuech-Michels-Mingarelli-Carnevale (3MC) patients. The identification of a novel 3MC patient carrying a previously unidentified MASP-3 G665S mutation prompted us to develop an analytical isoelectric focusing technique that resolves endogenous FD variants in complex samples. This enabled us to show that although 3MC patients predominantly contain pro-FD, they also contain detectable levels of mature FD. Moreover, using isoelectric focusing analysis, we show that both pro-FD and FD are present in the circulation of healthy donors. We characterized the naturally occurring 3MC-associated MASP-3 mutants and found that they all yielded enzymatically inactive proteins. Using MASP-3-depleted human serum, serum from 3MC patients, and Masp1/3(-/-) mice, we found that lack of enzymatically active MASP-3, or complete MASP-3 deficiency, compromises the conversion of pro-FD to FD. In summary, our observations emphasize that MASP-3 acts as an important maturase in the AP of complement, while also highlighting that there exists MASP-3-independent pro-FD maturation in 3MC patients. AU - Pihl, R.* AU - Jensen, L.* AU - Hansen, A.G.* AU - Thøgersen, I.B.* AU - Andres, S.* AU - Dagnæs-Hansen, F.* AU - Oexle, K. AU - Enghild, J.J.* AU - Thiel, S.* C1 - 51704 C2 - 43341 CY - Bethesda SP - 2158-2170 TI - Analysis of factor D isoforms in Malpuech-Michels-Mingarelli-Carnevale patients highlights the role of MASP-3 as a maturase in the alternative pathway of complement. JO - J. Immunol. VL - 199 IS - 6 PB - Amer Assoc Immunologists PY - 2017 SN - 0022-1767 ER - TY - JOUR AB - The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs. AU - Schuh, E.* AU - Musumeci, A.* AU - Thaler, F.S.* AU - Laurent, S.A.* AU - Ellwart, J.W. AU - Hohlfeld, R.* AU - Krug, A.* AU - Meinl, E.* C1 - 50693 C2 - 42826 SP - 3081-3088 TI - Human plasmacytoid dendritic cells display and shed B cell maturation antigen upon TLR engagement. JO - J. Immunol. VL - 198 IS - 8 PY - 2017 SN - 0022-1767 ER - TY - JOUR AU - Boeckl, K.* AU - Körber, N. AU - Barabas, S.* AU - Demi, L.* AU - Bauer, T. C1 - 49318 C2 - 33275 CY - Bethesda TI - An optimized IFN-gamma ELISpot assay using T-activated (R) proteins for determination of CMV-specific cell-mediated immunity. JO - J. Immunol. VL - 196 PB - Amer Assoc Immunologists PY - 2016 SN - 0022-1767 ER - TY - JOUR AB - The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs. Upon activation, mature skin dendritic cells fail to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response. AU - Foerster, R.* AU - Schubel, A.* AU - Breitfeld, D.* AU - Kremmer, E. AU - Renner-Mueller, I.* AU - Wolf, E.* AU - Lipp, M.* C1 - 47636 C2 - 39465 SP - 5-15 TI - CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs (Reprinted from Cell, vol 99, pg 23-33, 1999). JO - J. Immunol. VL - 196 IS - 1 PY - 2016 SN - 0022-1767 ER - TY - JOUR AU - Reissig, S.* AU - Nikolaev, A.* AU - Weigmann, B.* AU - Gerlach, K.* AU - Glasmacher, E. AU - Hoevelmeyer, N.* AU - Waisman, A.* C1 - 49319 C2 - 33278 CY - Bethesda TI - The transcriptional regulator Bcl-3 impaires the function of regulatory T cells leading to the development of intestinal inflammation. JO - J. Immunol. VL - 196 PB - Amer Assoc Immunologists PY - 2016 SN - 0022-1767 ER - TY - JOUR AB - Autoreactive CD4(+) T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4(+) T cells, and TCR α-chain next-generation sequencing to bulk memory CD4(+) T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4(+) T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer(+) cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer(+) CD4(+) T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001-1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies. AU - Eugster, A.* AU - Lindner, A.* AU - Catani, M.* AU - Heninger, A.K.* AU - Dahl, A.* AU - Klemroth, S.* AU - Kühn, D.* AU - Dietz, S.* AU - Bickle, M.* AU - Ziegler, A.-G. AU - Bonifacio, E. C1 - 43299 C2 - 36568 CY - Bethesda SP - 2531-2538 TI - High diversity in the TCR repertoire of GAD65 autoantigen-specific human CD4+ T cells. JO - J. Immunol. VL - 194 IS - 6 PB - Amer Assoc Immunologists PY - 2015 SN - 0022-1767 ER - TY - JOUR AB - Elevated levels of the transcription factor c-myc are strongly associated with various cancers, and in particular B cell lymphomas. Although many of c-MYC's functions have been elucidated, its effect on the presentation of Ag through the HLA class II pathway has not been reported previously. This is an issue of considerable importance, given the low immunogenicity of many c-MYC-positive tumors. We report in this paper that increased c-MYC expression has a negative effect on the ability of B cell lymphomas to functionally present Ags/peptides to CD4(+) T cells. This defect was associated with alterations in the expression of distinct cofactors as well as interactions of antigenic peptides with class II molecules required for the presentation of class II-peptide complexes and T cell engagement. Using early passage Burkitt's lymphoma (BL) tumors and transformed cells, we show that compared with B lymphoblasts, BL cells express decreased levels of the class II editor HLA-DM, lysosomal thiol-reductase GILT, and a 47-kDa enolase-like protein. Functional Ag presentation was partially restored in BL cells treated with a c-MYC inhibitor, demonstrating the impact of this oncogene on Ag recognition. This restoration of HLA class II-mediated Ag presentation in early passage BL tumors/cells was linked to enhanced HLA-DM expression and a concurrent decrease in HLA-DO in BL cells. Taken together, these results reveal c-MYC exerts suppressive effects at several critical checkpoints in Ag presentation, which contribute to the immunoevasive properties of BL tumors. AU - God, J.M.* AU - Cameron, C.* AU - Figueroa, J.* AU - Amria, S.* AU - Hossain, A.* AU - Kempkes, B. AU - Bornkamm, G.W. AU - Stuart, R.K.* AU - Blum, J.S.* AU - Haque, A.* C1 - 43357 C2 - 36357 CY - Bethesda SP - 1434-1445 TI - Elevation of c-MYC disrupts HLA class II-mediated immune recognition of human B cell tumors. JO - J. Immunol. VL - 194 IS - 4 PB - Amer Assoc Immunologists PY - 2015 SN - 0022-1767 ER - TY - JOUR AB - BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF-APRIL receptor TACI, was applied in clinical trials. However, disease activity in multiple sclerosis unexpectedly increased, whereas in systemic lupus erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-κB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF-APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted autoimmune disease multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation. AU - Hoffmann, F.S.* AU - Kühn, P.* AU - Laurent, S.A.* AU - Hauck, S.M. AU - Berer, K.* AU - Wendlinger, S.A.* AU - Krumbholz, M.* AU - Khademi, M.* AU - Olsson, T.* AU - Dreyling, M.* AU - Pfister, H.* AU - Alexander, T.* AU - Hiepe, F.* AU - Kümpfel, T.* AU - Crawford, H.C.* AU - Wekerle, H.* AU - Hohlfeld, R.* AU - Lichtenthaler, S.F.* AU - Meinl, E.* C1 - 43046 C2 - 35954 SP - 542-552 TI - The immunoregulator soluble TACI is released by ADAM10 and reflects B cell activation in autoimmunity. JO - J. Immunol. VL - 194 IS - 2 PY - 2015 SN - 0022-1767 ER - TY - JOUR AB - Efficient leukocyte migration is important for an effective host response to viral infection and the development of adaptive immunity. The poxvirus strain modified vaccinia virus Ankara (MVA), a safe and efficient viral vector, rapidly induces chemokine expression and respiratory recruitment of leukocytes, which is unique among vaccinia viruses. In addition to chemokines, the complement system contributes to the attraction and activation of different types of leukocytes. Using a murine model of intranasal infection, we show in this study that MVA-induced neutrophil recruitment depends on complement component C5. Remarkably, we find that C5 mediates neutrophil recruitment to the lung, even in the absence of the central complement component C3. Our findings argue for complement C5 activation during MVA infection of the lung via a C3-independent pathway, which enables rapid recruitment of neutrophils. AU - Price, P.J.* AU - Bánki, Z.* AU - Scheideler, A. AU - Stoiber, H.* AU - Verschoor, A.* AU - Sutter, G.* AU - Lehmann, M.H.* C1 - 43026 C2 - 35929 CY - Bethesda SP - 1164-1168 TI - Complement component C5 recruits neutrophils in the absence of C3 during respiratory infection with modified vaccinia virus Ankara. JO - J. Immunol. VL - 194 IS - 3 PB - Amer Assoc Immunologists PY - 2015 SN - 0022-1767 ER - TY - JOUR AB - Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and β-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections. AU - Sun, C.* AU - Schattgen, S.A.* AU - Pisitkun, P.* AU - Jorgensen, J.P.* AU - Hilterbrand, A.T.* AU - Wang, L.J.* AU - West, J.A.* AU - Hansen, K.* AU - Horan, K.A.* AU - Jakobsen, M.R.* AU - O'Hare, P.* AU - Adler, H. AU - Sun, R.* AU - Ploegh, H.L.* AU - Damania, B.* AU - Upton, J.W.* AU - Fitzgerald, K.A.* AU - Paludan, S.R.* C1 - 43124 C2 - 36026 CY - Bethesda SP - 1819-1831 TI - Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection. JO - J. Immunol. VL - 194 IS - 4 PB - Amer Assoc Immunologists PY - 2015 SN - 0022-1767 ER - TY - JOUR AB - V(D)J recombination assembles Ag receptor genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part by increasing chromatin accessibility of the locus, to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. In this article, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Dβ-Jβ-Cβ gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCβ clusters are highly enriched for Ser(5)-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Of interest, these features are shared with few other tissue-specific genes. We propose that the entire DJCβ regions behave as transcription "initiation" platforms, therefore linking a specialized mechanism of Pol II transcription with extended H3K4 trimethylation and highly accessible Dβ and Jβ gene segments. AU - Zacarias-Cabeza, J.* AU - Belhocine, M.* AU - Vanhille, L.* AU - Cauchy, P.* AU - Koch, F.* AU - Pekowska, A.* AU - Fenouil, R.* AU - Bergon, A.* AU - Gut, M.* AU - Gut, I.* AU - Eick, D. AU - Imbert, J.* AU - Ferrier, P.* AU - Andrau, J.C.* AU - Spicuglia, S.* C1 - 43554 C2 - 36670 CY - Bethesda SP - 3432-3443 TI - Transcription-dependent generation of a specialized chromatin structure at the TCRβ locus. JO - J. Immunol. VL - 194 IS - 7 PB - Amer Assoc Immunologists PY - 2015 SN - 0022-1767 ER - TY - JOUR AB - Immunoevasive proteins ("evasins") of human CMV (HCMV) modulate stability and localization of MHC class I (MHC I) molecules, and their supply of antigenic peptides. However, it is largely unknown to what extent these evasins interfere with recognition by virus-specific CD8 T cells. We analyzed the recognition of HCMV-infected cells by a panel of CD8 T cells restricted through one of nine different MHC I allotypes. We employed a set of HCMV mutants deleted for three or all four of the MHC I modulatory genes US2, US3, US6, and US11. We found that different HCMV evasins exhibited different allotype-specific patterns of interference with CD8 T cell recognition of infected cells. In contrast, recognition of different epitopes presented by the same given MHC I allotype was uniformly reduced. For some allotypes, single evasins largely abolished T cell recognition; for others, a concerted action of evasins was required to abrogate recognition. In infected cells whose Ag presentation efficiency had been enhanced by IFN-γ pretreatment, HCMV evasins cooperatively impared T cell recognition for several different MHC I allotypes. T cell recognition and MHC I surface expression under influence of evasins were only partially congruent, underscoring the necessity to probe HCMV immunomodulation using specific T cells. We conclude that the CD8 T cell evasins of HCMV display MHC I allotype specificity, complementarity, and cooperativity. AU - Ameres, S. AU - Besold, K.* AU - Plachter, B.* AU - Moosmann, A. C1 - 31326 C2 - 34341 CY - Bethesda SP - 5894-5905 TI - CD8 T cell-evasive functions of human cytomegalovirus display pervasive MHC allele specificity, complementarity, and cooperativity. JO - J. Immunol. VL - 192 IS - 12 PB - Amer Assoc Immunologists PY - 2014 SN - 0022-1767 ER - TY - JOUR AB - The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. AU - Lehnert, C.* AU - Weiswange, M.* AU - Jeremias, I. AU - Bayer, C.* AU - Grunert, M. AU - Debatin, K.M.* AU - Strauss, G.P.* C1 - 32183 C2 - 35436 CY - Bethesda SP - 4021-4031 TI - TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation. JO - J. Immunol. VL - 193 IS - 8 PB - Amer Assoc Immunologists PY - 2014 SN - 0022-1767 ER - TY - JOUR AB - CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs. AU - Luteijn, R.D.* AU - Hoelen, H.* AU - Kruse, E.* AU - van Leeuwen, W.F.* AU - Grootens, J.* AU - Horst, D.* AU - Koorengevel, M.* AU - Drijfhout, J.W.* AU - Kremmer, E. AU - Früh, K.* AU - Neefjes, J.J.* AU - Killian, A.* AU - Lebbink, R.J.* AU - Ressing, M.E.* AU - Wiertz, E.J.* C1 - 31776 C2 - 34730 CY - Bethesda SP - 1578-1589 TI - Cowpox virus protein CPXV012 eludes CTLs by blocking ATP binding to TAP. JO - J. Immunol. VL - 193 IS - 4 PB - Amer Assoc Immunologists PY - 2014 SN - 0022-1767 ER - TY - JOUR AB - Ag recognition via the TCR is necessary for the expansion of specific T cells that then contribute to adaptive immunity as effector and memory cells. Because CD4(+) and CD8(+) T cells differ in terms of their priming APCs and MHC ligands we compared their requirements of Ag persistence during their expansion phase side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal mouse T cells were thus analyzed after transient and continuous TCR signals. Following equally strong stimulation, CD4(+) T cell proliferation depended on prolonged Ag presence, whereas CD8(+) T cells were able to divide and differentiate into effector cells despite discontinued Ag presentation. CD4(+) T cell proliferation was neither affected by Th lineage or memory differentiation nor blocked by coinhibitory signals or missing inflammatory stimuli. Continued CD8(+) T cell proliferation was truly independent of self-peptide/MHC-derived signals. The subset divergence was also illustrated by surprisingly broad transcriptional differences supporting a stronger propensity of CD8(+) T cells to programmed expansion. These T cell data indicate an intrinsic difference between CD4(+) and CD8(+) T cells regarding the processing of TCR signals for proliferation. We also found that the presentation of a MHC class II-restricted peptide is more efficiently prolonged by dendritic cell activation in vivo than a class I bound one. In summary, our data demonstrate that CD4(+) T cells require continuous stimulation for clonal expansion, whereas CD8(+) T cells can divide following a much shorter TCR signal. AU - Rabenstein, H.* AU - Behrendt, A.C.* AU - Ellwart, J.W. AU - Naumann, R.* AU - Horsch, M. AU - Beckers, J. AU - Obst, R.* C1 - 31296 C2 - 34321 CY - Bethesda SP - 3507-3517 TI - Differential kinetics of antigen dependency of CD4+ and CD8+ T cells. JO - J. Immunol. VL - 192 IS - 8 PB - Amer Assoc Immunologists PY - 2014 SN - 0022-1767 ER - TY - JOUR AB - Autoantibodies targeting conformationally intact myelin oligodendrocyte glycoprotein (MOG) are found in different inflammatory diseases of the CNS, but their antigenic epitopes have not been mapped. We expressed mutants of MOG on human HeLa cells and analyzed sera from 111 patients (104 children, 7 adults) who recognized cell-bound human MOG, but had different diseases, including acute disseminated encephalomyelitis (ADEM), one episode of transverse myelitis or optic neuritis, multiple sclerosis (MS), anti-aquaporin-4 (AQP4)-negative neuromyelitis optica (NMO), and chronic relapsing inflammatory optic neuritis (CRION). We obtained insight into the recognition of epitopes in 98 patients. All epitopes identified were located at loops connecting the beta strands of MOG. The most frequently recognized MOG epitope was revealed by the P42S mutation positioned in the CC'-loop. Overall, we distinguished seven epitope patterns, including the one mainly recognized by mouse mAbs. In half of the patients, the anti-MOG response was directed to a single epitope. The epitope specificity was not linked to certain disease entities. Longitudinal analysis of 11 patients for up to 5 y indicated constant epitope recognition without evidence for intramolecular epitope spreading. Patients who rapidly lost their anti-MOG IgG still generated a long-lasting IgG response to vaccines, indicating that their loss of anti-MOG reactivity did not reflect a general lack of capacity for long-standing IgG responses. The majority of human anti-MOG Abs did not recognize rodent MOG, which has implications for animal studies. Our findings might assist in future detection of potential mimotopes and pave the way to Ag-specific depletion. AU - Mayer, M.C.* AU - Breithaupt, C.* AU - Reindl, M.* AU - Schanda, K.* AU - Rostasy, K.* AU - Berger, T.* AU - Dale, R.C.* AU - Brilot, F.* AU - Olsson, T.* AU - Jenne, D. AU - Pröbstel, A.K.* AU - Dornmair, K.* AU - Wekerle, H.* AU - Hohlfeld, R.* AU - Banwell, B.* AU - Bar-Or, A.* AU - Meinl, E.* C1 - 27886 C2 - 32846 SP - 3594-3604 TI - Distinction and temporal stability of conformational epitopes on myelin oligodendrocyte glycoprotein recognized by patients with different inflammatory central nervous system diseases. JO - J. Immunol. VL - 191 IS - 7 PB - Amer. Assoc. Immunologists PY - 2013 SN - 0022-1767 ER - TY - JOUR AB - Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1', P2', P3, P4, and P2 positions were tested. This enabled us to construct an α1-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils. AU - Perera, N.C. AU - Wiesmüller, K.H.* AU - Larsen, M.T.* AU - Schacher, B.* AU - Eickholz, P.* AU - Borregaard, N.* AU - Jenne, D. C1 - 27487 C2 - 32694 SP - 2700-2707 TI - NSP4 is stored in azurophil granules and released by activated neutrophils as active endoprotease with restricted specificity. JO - J. Immunol. VL - 191 IS - 5 PB - Amer. Assoc. Immunologists PY - 2013 SN - 0022-1767 ER - TY - JOUR AB - Transplacental immune regulation refers to the concept that during pregnancy, significant cross-talk occurs between the maternal and fetal immune system with potential long-term effects for both the mother and child. In this study, we made the surprising observation that there is a strong correlation of peripheral blood regulatory T (Treg) cells between the mother and the fetus. In contrast, there is no significant Treg cell correlation between paternal fetal dyads (pairs), suggesting that the specific context of pregnancy, rather than the genetic parental similarity to the fetus, is responsible for this correlation. Gene microarray analysis of Treg cells identified a typical IL-10-dependent signature in maternal and fetal Treg cells. In addition, a direct correlation of serum IL-10 protein levels between maternal fetal dyads was observed. Furthermore, we show that maternal serum IL-10 levels correlate with serum estradiol and estriol, implicating hormonal involvement in this alignment. Interestingly, we show that Treg cells possess higher expression of IL-10 receptor a and that Treg cell IL-10 receptor a expression directly correlates with their Bcl-2 expression. Indeed, in vitro data in both humans and mice demonstrate that IL-10 upregulates Bcl-2 specifically in Treg cells but not non-Treg cells. Our results provide evidence for transplacental regulation of cellular immunity and suggest that IL-10 may influence Treg cell homeostasis through its effect on Treg cell Bcl-2 expression. These novel findings have important implications on immune tolerance in pregnancy and beyond in areas of autoimmunity, allergy, and transplantation. AU - Santner-Nanan, B.* AU - Straubinger, K.* AU - Hsu, P.* AU - Parnell, G.* AU - Tang, B.* AU - Xu, B.* AU - Makris, A.* AU - Hennessy, A.* AU - Peek, M.J.* AU - Busch, D.H. AU - da Costa, C.P.* AU - Nanan, R.* C1 - 26280 C2 - 32163 SP - 145-153 TI - Fetal-maternal alignment of regulatory T cells correlates with IL-10 and bcl-2 upregulation in pregnancy. JO - J. Immunol. VL - 191 IS - 1 PB - Amer. Assoc. Immunologists PY - 2013 SN - 0022-1767 ER - TY - JOUR AB - Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock. AU - Siegfried, A.* AU - Berchtold, S.* AU - Manncke , B.* AU - Deuschle, E.* AU - Reber, J.* AU - Ott, T.* AU - Weber, M.* AU - Kalinke, U.* AU - Hofer, M.J.* AU - Hatesuer, B.* AU - Schughart, K.* AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Weber, F.* AU - Hornef, M.W.* AU - Autenrieth, I.B.* AU - Bohn, E.* C1 - 27741 C2 - 32797 SP - 3913-3921 TI - IFIT2 is an effector protein of type 1 IFN-mediated amplification of lipopolysaccharide (LPS)-induced TNF-α secretion and LPS-induced endotoxin shock. JO - J. Immunol. VL - 191 IS - 7 PB - Amer. Assoc. Immunologists PY - 2013 SN - 0022-1767 ER - TY - JOUR AB - Because of the numerous targets of microRNAs (miRNAs), functional dissection of specific miRNA/mRNA interactions is important to understand the complex miRNA regulatory mechanisms. Glycoprotein A repetitions predominant (GARP) is specifically expressed on regulatory CD25(+) CD4 T cells upon their activation. GARP has a long 39 untranslated region containing five highly conserved regions suggesting miRNA regulation of its expression. Although GARP is physiologically expressed on a cell subset characterized by stringent control of proliferation, amplification of the GARP gene has been found in many tumors characterized by uncontrolled proliferation. In this study, we investigated in detail miRNA regulation of GARP expression, in particular by miR-142-3p, and dissected the functional outcome of miR-142-3p/GARP mRNA interaction. We demonstrate that miR-142-3p binds directly to the 39 untranslated region of GARP and represses GARP protein expression by Argonaute 2-associated degradation of GARP mRNA. Functionally, miR-142-3p-mediated regulation of GARP is involved in the expansion of CD25(+) CD4 T cells in response to stimulation. The data indicate that miR-142-3p regulates GARP expression on CD25(+) CD4 T cells and, as a result, their expansion in response to activation. Our data provide novel insight into the molecular mechanisms controlling regulatory T cell expansion. They may also have implications for understanding tumor cell biology. AU - Zhou, Q.H.* AU - Haupt, S.* AU - Prots, I.* AU - Thummler, K.* AU - Kremmer, E. AU - Lipsky, P.E.* AU - Schulze-Koops, H.* AU - Skapenko, A.* C1 - 25912 C2 - 31988 SP - 6579-6588 TI - miR-142-3p is involved in CD25+ CD4 T cell proliferation by targeting the expression of glycoprotein A repetitions predominant. JO - J. Immunol. VL - 190 IS - 12 PB - Amer. Assoc. Immunologists PY - 2013 SN - 0022-1767 ER - TY - JOUR AB - Systemic bacterial infection is rapidly recognized as an emergency state leading to neutrophil release into the circulation and increased myeloid cell production within the bone marrow. However, the mechanisms of sensing infection and subsequent translation into emergency myelopoiesis have not been defined. In this study, we demonstrate in vivo in mice that, surprisingly, selective TLR4 expression within the hematopoietic compartment fails to induce LPS-driven emergency myelopoiesis. In contrast, TLR4-expressing nonhematopoietic cells are indispensable for LPS-induced, G-CSF-mediated myelopoietic responses. Furthermore, LPS-induced emergency myelopoiesis is independent of intact IL-1RI signaling and, thus, does not require inflammasome activation. Collectively, our findings reveal a key and nonredundant role for nonhematopoietic compartment pathogen sensing that is subsequently translated into cytokine release for enhanced, demand-adapted myeloid cell production. AU - Boettcher, S.* AU - Ziegler, P.* AU - Schmid, M.A.* AU - Takizawa, H.* AU - van Rooijen, N.* AU - Kopf, M.* AU - Heikenwälder, M. AU - Manz, M.G.* C1 - 7568 C2 - 29858 SP - 5824-5828 TI - Cutting edge: LPS-induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells. JO - J. Immunol. VL - 188 IS - 12 PB - Amer. Assoc. Immunologists PY - 2012 SN - 0022-1767 ER - TY - JOUR AB - The ubiquitin-editing enzyme A20 (TNFAIP3) and the deubiquitinase CYLD are central negative regulators of NF-κB signaling. Both can act by removing nonproteolytic K63-linked polyubiquitin chains from an overlapping set of signaling molecules. In B cells, A20 deficiency results in hyperactivity, loss of immune homeostasis, inflammation, and autoimmunity. The reported consequences of CYLD deficiency are controversial, ranging from an absence of effects to dramatic B cell hyperplasia. These differences could be due to varying compensation for the loss of CYLD function by A20. Therefore, to explore potential overlapping physiological functions between A20 and CYLD, we generated and characterized A20/CYLD double-deficient B cells. Interestingly, the lack of both A20 and CYLD did not exacerbate the developmental defects and hyperresponsive activity of A20-deficient B cells. In addition, the extent of B cell activation after in vitro stimulation with anti-CD40, LPS, and CpG was comparable in B cells lacking A20/CYLD and A20 alone. However, in response to BCR cross-linking, we observed small but reproducible additive effects of the lack of A20 and CYLD. Taken together, our results demonstrate that A20 and CYLD do not share significant functions during B cell development and activation. AU - Chu, Y.* AU - Soberon, V.* AU - Glockner, L. AU - Beyaert, R.* AU - Massoumi, R.* AU - van Loo, G.* AU - Krappmann, D. AU - Schmidt-Supprian, M.* C1 - 10544 C2 - 30288 SP - 4437-4443 TI - A20 and CYLD do not share significant overlapping functions during B cell development and activation. JO - J. Immunol. VL - 189 IS - 9 PB - Williams & Wilkins PY - 2012 SN - 0022-1767 ER - TY - JOUR AB - Although microRNA (miRNA) regulation of TLR signaling is well established, this has not yet been observed for NLR proteins or the inflammasomes they form. We have now validated a highly conserved miR-223 target site in the NLRP3 3'-untranslated region. miR-223 expression decreases as monocytes differentiate into macrophages, whereas NLRP3 protein increases during this time. However, overexpression of miR-223 prevents accumulation of NLRP3 protein and inhibits IL-1 beta production from the inflammasome. Virus inhibition of the inflammasome is an emerging theme, and we have also identified an EBV miRNA that can target the miR-223 binding site in the NLRP3 3'-untranslated region. Furthermore, this virus miRNA can be secreted from infected B cells via exosomes to inhibit the NLRP3 inflammasome in noninfected cells. Therefore, we have identified both the first endogenous miRNA that limits NLRP3 inflammatory capacity during myeloid cell development and also a viral miRNA that takes advantage of this, limiting inflammation for its own purposes. The Journal of Immunology, 2012, 189: 3795-3799. AU - Haneklaus, M.* AU - Gerlic, M.* AU - Kurowska-Stolarska, M.* AU - Rainey, A.A.* AU - Pich, D. AU - McInnes, I.B.* AU - Hammerschmidt, W. AU - O'Neill, L.A.J.* AU - Masters, S.L.* C1 - 10876 C2 - 30442 SP - 3795-3799 TI - Cutting edge: MiR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1β production. JO - J. Immunol. VL - 189 IS - 8 PB - Amer. Assoc. Immunologists PY - 2012 SN - 0022-1767 ER - TY - JOUR AB - Stimulation of the immune system by pathogens, allergens, or autoantigens leads to differentiation of CD4(+) T cells with pro- or anti-inflammatory effector cell functions. Based on functional properties and expression of characteristic cytokines and transcription factors, effector CD4(+) T cells have been grouped mainly into Th1, Th2, Th17, and regulatory T (Treg) cells. At least some of these T cell subsets remain responsive to external cues and acquire properties of other subsets, raising the hope that this functional plasticity might be exploited for therapeutic purposes. In this study, we used an Ag-specific adoptive transfer model and determined whether in vitro-polarized or ex vivo-isolated Th1, Th17, or Treg cells can be converted into IL-4-expressing Th2 cells in vivo by infection of mice with the gastrointestinal helminth Nippostrongylus brasiliensis. Th1 and Th17 cells could be repolarized to acquire the expression of IL-4 and lose the expression of their characteristic cytokines IFN-γ and IL-17A, respectively. In contrast, both in vitro-generated and ex vivo-isolated Treg cells were largely resistant to repolarization. The helminth-induced conversion of Th1 or Th17 cells into Th2 cells may partially explain the inverse correlation between helminth infection and protection against autoimmune disorders. AU - Panzer, M.* AU - Sitte, S.* AU - Wirth, S.* AU - Drexler, I. AU - Sparwasser, T.* AU - Voehringer, D.* C1 - 7190 C2 - 29535 SP - 615-623 TI - Rapid in vivo conversion of effector T cells into Th2 cells during helminth infection. JO - J. Immunol. VL - 188 IS - 2 PB - Amer. Assoc. Immunologists PY - 2012 SN - 0022-1767 ER - TY - JOUR AB - CD8+ tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-gamma production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase C gamma) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (I κ B, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8+ T cells infiltrating non-tumor kidney areas (CD8+ non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-alpha (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8+ T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy. AU - Prinz, P.U. AU - Mendler, A.N. AU - Masouris, I. AU - Durner, L.* AU - Oberneder, R.* AU - Nößner, E. C1 - 7569 C2 - 30168 SP - 5990-6000 TI - High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention. JO - J. Immunol. VL - 188 IS - 12 PB - Amer. Assoc. Immunologists PY - 2012 SN - 0022-1767 ER - TY - JOUR AU - Schendel, D.J. AU - Wilde, S. AU - Leisegang, M.* AU - Uckert, W.* C1 - 10877 C2 - 30485 SP - 3787-3788 TI - Response to comment on "Human antitumor CD8+ T cells producing Th1 polycytokines show superior antigen sensitivity and tumor recognition". JO - J. Immunol. VL - 189 IS - 8 PB - Amer. Assoc. Immunologists PY - 2012 SN - 0022-1767 ER - TY - JOUR AB - Adoptive transfer of T cells expressing transgenic TCR with antitumor specificity provides a hopeful new therapy for patients with advanced cancer. To fulfill a large need for TCR with high affinity and specificity for various tumor entities, we sought to identify parameters for rapid selection of CTL clones with suitable characteristics. Twelve CTL clones displaying different Ag sensitivities for the same peptide-MHC epitope of the melanoma-associated Ag tyrosinase were analyzed in detail. Better MHC-multimer binding and slower multimer release are thought to reflect stronger TCR-peptide-MHC interactions; thus, these parameters would seem well suited to identify higher avidity CTL. However, large disparities were found comparing CTL multimer binding with peptide sensitivity. In contrast, CD8(+) CTL with superior Ag sensitivity mediated good tumor cytotoxicity and also secreted the triple combination of IFN-gamma, IL-2, and TNF-alpha, representing a Th1 pattern often missing in lower avidity CTL. Furthermore, recipient lymphocytes were imbued with high Ag sensitivity, superior tumor recognition, as well as capacity for Th1 polycytokine secretion after transduction with the TCR of a high-avidity CTL. Thus, Th1 polycytokine secretion served as a suitable parameter to rapidly demark cytotoxic CD8(+) T cell clones for further TCR evaluation. The Journal of Immunology, 2012, 189: 598-605. AU - Wilde, S. AU - Sommermeyer, D.* AU - Leisegang, M.* AU - Frankenberger, B. AU - Mosetter, B. AU - Uckert, W.* AU - Schendel, D.J. C1 - 8233 C2 - 30035 SP - 598-605 TI - Human antitumor CD8+ T cells producing Th1 polycytokines show superior antigen sensitivity and tumor recognition. JO - J. Immunol. VL - 189 IS - 2 PB - Amer. Assoc Immunologists PY - 2012 SN - 0022-1767 ER - TY - JOUR AB - EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host. AU - Horst, D.* AU - Favaloro, V.* AU - Vilardi, F.* AU - van Leeuwen, H.C.* AU - Garstka, M.A.* AU - Hislop, A.D.* AU - Rabu, C.* AU - Kremmer, E. AU - Rickinson, A.B.* AU - High, S.* AU - Dobberstein, B.* AU - Ressing, M.E.* AU - Wiertz, E.J.* C1 - 6425 C2 - 28656 SP - 3594-3605 TI - EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP. JO - J. Immunol. VL - 186 IS - 6 PB - Amer Assoc Immunologists PY - 2011 SN - 0022-1767 ER - TY - JOUR AB - Regulatory T cells (Treg) are key players in maintaining immune homeostasis but have also been shown to regulate immune responses against infectious pathogens. Therefore, Treg are a promising target for modulating immune responses to vaccines to improve their efficacy. Using a viral vector system, we found that Treg act on the developing immune response early postinfection by reducing the extent of dendritic cell costimulatory molecule expression. Due to this change and the lower IL-2 production that results, a substantial fraction of CD8(+) effector T cells lose CD25 expression several days after activation. Surprisingly, such Treg-dependent limitations in IL-2 signaling by Ag-activated CD8(+) T cells prevent effector differentiation without interfering with memory cell formation. In this way, Treg fine-tune the numbers of effector T cells generated while preserving the capacity for a rapid recall response upon pathogen re-exposure. This selective effect of Treg on a subpopulation of CD8(+) T cells indicates that although manipulation of the Treg compartment might not be optimal for prophylactic vaccinations, it can be potentially exploited to optimize vaccine efficacy for therapeutic interventions. AU - Kastenmüller, W.* AU - Gasteiger, G. AU - Subramanian, N.* AU - Sparwasser, T.* AU - Busch, D.H. AU - Belkaid, Y.* AU - Drexler, I. AU - Germain, R.N.* C1 - 6489 C2 - 28780 SP - 3186-3197 TI - Regulatory T cells selectively control CD8+ T cell effector pool size via IL-2 restriction. JO - J. Immunol. VL - 187 IS - 6 PB - American Assoc. of Immunologists PY - 2011 SN - 0022-1767 ER - TY - JOUR AB - Plasmacytoid dendritic cells (PDCs) are capable of presenting Ags to T cells in a tolerogenic or immunogenic manner depending on the formulation of the Ag and the mode of stimulation. It has not been investigated whether effective adaptive immune responses useful for vaccination can be induced by Ab-mediated Ag targeting to PDCs in vivo. In this study, we show that Ag delivered to murine PDCs via bone marrow stromal cell Ag 2 (BST2)/CD317 in combination with TLR agonists as adjuvants is specifically presented by PDCs in vivo and elicits strong cellular and humoral immune responses. These include IFN-γ production by CD4(+) T cells and high Ab titers with a broad range of IgG isotypes. In addition, BST2-mediated Ag delivery in the presence of polyinosinic-polycytidylic acid as adjuvant induces cytotoxic T lymphocytes that are functional in vivo. A single immunization with Ag-fused anti-BST2 Ab together with polyinosinic-polycytidylic acid as adjuvant is sufficient to trigger protective immunity against subsequent viral infection and tumor growth. We conclude that despite the potential tolerogenic properties of PDCs, Ag targeting to PDCs in combination with TLR agonists as adjuvants is an effective vaccination strategy. AU - Loschko, J.* AU - Schlitzer, A.* AU - Dudziak, D.* AU - Drexler, I. AU - Sandholzer, N.* AU - Bourquin, C.* AU - Reindl, W.* AU - Krug, A.B.* C1 - 6387 C2 - 28550 SP - 6718-6725 TI - Antigen delivery to plasmacytoid dendritic cells via BST2 induces protective T cell-mediated immunity. JO - J. Immunol. VL - 186 IS - 12 PB - Amer Assoc Immunologists PY - 2011 SN - 0022-1767 ER - TY - JOUR AB - The B lymphocyte adaptor molecule of 32 kDa (Bam32) is strongly induced during the maturation of dendritic cells (DC). Most known functions of Bam32 are related to the signaling of the B cell receptor for Ag. Because DC do not express receptors specific for Ags, we aim at characterizing the role of Bam32 in human monocyte-derived DC in this study. Our results show that binding of allogeneic T cells to mature DC causes accumulation of Bam32 on the contact sites and that this translocation is mimicked by Ab-mediated engagement of MHC class I. Silencing of Bam32 in mature monocyte-derived DC results in an enhanced proliferation of CD8(+) T cells in an Ag-specific T cell proliferation assay. Further studies identify galectin-1 as an intracellular binding partner of Bam32. Regulating immune responses via regulatory T cell (Treg) modulation is one of the many immunological activities attributed to galectin-1. Therefore, we assayed mixed leukocyte reactions for Treg expansion and found fewer Treg in reactions stimulated with DC silenced for Bam32 compared to reactions stimulated with DC treated with a nontarget control. Based on our findings, we propose a role for Bam32 in the signaling of MHC class I molecules in professional Ag-presenting DC for the regulation of CD8(+) T cell activation. It is distinct from that of MHC class I recognized by CD8(+) T cells leading to T cell death. Thus, our data pinpoint a novel level of T cell regulation that may be of biological relevance. AU - Ortner, D.* AU - Grabher, D.* AU - Hermann, M.* AU - Kremmer, E. AU - Hofer, S.* AU - Heufler, C.* C1 - 6685 C2 - 29122 SP - 3972-3978 TI - The adaptor protein Bam32 in human dendritic cells participates in the regulation of MHC class I-induced CD8+ T cell activation. JO - J. Immunol. VL - 187 IS - 8 PB - Amer Assoc Immunologists PY - 2011 SN - 0022-1767 ER - TY - JOUR AU - Adler, H. C1 - 1167 C2 - 27307 SP - 1350-1351 TI - Comment on "Induction of TGF-β1, not regulatory T cells, impairs antiviral immunity in the lung following bone marrow transplant". JO - J. Immunol. VL - 185 IS - 3 PB - American Assoc. of Immunologists PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - The process of Th cell differentiation toward polarized effector T cells tailors specific immunity against invading pathogens while allowing tolerance against commensal microorganisms, harmless allergens, or autologous Ags. Identification of the mechanisms underlying this polarization process is therefore central to understand how the immune system confers immunity and tolerance. The present study demonstrates that retinoic acid receptor-related orphan receptor C2 (RORC2), a key transcription factor in Th17 cell development, inhibits FOXP3 expression in human T cells. Although overexpression of RORC2 in naive T cells reduces levels of FOXP3, small interfering RNA-mediated knockdown of RORC2 enhances its expression. RORC2 mediates this inhibition at least partially by binding to two out of four ROR-responsive elements on the FOXP3 promoter. Knockdown of RORC2 promotes high FOXP3 levels and decreased expression of proinflammatory cytokines beta form of pro-IL-1, IL-6, IL-17A, IFN-gamma, and TNF-alpha in differentiating naive T cells, suggesting that the role of RORC2 in Th17 cell development involves not only induction of Th17-characteristic genes, but also suppression of regulatory T cell-specific programs. Together, this study identifies RORC2 as a polarizing factor in transcriptional cross-regulation and provides novel viewpoints on the control of immune tolerance versus effector immune responses. AU - Burgler, S.* AU - Mantel, P.Y.* AU - Bassin, C.* AU - Ouaked, N.* AU - Akdis, C.A.* AU - Schmidt-Weber, C.B. C1 - 3407 C2 - 27906 SP - 6161-6169 TI - RORC2 is involved in T cell polarization through interaction with the FOXP3 promoter. JO - J. Immunol. VL - 184 IS - 11 PB - Amer. Assoc. Immunologists PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - Allergic contact dermatitis is a common disease caused by an exaggerated T cell-mediated immune response to skin-applied haptens. We show in this study that NK cells affect skin immune responses to haptens by releasing type 1 cytokines and inducing keratinocytes apoptosis. Immunohistochemical stainings demonstrated that NK lymphocytes constitute approximately 10% of the inflammatory infiltrate mostly distributed in the superficial dermis and in the epidermis at the site of intense spongiotic changes. More than 90% of NK cells isolated from allergic contact dermatitis skin showed a CD3-CD56(high)CD16- phenotype by FACS analysis. In addition, they uniformly expressed NKG2A, intermediate to high levels of perforin, and the activating receptors, NKG2D, NKp44, and NKp46, but lacked NKp30 and killer Ig-related receptors. Skin NK lymphocytes displayed a CXCR3+CCR6+CCR5+ chemokine receptor asset for homing into inflamed skin, but not CD62L and CCR7 for lymph node homing. When NK cells from nickel-allergic donors were exposed in vitro to the metal, they failed to proliferate, to upregulate CD69, and to release IFN-gamma, thus indicating that NK lymphocytes do not exhibit memory-like properties to haptens. However, IL-2 released by hapten-driven T lymphocytes rapidly induced the release of IFN-gamma by NK cells and promoted the NK-mediated apoptosis of autologous keratinocytes in a hapten-independent manner. Our findings underline the importance of the interaction between innate and adaptive immune mechanisms for amplification of skin allergic responses to haptens and full expression of allergic contact dermatitis. AU - Carbone, T.* AU - Nasorri, F.* AU - Pennino, D.* AU - Eyerich, K. AU - Foerster, S. AU - Cifaldi, L.* AU - Traidl-Hoffmann, C. AU - Behrendt, H. AU - Cavani, A.* C1 - 5267 C2 - 27879 SP - 1102-1110 TI - CD56highCD16-CD62L- NK cells accumulate in allergic contact dermatitis and contribute to the expression of allergic responses. JO - J. Immunol. VL - 184 IS - 2 PB - American Assoc. of Immunologists PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2(369)) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCR alpha-chain of this TCR specifically recognized HER2(369) not only in combination with the original beta-chain but also with four other beta-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one beta-chain derived from another HER2(369)-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells. AU - Liang, X. AU - Weigand, L.U. AU - Schuster, I.G. AU - Eppinger, E. AU - van der Griendt, J.C. AU - Schub, A. AU - Leisegang, M.* AU - Sommermeyer, D.* AU - Anderl, F.* AU - Han, Y.Y. AU - Ellwart, J.W. AU - Moosmann, A. AU - Busch, D.H. AU - Uckert, W.* AU - Peschel, C.* AU - Krackhardt, A.M. C1 - 1621 C2 - 27214 SP - 1617-1629 TI - A single TCRα-chain with dominant peptide recognition in the allorestricted HER2/neu-specific T cell repertoire. JO - J. Immunol. VL - 184 IS - 3 PB - American Assoc. of Immunologists PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - Although adoptive immunotherapy with CD8(+) CTL is providing clinically relevant results against EBV-driven malignancies, the effector role of CD4(+) T cells has been poorly investigated. We addressed this issue in a lymphoblastoid cell line-induced mouse model of posttransplant lymphoproliferative disease (PTLD) by comparing the therapeutic efficacy of EBV-specific CD4(+) and CD8(+) T cell lines upon adoptive transfer. CD4(+) T cells disclosed a long-lasting and stronger proliferative potential than CD8(+) T cells, had a similar activation and differentiation marker profile, efficiently killed their targets in a MHC class II-restricted manner, and displayed a lytic machinery comparable to that of cognate CD8(+) T cells. A detailed analysis of Ag specificity revealed that CD4(+) T cells potentially target EBV early lytic cycle proteins. Nonetheless, when assessed for the relative therapeutic impact after in vivo transfer, CD4(+) T cells showed a reduced activity compared with the CD8(+) CTL counterpart. This feature was apparently due to a strong and selective downmodulation of MHC class II expression on the tumor cells surface, a phenomenon that could be reverted by the demethylating agent 5-aza-2'-deoxycytidine, thus leading to restoration of lymphoblastoid cell line recognition and killing by CD4(+) T cells, as well as to a more pronounced therapeutic activity. Conversely, immunohistochemical analysis disclosed that HLA-II expression is fully retained in human PTLD samples. Our data indicate that EBV-specific cytotoxic CD4(+) T cells are therapeutic in mice bearing PTLD-like tumors, even in the absence of CD8(+) T cells. These findings pave the way to use cultures of pure CD4(+) T cells in immunotherapeutic approaches for EBV-related malignancies. AU - Merlo, A.* AU - Turrini, R.* AU - Bobisse, S.* AU - Zamarchi, R.* AU - Alaggio, R.* AU - Dolcetti, R.* AU - Mautner, J. AU - Zanovello, P.* AU - Amadori, A.* AU - Rosato, A.* C1 - 3118 C2 - 27960 SP - 5895-5902 TI - Virus-specific cytotoxic CD4⁺ T cells for the treatment of EBV-related tumors. JO - J. Immunol. VL - 184 IS - 10 PB - American Assoc. of Immunologists PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - Although semaphorins were-originally identified as axonal guidance molecules during neuronal development, it is emerging that several semaphorins play crucial roles in various phases of immune responses. Sema4D/CD100, a class IV semaphorin, has been shown to be involved in the nervous and immune systems through its receptors plexin-B1 and CD72, respectively. However, the involvement of Sema4D in neuroinflammation still remains unclear. We found that Sema4D promoted inducible NO synthase expression by primary mouse microglia, the effects of which were abolished in plexin-B1-deficient but not in CD72-deficient microglia. In addition, during the development of experimental autoimmune encephalomyelitis (EAE), which was induced by immunization with myelin oligodendrocyte glycoprotein-derived peptides, we observed that the expression of Sema4D and plexin-B1 was induced in infiltrating mononuclear cells and microglia, respectively. Consistent with these expression profiles, when myelin oligodendrocyte glycoprotein-specific T cells derived from wild-type mice were adoptively transferred into plexin-B1-deficient mice or bone marrow chimera mice with plexin-B1-deficient CNS resident cells, the development of EAE was considerably attenuated. Furthermore, blocking Abs against Sema4D significantly inhibited neuroinflammation during EAE development. Collectively, our findings demonstrate the role of Sema4D-plexin-B1 interactions in the activation of microglia and provide their pathologic significance in neuroinflammation. AU - Okuno, T.* AU - Nakatsuji, Y.* AU - Moriya, M.* AU - Takamatsu, H.* AU - Nojima, S.* AU - Takegahara, N.* AU - Toyofuku, T.* AU - Nakagawa, Y.* AU - Kang, S.* AU - Friedel, R.H. AU - Sakoda, S.* AU - Kikutani, H.* AU - Kumanogoh, A.* C1 - 5093 C2 - 28122 CY - Bethesda SP - 1499-1506 TI - Roles of Sema4D-plexin-B1 interactions in the central nervous system for pathogenesis of experimental autoimmune encephalomyelitis. JO - J. Immunol. VL - 184 IS - 3 PB - Amer. Assoc. Immunologists PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - Th17 is a newly identified lineage of effector T cells involved in autoimmunity and immune responses to pathogens. We demonstrate in this study the pathogenic role of IL-17-producing CD4(+) T lymphocytes in allergic contact dermatitis (ACD) to skin-applied chemicals. IL-17(+) T cells infiltrate ACD reactions and predominantly distribute at the site of heavy spongiosis. Skin IL-17(+) T cells were functionally and phenotypically heterogeneous: although pure Th17 prevailed in ACD skin, hapten responsiveness was restricted to Th1/IL-17 (IFN-gamma(+)IL-17(+)) and Th0/IL-17 (IFN-gamma(+)IL-17(+)IL-4(+)) fractions, and to lesser extent Th2/IL-17 cells. In the IFN-gamma-dominated ACD environment, IL-17-releasing T cells affect immune function of keratinocytes by promoting CXCL8, IL-6, and HBD-2 production. In addition, compared with Th1, supernatants from Th1/IL-17 T cells were much more efficient in inducing ICAM-1 expression on keratinocytes and keratinocyte-T cell adhesiveness in vitro. As a consequence, exposure to combined IFN-gamma and IL-17 rendered keratinocytes susceptible to ICAM-1-dependent Ag nonspecific T cell killing. Thus, IL-17 efficiently amplifies the allergic reaction by rendering virtually all of the T lymphocytes recruited at the site of skin inflammation capable to directly contribute to tissue damage. AU - Pennino, D.* AU - Eyerich, K.* AU - Scarponi, C.* AU - Carbone, T.* AU - Eyerich, S. AU - Nasorri, F.* AU - Garcovich, S.* AU - Traidl-Hoffmann, C. AU - Albanesi, C.* AU - Cavani, A.* C1 - 3139 C2 - 27898 SP - 4880-4888 TI - IL-17 amplifies human contact hypersensitivity by licensing hapten nonspecific Th1 cells to kill autologous keratinocytes. JO - J. Immunol. VL - 184 IS - 9 PB - American Associaton of Immunologists, Inc. PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - In this paper, we describe a new method for preparation of human dendritic cells (DCs) that secrete bioactive IL-12(p70) using synthetic immunostimulatory compounds as TLR7/8 agonists. Monocyte-derived DCs were generated using a procedure that provided mature cells within 3 d. Several maturation mixtures that contained various cytokines, IFN-gamma, different TLR agonists, and PGE(2) were compared for impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation. Mixtures that included the TLR7/8 agonists R848 or CL075, combined with the TLR3 agonist polyinosinic:polycytidylic acid, yielded 3-d mature DCs that secreted high levels of IL-12(p70), showed strong chemotaxis to CCR7 ligands, and had a positive costimulatory potential. They also had excellent capacity to activate NK cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-gamma and to induce T cell-mediated cytotoxic function. Thereby, mature DCs prepared within 3 d using such maturation mixtures displayed optimal functions required for vaccine development. AU - Spranger, S. AU - Javorovic, M. AU - Bürdek, M. AU - Wilde, S. AU - Mosetter, B. AU - Tippmer, S. AU - Bigalke, I. AU - Geiger, C. AU - Schendel, D.J. AU - Frankenberger, B. C1 - 5654 C2 - 27733 SP - 738-747 TI - Generation of Th1-polarizing dendritic cells using the TLR7/8 agonist CL075. JO - J. Immunol. VL - 185 IS - 1 PB - American Assoc. of Immunologistis PY - 2010 SN - 0022-1767 ER - TY - JOUR AB - Helicobacter pylori rapidly activates MAPKs and transcription factors, NF-kappaB and AP-1, in gastric epithelial cells following host attachment. Activation of these signal transducers is largely dependent on the cag pathogenicity island (cagPAI)-encoded Type IV Secretion System. H. pylori was shown to translocate peptidoglycan through the Type IV Secretion System, which is recognized by the pathogen recognition molecule, NOD1, thus resulting in NF-kappaB activation. The mechanisms of H. pylori-induced MAPK and AP-1 activation, however, are less well defined and therefore, we assessed the contribution of NOD1 to their activation. For this, we used gastric epithelial cell lines, stably expressing siRNA to either NOD1 or a control gene. In siNOD1-expressing cells stimulated with cagPAI(+) H. pylori, we observed significant reductions in p38 and ERK phosphorylation (p < 0.05), whereas the levels of Jnk phosphorylation remained unchanged. Consistent with a previous report, however, we were able to demonstrate NOD1-dependent Jnk phosphorylation by the invasive pathogen Shigella flexneri, highlighting pathogen-specific host responses to infection. We also show that NOD1 was essential for H. pylori induction of not only NF-kappaB, but also AP-1 activation, implying that NOD1 induces robust proinflammatory responses, in an attempt to rapidly control infection. Pharmacological inhibition of p38 and ERK activity significantly reduced IL-8 production in response to H. pylori, further emphasizing the importance of MAPKs in innate immune responses to the pathogen. Thus, for the first time we have shown the important role for NOD1 in MAPK and AP-1 activation in response to cagPAI(+) H. pylori. AU - Allison, C.C.* AU - Kufer, T.A.* AU - Kremmer, E. AU - Kaparakis, M.* AU - Ferrero, R.L.* C1 - 651 C2 - 26686 CY - United States SP - 8099-9109 TI - Helicobacter pylori induces MAPK phosphorylation and AP-1 activation via a NOD1-dependent mechanism. JO - J. Immunol. VL - 183 IS - 12 PB - American Assoc. of Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN-gamma production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants. AU - Bourquin, C.* AU - Schmidt, L.* AU - Lanz, A.-L.* AU - Storch, B.* AU - Wurzenberger, C.* AU - Anz, D.* AU - Sandholzer, N.* AU - Mocikat, R. AU - Berger, M.* AU - Poeck, H.* AU - Hartmann, G.* AU - Hornung, V.* AU - Endres, S.* C1 - 2408 C2 - 26573 CY - Bethesda SP - 6078-6086 TI - Immunostimulatory RNA oligonucleotides induce an effective antitumoral NK cell response through the TLR7. JO - J. Immunol. VL - 183 IS - 10 PB - American Assoc. of Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - The Carmal-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical I kappa B kinase (IKK)/NF-kappa B pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappa B response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappa B signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappa B signaling. AU - Düwel, M. AU - Welteke, V. AU - Oeckinghaus, A. AU - Baens, M.* AU - Kloo, B. AU - Ferch, U.* AU - Darnay, B.G.* AU - Ruland, J.* AU - Marynen, P.* AU - Krappmann, D. C1 - 1106 C2 - 26162 SP - 7718-7728 TI - A20 negatively regulates T cell receptor signaling to NF-κB by cleaving Malt1 ubiquitin chains. JO - J. Immunol. VL - 182 IS - 12 PB - Amer Assoc Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - In a humid milieu such as mucosal surfaces, pollen grains do not only release allergens but also proinflammatory and immunomodulatory lipids, termed pollen-associated lipid mediators. Among these, the E(1)-phytoprostanes (PPE(1)) were identified to modulate dendritic cell (DC) function: PPE(1) inhibit the DC's capacity to produce IL-12 and enhance DC mediated T(H)2 polarization of naive T cells. The mechanism(s) by which PPE(1) act on DC remained elusive. We thus analyzed candidate signaling elements and their role in PPE(1)-mediated regulation of DC function. Aqueous birch pollen extracts induced a marked cAMP response in DC that could be blocked partially by EP2 and EP4 antagonists. In contrast, PPE(1) hardly induced cAMP and the inhibitory effect on IL-12 production was mostly independent of EP2 and EP4. Instead, PPE(1) inhibited the LPS-induced production of IL-12 p70 by a mechanism involving the nuclear receptor PPAR-gamma. Finally, PPE(1) efficiently blocked NF-kappaB signaling in DCs by inhibiting IkappaB-alpha degradation, translocation of p65 to the nucleus, and binding to its target DNA elements. We conclude that pollen-derived PPE(1) modulate DC function via PPAR-gamma dependent pathways that lead to inhibition of NFkappaB activation and result in reduced DC IL-12 production and consecutive T(H)2 polarization. AU - Gilles, S. AU - Mariani, V.* AU - Bryce, M. AU - Mueller, M.J.* AU - Ring, J.* AU - Jakob, T.* AU - Pastore, S.* AU - Behrendt, H. AU - Traidl-Hoffmann, C. C1 - 48569 C2 - 41190 SP - 6653-6658 TI - Pollen-derived E1-phytoprostanes signal via PPAR-γ and NF-κB-dependent mechanisms. JO - J. Immunol. VL - 182 IS - 11 PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - EBV persists for life in the human host while facing vigorous antiviral responses that are induced upon primary infection. This persistence supports the idea that herpesviruses have acquired dedicated functions to avoid immune elimination. The recently identified EBV gene product BNLF2a blocks TAP. As a result, reduced amounts of peptides are transported by TAP from the cytoplasm into the endoplasmic reticulum (ER) lumen for binding to newly synthesized HLA class I molecules. Thus, BNLF2a perturbs detection by cytotoxic T cells. The 60-aa-long BNLF2a protein prevents the binding of both peptides and ATP to TAP, yet further mechanistic insight is, to date, lacking. In this study, we report that EBV BNLF2a represents a membrane-associated protein that colocalizes with its target TAP in subcellular compartments, primarily the ER. In cells devoid of TAP, expression levels of BNLF2a protein are greatly diminished, while ER localization of the remaining BNLF2a is retained. For interactions of BNLF2a with the HLA class I peptide-loading complex, the presence of TAP2 is essential, whereas tapasin is dispensible. Importantly, we now show that in B cells supporting EBV lytic replication, the BNLF2a protein is expressed early in infection, colocalizing and associating with the peptide-loading complex. These results imply that, during productive EBV infection, BNLF2a contributes to TAP inhibition and surface HLA class I down-regulation. In this way, EBV BNLF2a-mediated evasion from HLA class I-restricted T cell immunity contributes to creating a window for undetected virus production. AU - Horst, D.* AU - van Leeuwen, D.* AU - Croft, N.P.* AU - Garstka, M.A.* AU - Hislop, A.D.* AU - Kremmer, E. AU - Rickinson, A.B.* AU - Wiertz, E.J.* AU - Ressing, M.E.* C1 - 1619 C2 - 26852 SP - 2313-2324 TI - Specific targeting of the EBV lytic phase protein BNLF2a to the transporter associated with antigen processing results in impairment of HLA class I-restricted antigen presentation. JO - J. Immunol. VL - 182 IS - 4 PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - The size of the adaptive immune system is considered to be kept constant by the attrition of pre-existing memory. However, recently it was shown that the CD8 memory compartment can grow in size and the number of pre-existing memory is largely preserved, predicting that pre-existing immunity should be maintained (Vezys et al.; Nature 457: 196-199). Experimental proof for this assumption is still lacking. We address this question in the Listeria monocytogenes (L. m.) infection model and confirm the growth of size of the memory compartment by subsequent vaccination with modified vaccinia virus Ankara. We also find only modest attrition of pre-existing L.m.-specific memory CD8 T cells. However, preexisting protective immunity toward L.m. is not preserved. Pre-existing L.m.-specific effector-memory cells, in contrast to central memory cells, become altered, and this results in a significant loss of pre-existing protective immunity. Our findings are clinically relevant for vaccines introducing new CD memory cells in high numbers, as this might influence pre-existing immunity. AU - Huster, K.M. AU - Stemberger, C. AU - Gasteiger, G. AU - Kastenmüller, W. AU - Drexler, I. AU - Busch, D.H. C1 - 2957 C2 - 26733 SP - 6898-6902 TI - Cutting edge: Memory CD8 T cell compartment grows in size with immunological experience but nevertheless can lose function. JO - J. Immunol. VL - 183 IS - 11 PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - A major problem of current vaccines is storage stability, often requiring strict maintenance of cold chains. In the course of the eradication of smallpox, a freeze-dried vaccinia virus (Dryvax), which proved to be very stable, was used to overcome this limitation. However, Dryvax needs to be reconstituted before usage and is administered using a bifurcated needle, procedures that pose a number of additional health risks. We report in this study that a stable, lyophilized, modified vaccinia virus Ankara (MVA) vaccine can be directly applied to the nostrils of mice without previous reconstitution. This direct mucosal application induced systemic Ab and T cell responses comparable to those achieved by i.m. administration. Importantly, mucosal application of lyophilized MVA induced long-lasting protective immunity against lethal bacterial and viral challenges. These data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized MVA. AU - Kastenmüller, W. AU - Gasteiger, G. AU - Stross, L. AU - Busch, D.H. AU - Drexler, I. C1 - 549 C2 - 26153 SP - 2573-2577 TI - Cutting edge: Mucosal application of a lyophilized viral vector vaccine confers systemic and protective immunity toward intracellular pathogens. JO - J. Immunol. VL - 182 IS - 5 PB - Amer Assoc Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - Murine gamma-herpes virus 68 is a natural rodent pathogen closely related to the human gamma-herpes viruses Kaposi's sarcoma-associated herpes virus and EBV. By intranasally infecting wild-type and CCR7-deficient mice, we investigated whether CCR7 is necessary for viral clearance from the lung and the establishment of latency. We found during the lytic phase of infection that inflammation in lungs of CCR7(-/-) mice was more severe and viral load significantly higher compared with wild-type littermates. In addition, activation of T cells was delayed and clearance of the inflammation was retarded in mutant lungs, demonstrating that CCR7 is necessary for a rapid and efficient immune response. However, for the establishment of splenomegaly and latency, the presence of CCR7 was dispensable. Finally, by microdissecting BALT, we could demonstrate that these ectopic lymphoid structures are a place in the lung where virus resides during latency. AU - Kocks, J.R.* AU - Adler, H. AU - Danzer, H.* AU - Hoffmann, K.* AU - Jonigk, D.* AU - Lehmann, U.* AU - Forster, R.* C1 - 2052 C2 - 26175 SP - 6861-6869 TI - Chemokine receptor CCR7 contributes to a rapid and efficient clearance of lytic murine γ-herpes virus 68 from the lung, whereas bronchus-associated lymphoid tissue harbors virus during latency. JO - J. Immunol. VL - 182 IS - 11 PB - Amer Assoc Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - The MAPKs ERK, JNK, and p38 control diverse aspects of the immune response, including regulation of cytotoxin biology in NK cells and CTL. The chemokine CCL5 is coreleased with the cytotoxins, perforin, the granzymes, and granulysin, during the lethal hit administered by cytotoxic CD8(+) T cells (CTL). CCL5 expression is up-regulated relatively late in CTL coincident with their functional maturation 3-7 days after activation. Unlike T cells, NK cells have the ability to kill virally infected or transformed cells when directly isolated from the peripheral circulation. In this study, we show that in contrast to T cells, peripheral blood NK cells express CCL5 constitutively. The use of specific inhibitors of the JNK, ERK, and p38 MAPK pathways showed that the JNK pathway controls expression of CCL5 by NK cells. Promoter-reporter assays identified a compact region of the CCL5 promoter responsible for the constitutive transcription of CCL5 by NK cells. EMSA, chromatin immune precipitation, the use of heterologous promoters, and site-directed mutagenesis demonstrated that transcription in NK cells is largely controlled through binding of the transcription factor specificity protein 1 to a region -75 to -56 upstream of the site of transcriptional initiation. Specificity protein 1 expression, and in turn the constitutive expression of CCL5, was found to be controlled through constitutive activation of the JNK[MAPK pathway in peripheral blood NK cells. AU - Kumar, D.* AU - Hosse, J. AU - von Toerne, C.* AU - Nößner, E. AU - Nelson, P.J.* C1 - 2399 C2 - 26891 SP - 1011-1020 TI - JNK MAPK pathway regulates constitutive transcription of CCL5 by human NK cells through SP1. JO - J. Immunol. VL - 182 IS - 2 PB - Amer Assoc Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - For the efficient stimulation of T cells by tumor Ag, tumor-derived material has to be presented by dendritic cells (DC). This very likely involves the uptake of dead tumor cells by DC. Cell death in tumors often occurs through apoptosis, but necrotic cell death may also be prevalent. This distinction is relevant because numerous studies have proposed that apoptotic cells have immunosuppressive effects while necrosis may be stimulatory. However, a system has been lacking that would allow the induction of apoptosis or necrosis without side effects by the death stimuli used experimentally. In this study, we present such a system and test its effects on immune cells in vitro. B16 mouse melanoma cells were generated and underwent cell death through the doxycycline-inducible induction of death proteins. In one cell line, the induction of Bim(S), induced rapid apoptosis, in the other line the induction of the FADD death domain induced nonapoptotic/necrotic cell death. Bim(S)-induced apoptosis was associated with the typical morphological and biochemical changes. FADD death domain induced necrosis occurred through a distinct pathway involving RIP1 and the loss of membrane integrity in the absence of apoptotic changes. Apoptotic and necrotic cells were taken up with comparable efficiency by DC. OVA expressed in cells dying by either apoptosis or necrosis was cross-presented to OT-1 T cells and induced their proliferation. These results argue that it is not the form of cell death but its circumstances that decide the question whether cell death leads to a productive T cell response. AU - Lohmann, C.* AU - Muschaweckh, A.* AU - Kirschnek, S.* AU - Jennen, L. AU - Wagner, H.* AU - Häcker, G.* C1 - 1513 C2 - 26799 SP - 4538-4546 TI - Induction of tumor cell apoptosis or necrosis by conditional expression of cell death proteins: Analysis of cell death pathways and in vitro immune stimulatory potential. JO - J. Immunol. VL - 182 IS - 8 PB - Amer Assoc Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN-gamma and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory. AU - Schub, A. AU - Schuster, I.G. AU - Hammerschmidt, W. AU - Moosmann, A. C1 - 421 C2 - 27224 SP - 6819-6830 TI - CMV-specific TCR-transgenic T cells for immunotherapy. JO - J. Immunol. VL - 183 IS - 10 PB - American Assoc. of Immunologists PY - 2009 SN - 0022-1767 ER - TY - JOUR AB - The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that alpha(4)beta(7) integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, alpha(4)beta(1) integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site. AU - Berberich, S.* AU - Dähne, S.* AU - Schippers, A.* AU - Peters, T.* AU - Müller, W.* AU - Kremmer, E. AU - Forster, R.* AU - Pabst, O.* C1 - 3206 C2 - 25690 SP - 2196-2203 TI - Differential molecular and anatomical basis for B cell migration into the peritoneal cavity and omental milky spots. JO - J. Immunol. VL - 180 IS - 4 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2(369-377) epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-gamma treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2(369-377)-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3(356-364) and HER4(361-369) were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies. AU - Conrad, H. AU - Gebhard, K. AU - Krönig, H.* AU - Neudorfer, J.* AU - Busch, D.H. AU - Peschel, C.* AU - Bernhard, H. C1 - 4537 C2 - 25608 SP - 8135-8145 TI - CTLs directed against HER2 specifically cross-react with HER3 and HER4. JO - J. Immunol. VL - 180 IS - 12 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - The human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9(-/-) mice secreted reduced levels of IL-12, IFN-alpha, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9(-/-) and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9(-/-) mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity. AU - Guggemoos, S. AU - Hangel, D.* AU - Hamm, S.* AU - Heit, A.* AU - Bauer, S. AU - Adler, H. C1 - 3791 C2 - 24973 SP - 438-443 TI - TLR9 contributes to antiviral immunity during gammaherpesvirus infection. JO - J. Immunol. VL - 180 IS - 1 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - Various inflammatory diseases are characterized by tissue infiltration of neutrophils. Chemokines recruit and activate leukocytes, but neutrophils are traditionally known to be restricted in their chemokine receptor (CR) expression repertoire. Neutrophils undergo phenotypic and functional changes under inflammatory conditions, but the mechanisms regulating CR expression of infiltrated neutrophils at sites of chronic inflammation are poorly defined. Here we show that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis highly express CR on their surface that are absent or only marginally expressed on circulating neutrophils, i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4, as measured by flow cytometry, immunohistochemistry, and confocal microscopy. The induction of CR surface expression on infiltrated neutrophils was functionally relevant, because receptor activation by chemokine ligands ex vivo modulated neutrophil effector functions such as respiratory burst activity and bacterial killing. In vitro studies with isolated neutrophils demonstrated that the surface expression of CR was differentially induced in a cytokine-mediated, protein synthesis-dependent manner (CCR1, CCR3), through Toll-like (CXCR3) or NOD2 (CCR5) receptor engagement, through neutrophil apoptosis (CCR5, CXCR4), and/or via mobilization of intracellular CD63(+) granules (CXCR3). CR activation on infiltrated neutrophils may represent a key mechanism by which the local inflammatory microenvironment fine-tunes neutrophil effector functions in situ. Since the up-regulation of CR was exclusively found on infiltrated neutrophils at inflammatory sites in situ, the targeting of these G protein-coupled receptors may have the potential to site-specifically target neutrophilic inflammation. AU - Hartl, D. AU - Krauss-Etschmann, S. AU - Koller, B.* AU - Hordijk, P.L.* AU - Kuijpers, T.W.* AU - Hoffmann, F.* AU - Hector, A.* AU - Eber, E.* AU - Marcos, V.* AU - Bittmann, I.* AU - Eickelberg, O. AU - Griese, M.* AU - Roos, D.* C1 - 3715 C2 - 25926 SP - 8053-8067 TI - Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases. JO - J. Immunol. VL - 181 IS - 11 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-kappaB, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-kappaB but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin. AU - Kalali, B.N.* AU - Köllisch, G.V. AU - Mages, J.* AU - Müller, T.* AU - Bauer, S.* AU - Wagner, H.* AU - Ring, J. AU - Lang, R.* AU - Mempel, M.* AU - Ollert, M.* C1 - 1788 C2 - 25593 SP - 2694-2704 TI - Double-stranded RNA induces an antiviral defense status in epidermal keratinocytes through TLR3-, PKR-, and MDA5/RIG-I-mediated differential signaling. JO - J. Immunol. VL - 181 IS - 4 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines. AU - Meyer, V.S.* AU - Kastenmüller, W. AU - Gasteiger, G. AU - Franz-Wachtel, M.* AU - Lamkemeyer, T.* AU - Rammensee, H.-G.* AU - Stevanovic, S.* AU - Siguardardottir, D.* AU - Drexler, I. C1 - 3274 C2 - 25933 SP - 6371-6383 TI - Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling. JO - J. Immunol. VL - 181 IS - 9 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-alpha production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system. AU - Vega, V.L.* AU - Rodríguez-Silva, M.* AU - Frey, T.* AU - Gehrmann, M. AU - Diaz, J.C.* AU - Steinem, C.* AU - Multhoff, G. AU - Arispe, N.* AU - de Maio, A.* C1 - 2660 C2 - 25203 SP - 4299-4307 TI - Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. JO - J. Immunol. VL - 180 IS - 6 PB - American Assoc. of Immunologists PY - 2008 SN - 0022-1767 ER - TY - JOUR AB - Dendritic cells (DC) are able to capture, process, and present exogenous Ag to CD8(+) T lymphocytes through MHC class I, a process referred to as cross-presentation. In this study, we demonstrate that CD103(+) (CD11c(high)CD11b(low)) and CD103(-) (CD11c(int)CD11b(high)) DC residing in the lung-draining bronchial lymph node (brLN) have evolved to acquire opposing functions in presenting innocuous inhaled Ag. Thus, under tolerogenic conditions, CD103(-) DC are specialized in presenting innocuous Ag to CD4(+) T cells, whereas CD103(+) DC, which do not express CD8alpha, are specialized in presenting Ag exclusively to CD8(+) T cells. In CCR7-deficient but not in plt/plt mice, Ag-carrying CD103(+) DC are largely absent in the brLN, although CD103(+) DC are present in the lung of CCR7-deficient mice. As a consequence, adoptively transferred CD8(+) T cells can be activated under tolerizing conditions in plt/plt but not in CCR7-deficient mice. These data reveal that CD103(+) brLN DC are specialized in cross-presenting innocuous inhaled Ag in vivo. Because these cells are largely absent in CCR7(-/-) mice, our findings strongly suggest that brLN CD103(+) DC are lung-derived and that expression of CCR7 is required for their migration from the lung into its draining lymph node. AU - del Rio, M.-L.* AU - Rodriguez-Barbosa, J.-I.* AU - Kremmer, E. AU - Forster, R.* C1 - 2124 C2 - 24802 SP - 6861-6866 TI - CD103- and CD103+ bronchial lymph node dendritic cells are specialized in presenting and cross-presenting innocuous antigen to CD4+ and CD8+ T cells. JO - J. Immunol. VL - 178 IS - 11 PB - American Assoc. of Immunologists PY - 2007 SN - 0022-1767 ER - TY - JOUR AB - The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease. AU - Elsner, L.* AU - Muppala, V.* AU - Gehrmann, M. AU - Lozano, J.* AU - Malzahn, D.* AU - Bickeböller, H.* AU - Brunner, E.* AU - Zientkowska, M.* AU - Herrmann, T.* AU - Walter, L.* AU - Alves, F.* AU - Multhoff, G. AU - Dressel, R.* C1 - 2682 C2 - 24692 SP - 5523-5533 TI - The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands. JO - J. Immunol. VL - 179 IS - 8 PB - American Assoc. of Immunologists PY - 2007 SN - 0022-1767 ER - TY - JOUR AB - Prion diseases are fatal neurodegenerative diseases that are characterized by the conformational conversion of the normal, mainly {alpha}-helical cellular prion protein (PrP) into the abnormal beta-sheet-rich infectious isoform (PrPSc). The immune system neither shows reaction against cellular PrP nor PrPSc, most likely due to profound self-tolerance. In previous studies, we were able to partly overcome self-tolerance using recombinantly expressed dimeric PrP (tandem PrP (tPrP)), in association with different adjuvants. Proof of principle for antiprion efficacy was obtained in vitro and in vivo. In this study, we demonstrate the induction of a specific Th1 T cell response in wild-type mice immunized with tPrP and CpG-oligonucleotide (ODN). Biochemical influences such as refolding conditions, ionic strength, pH, and interaction with CpG-ODN affected antigenic structure and thus improved immunogenicity. Furthermore, s.c. immunization with tPrP and CpG-ODN coencapsulated in biodegradable polylactide-coglycolide microspheres (PLGA-MS) enhanced CD4 T cell responses and, more prominent, the induction of CD8 T cells. In this vaccination protocol, PLGA-MS function as endosomal delivery device of Ag plus CpG-ODN to macrophages and dendritic cells. In contrast, PLGA-MS-based DNA vaccination approaches with a tPrP construct generated poor humoral and T cell responses. Our data show that prophylactic and therapeutic immunization approaches against prion infections might be feasible using tPrP Ag and CpG-ODN adjuvant without detectable side effects. AU - Kaiser-Schulz, G.* AU - Heit, A.* AU - Quintanilla-Martinez, L. AU - Hammerschmidt, F.* AU - Hess, S.* AU - Jennen, L. AU - Rezaei, H.* AU - Wagner, H.* AU - Schatzl, H.M.* C1 - 5859 C2 - 24539 SP - 2797-2807 TI - Polylactide-Coglycolide Microspheres CoEncapsulating Recombinant Tandem Prion Protein with CpG-Oligonucleotide Break Self-Tolerance to Prion Protein in Wild-Type Mice and Induce CD4 and CD8 T Cell Responses. JO - J. Immunol. VL - 179 PB - American Assoc. of Immunologists PY - 2007 SN - 0022-1767 ER - TY - JOUR AB - The immune response of atopic individuals against allergens is characterized by increased levels of Th2 cytokines and chemokines. However, the way in which the cytokine/chemokine profile is matched to the type of invading allergen, and why these profiles sometimes derail and lead to disease, is not well understood. We recently demonstrated that pollen modulates dendritic cell (DC) function in a way that results in an enhanced capacity to initiate Th2 responses in vitro. Here, we examined the effects of aqueous birch pollen extracts (Bet.-APE) on chemokine receptor expression and chemokine production by human monocyte-derived DCs. Bet.-APE strongly induced expression and function of CXCR4 and reduced CCR1 and CCR5 expression on immature DCs. In addition, DC treatment with Bet.-APE significantly reduced LPS-induced production of CXCL10/IP-10, CCL5/RANTES; induced CCL22/macrophage-derived chemokine; and did not significantly change release of CCL17/thymus and activation-regulated chemokine. At a functional level, Bet.-APE increased the capacity of LPS-stimulated DCs to attract Th2 cells, whereas the capacity to recruit Th1 cells was reduced. Bet.-APE significantly and dose-dependently enhanced intracellular cAMP, suggesting that water-soluble factors from pollen grains bind a G(alphas)-protein-coupled receptor. E(1)-Phytoprostanes were identified to be one player in the Th2-polarizing potential of aqueous pollen extracts. In summary, our results demonstrate that pollen itself releases regulatory mediators which generate a Th2-promoting micromilieu with preferential recruitment of Th2 cells to the site of pollen exposure. AU - Mariani, V. AU - Gilles, S. AU - Jakob, T. AU - Thiel, M. AU - Mueller, M.J.* AU - Ring, J. AU - Behrendt, H. AU - Traidl-Hoffmann, C. C1 - 1379 C2 - 24731 SP - 7623-7631 TI - Immunomodulatory mediators from pollen enhance the migratory capacity of dendritic cells and license them for Th2 attraction. JO - J. Immunol. VL - 178 IS - 12 PB - American Assoc. of Immunologists PY - 2007 SN - 0022-1767 ER - TY - JOUR AB - CD4+CD25(high) forkhead box P3+ regulatory T cells (Treg) are critical mediators of peripheral self-tolerance and immune homeostasis. Treg suppress proliferation and cytokine production of conventional T cells (Tcon). The exact mechanism of suppression, however, is still unknown. To gain a better understanding of Treg function, we investigated the kinetics of cytokine suppression in Tcon reisolated from cocultures with preactivated human Treg. Treg inhibited induction of Th1 cytokine mRNA as early as 1 h after stimulation, whereas induction/suppression of Th2 cytokines was delayed to 10-15 h. We show that immediate cytokine mRNA suppression in Tcon was neither dependent on TGF-beta/IL-10 or IL-2 consumption, nor on induction of the transcriptional-repressor forkhead box P3 or other anergy-related genes (e.g., gene related to anergy, transducer of ErbB-2, forkhead homolog-4, repressor of GATA, inducible cAMP early repressor). In contrast, lymphocyte activation gene 3, suppressor of cytokine signaling 1, and suppressor of cytokine signaling 3 mRNA were strongly up-regulated in Tcon in the presence of Treg. However, protein analysis did not confirm a role for these proteins in early suppression. Thus, the identification of a fast inhibitory mechanism in Tcon induced by Treg constitutes an important step for future efforts to unravel the entire elusive suppressive mechanism. AU - Oberle, N.* AU - Eberhardt, N.* AU - Falk, C.S. AU - Krammer, P.H.* AU - Suri-Payer, E.* C1 - 3153 C2 - 24828 SP - 3578-3587 TI - Rapid suppression of cytokine transcription in human CD4⁺CD25ˉ T cells by CD4⁺Foxp3⁺ regulatory T cells: Independence of IL-2 consumption, TGF-β, and various inhibitors of TCR signaling. JO - J. Immunol. VL - 179 IS - 6 PB - American Assoc. of Immunologists PY - 2007 SN - 0022-1767 ER - TY - JOUR AB - L-10 is an important immunosuppressive cytokine that can down-regulate expression of other cytokines and has been shown to down-regulate itself. We show, in this study, that treatment of human monocyte-derived macrophages with IL-10 induces IL-10 mRNA in a dose- and time-dependent manner with an optimum induction at 100 ng/ml and at 6 h, whereas IL-10-induced IL-10 protein can be detected at 18 h. In the same cells, IL-10 can partially suppress IL-10 mRNA induced by LPS, but only down to the level of IL-10-induced IL-10. An adenoviral luciferase reporter construct driven by the –195 IL-10 promoter, which contains a Stat motif, was readily induced by both IL-10 and LPS. Mutation of this Stat motif ablated IL-10 activation of this promoter, but not the LPS activation. Finally, we show that overexpression of a dominant-negative Stat3 protein will prevent IL-10 induction, but not LPS induction, of IL-10 mRNA. These data show that IL-10 induces IL-10 in monocyte-derived macrophages in an autocrine manner via activation of the transcription factor Stat3. AU - Staples, K.J.* AU - Smallie, T.* AU - Williams, L.M.* AU - Foey, A.* AU - Burke, B.* AU - Foxwell, B.M.J.* AU - Ziegler-Heitbrock, L. C1 - 3932 C2 - 24405 SP - 4779-4785 TI - IL-10 Induces IL-10 in Primary Human Monocyte-Derived Macrophages via the Transcription Factor Stat3. JO - J. Immunol. VL - 178 PB - American Assoc. of Immunologists PY - 2007 SN - 0022-1767 ER - TY - JOUR AB - Dendritic cells (DC) are key regulators of T cell immunity and tolerance. NKT cells are well-known enhancers of Th differentiation and regulatory T cell function. However, the nature of the DC directing T and NKT cell activation and polarization as well as the role of the respective CD1d Ags presented is still unclear. In this study, we show that peptide-specific CD4(+)IL-10(+) T cell-mediated full experimental autoimmune encephalomyelitis (EAE) protection by TNF-treated semimatured DCs was dependent on NKT cells recognizing an endogenous CD1d ligand. NKT cell activation by TNF-matured DCs induced high serum levels of IL-4 and IL-13 which are absent in NKT cell-deficient mice, whereas LPS plus anti-CD40-treated fully mature DCs induce serum IFN-gamma. In the absence of IL-4Ralpha chain signaling or NKT cells, no complete EAE protection was achieved by TNF-DCs, whereas transfer of NKT cells into Jalpha281(-/-) mice restored it. However, activation of NKT cells alone was not sufficient for EAE protection and early serum Th2 deviation. Simultaneous activation of NKT cells and CD4(+) T cells by the same DC was required for EAE protection. Blocking experiments demonstrated that NKT cells recognize an endogenous glycolipid presented on CD1d on the injected DC. Together, this indicates that concomitant and interdependent presentation of MHC II/self-peptide and CD1d/self-isoglobotrihexosylceramide to T and NKT cells by the same partially or fully matured DC determines protective and nonprotective immune responses in EAE. AU - Wiethe, C.* AU - Schiemann, M. AU - Busch, D. AU - Haeberle, L.* AU - Kopf, M.* AU - Schuler, G.* AU - Lutz, M.B.* C1 - 2651 C2 - 25031 SP - 4908-4916 TI - Interdependency of MHC class II/self-peptide and CD1d/self-glycolipid presentation by TNF-matured dendritic cells for protection from autoimmunity. JO - J. Immunol. VL - 178 IS - 8 PY - 2007 SN - 0022-1767 ER - TY - JOUR AU - Duluvio, L.* AU - Vollmer, S.* AU - Besgen, P.* AU - Ellwart, J.W. AU - Chimenti, S.* AU - Prinz, J.C.* C1 - 4091 C2 - 23562 SP - 7104-7111 TI - Identical TCR β-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris. JO - J. Immunol. VL - 176 PB - American Assoc. of Immunologists PY - 2006 SN - 0022-1767 ER - TY - JOUR AU - Saribasak, H. AU - Saribasak, N.N. AU - Ipek, F.M. AU - Ellwart, J.W. AU - Arakawa, H. AU - Buerstedde, J.-M. C1 - 4751 C2 - 23206 SP - 365-371 TI - Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations. JO - J. Immunol. VL - 176 PB - American Assoc. of Immunologists PY - 2006 SN - 0022-1767 ER - TY - JOUR AU - Schoetz, U. AU - Cervelli, M.* AU - Wang, Y.-D.* AU - Fiedler, P. AU - Buerstedde, J.-M. C1 - 1761 C2 - 24147 SP - 395-400 TI - E2A expression stimulates Ig hypermutation. JO - J. Immunol. VL - 177 PB - American Assoc. of Immunologists PY - 2006 SN - 0022-1767 ER - TY - JOUR AB - TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-kappaB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta. In this study, we have shown that a kappaB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-kappaB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPbeta-p65 complexes. Remarkably, in C/EBPbeta(-/-) cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPbeta overexpression reduced p65-mediated transactivation and that association of C/EBPbeta with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPbeta is an essential signaling component for inhibition of NF-kappaB-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis. AU - Zwergal, A.* AU - Quirling, M.* AU - Saugel, B.* AU - Huth, K.C.* AU - Sydlik, C.* AU - Poli, V.* AU - Neumeier, D.* AU - Ziegler-Heitbrock, L. AU - Brand, K.* C1 - 4937 C2 - 23898 SP - 665-672 TI - C/EBPß blocks p65 phosphorylation and thereby NF-kB-mediated transcription in TNF-tolerant cells. JO - J. Immunol. VL - 177 IS - 1 PB - American Assoc. of Immunologists PY - 2006 SN - 0022-1767 ER - TY - JOUR AU - Dudziak, D.* AU - Nimmerjahn, F.* AU - Bornkamm, G.W. AU - Laux, G. C1 - 3514 C2 - 22645 SP - 6672-6676 TI - Alternative splicing generates putative soluble CD83 proteins that inhibit T cell proliferation. JO - J. Immunol. VL - 174 PB - American Assoc. of Immunologists PY - 2005 SN - 0022-1767 ER - TY - JOUR AU - Heit, A.* AU - Schmitz, F.* AU - O'Keeffe, M.* AU - Staib, C.* AU - Busch, D.H. AU - Wagner, H.* AU - Huster, K.M. C1 - 4642 C2 - 23312 SP - 4373-4380 TI - Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines. JO - J. Immunol. VL - 174 PB - American Assoc. of Immunologists PY - 2005 SN - 0022-1767 ER - TY - JOUR AU - Mack, M.* AU - Schneider, M.A.* AU - Moll, C.* AU - Cihak, J.* AU - Brühl, H.* AU - Ellwart, J.W. AU - Hogarth, M.P.* AU - Stangassinger, M.* AU - Schlöndorff, D.* C1 - 1125 C2 - 22353 SP - 735-741 TI - Identification of antigen-capturing cells as basophils. JO - J. Immunol. VL - 174 PB - American Assoc. of Immunologists PY - 2005 SN - 0022-1767 ER - TY - JOUR AU - Rodriguez, N.* AU - Fend, F. AU - Jennen, L. AU - Schiemann, M.* AU - Wantia, N.* AU - Prazeres da Costa, C.U.* AU - Dürr, S.* AU - Heinzmann, U. AU - Wagner, H.* AU - Miethke, T.* C1 - 4199 C2 - 22705 SP - 4836-4844 TI - Polymorphonuclear neutrophils improve replication of Chlamydia pneumoniae in vivo upon MyD88-dependent attraction. JO - J. Immunol. VL - 174 PY - 2005 SN - 0022-1767 ER - TY - JOUR AU - Brühl, H.* AU - Cihak, J.* AU - Schneider, M.A.* AU - Plachy, J.* AU - Rupp, T.* AU - Wenzel, I.* AU - Shakarami, M.* AU - Milz, S.* AU - Ellwart, J.W. AU - Stangassinger, M.* AU - Schlöndorff, D.* C1 - 4101 C2 - 22352 SP - 890-898 TI - Dual role of CCR2 during initiation and progression of collagen-induced arthritis: Evidencefor regulatory activity of CCR2+ T cells. JO - J. Immunol. VL - 172 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AU - Fleischer, K.* AU - Schmidt, B.* AU - Kastenmüller, W.* AU - Busch, D. AU - Drexler, I. AU - Sutter, G. AU - Heike, M.* AU - Peschel, C.* AU - Bernhard, H.* C1 - 3710 C2 - 22056 SP - 162-169 TI - Melanoma-reactive class I-restricted cytotoxic T cell clones are stimulated by dendritic cells loaded with synthetic peptides, but fail to respond to dendritic cells pulsed with melanoma-derived heat shock proteins in vitro. JO - J. Immunol. VL - 172 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AB - Profiling of surface-bound proteins uncovers a tumor-selective heat shock protein 70 (Hsp70) membrane expression that provides a target structure for human NK cells. Hsp70 peptide TKD (TKDNNLLGRFELSG; aa 450-463) was found to enhance the cytolytic activity of NK cells. In this study, we demonstrate that TKD-activated CD3-CD56+CD94+ NK cells are selectively attracted by Hsp70 membrane-positive tumor cells, and supernatants derived thereof. Hsp70 membrane-negative tumors failed to attract these NK cells. The capacity to migrate was associated with a substantial lytic activity against Hsp70-positive tumor cells. Because NK cell migration was independent of cell-to-cell contact, the involvement of a soluble factor was assumed. Interestingly, synthetic Hsp70 protein and Hsp70 peptide TKD, mimicking surface-bound Hsp70, initiates migration of NK cells in a concentration-dependent (1-5 microg/ml), highly selective, and chemokine-independent manner. In summary, our results indicate that Hsp70 peptide TKD not only stimulates cytolysis but also chemotaxis in CD3-CD56+CD94+ NK cells. AU - Gastpar, R.* AU - Gross, C.* AU - Rossbacher, L.* AU - Ellwart, J.W. AU - Riegger, J.* AU - Multhoff, G.* C1 - 3477 C2 - 21680 SP - 972-980 TI - The cell surface-localized heat shock protein 70 epitope TKD induces migration and cytolytic activity selectively in human NK cells. JO - J. Immunol. VL - 172 IS - 2 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AU - Markel, G.* AU - Gruda, R.* AU - Achdout, H.* AU - Katz, G.* AU - Nechama, M.* AU - Blumberg, R.S.* AU - Kammerer, R. AU - Zimmermann, W.* AU - Mandelboim, O.* C1 - 3378 C2 - 22384 SP - 3732-3739 TI - The critical role of residues 43R and 44Q of carcinoembryonic antigen cell adhesion molecules-1 in the protection from killing by human NK cells. JO - J. Immunol. VL - 173 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AU - Roth, W.* AU - Sustmann, C.* AU - Kieslinger, M.* AU - Gilmozzi, A.* AU - Irmer, D.* AU - Kremmer, E. AU - Turck, C.* AU - Grosschedl, R.* C1 - 3370 C2 - 22329 SP - 6189-6199 TI - PIASy-deficient mice display modest defects in IFN and Wnt signaling. JO - J. Immunol. VL - 173 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AU - Siedlar, M.* AU - Frankenberger, M. AU - Benkhart, E. AU - Espevik, T.* AU - Quirling, M.* AU - Brand, K.* AU - Zembala, M.* AU - Ziegler-Heitbrock, L.* C1 - 4108 C2 - 21928 SP - 2736-2745 TI - Tolerance induced by the lipopeptide Pam3Cys is due to ablation of IL-1R-associated kinase-1. JO - J. Immunol. VL - 173 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AB - The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-{alpha}, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-{alpha}, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-{alpha}, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-{gamma} production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS. AU - Wagner, M.* AU - Poeck, H.* AU - Jahrsdoerfer, B.* AU - Rothenfusser, S.* AU - Prell, D.* AU - Bohle, B.* AU - Tuma, E.* AU - Giese, Th.* AU - Ellwart, J.W. AU - Endres, S.* AU - Hartmann, G.* C1 - 405 C2 - 21681 SP - 954-963 TI - IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA. JO - J. Immunol. VL - 172 IS - 2 PB - American Assoc. of Immunologists PY - 2004 SN - 0022-1767 ER - TY - JOUR AU - Bronte, V.* AU - Serafini, P.* AU - de Santo, C.* AU - Marigo, I.* AU - Tosello, V.* AU - Mazzoni, A.* AU - Segal, D.M.* AU - Staib, C. AU - Löwel, M. AU - Sutter, G. AU - Colombo, M.P.* AU - Zanovello, P.* C1 - 22259 C2 - 21026 SP - 270-278 TI - IL-4-Induced Arginase 1 Suppresses Alloreactive T Cells in Tumor-Bearing Mice. JO - J. Immunol. VL - 170 PY - 2003 SN - 0022-1767 ER - TY - JOUR AU - Lee, B.J.* AU - Koszinowski, U.H.* AU - Sarawar, S.R.* AU - Adler, H. C1 - 10076 C2 - 20651 SP - 243-251 TI - A Gammaherpesvirus G Protein-Coupled Receptor Homologue is Required for Increased Viral Replication in Response to Chemokines and Efficient Reactivation from Latency1,2. JO - J. Immunol. VL - 170 PY - 2003 SN - 0022-1767 ER - TY - JOUR AB - The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1(-/-) cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu(263) was comparable to that of TAP1(-/-) cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe(265) of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu(263) of the TAP1 protein, but not the Phe(265), is critical for proper TAP function. AU - Ritz, U.* AU - Drexler, I. AU - Sutter, D.* AU - Abele, R.* AU - Huber, C.* AU - Seliger, B.* C1 - 23867 C2 - 31371 SP - 941-946 TI - Impaired transporter associated with antigen processing (TAP) function attributable to a single amino acid alteration in the peptide TAP subunit TAP1. JO - J. Immunol. VL - 170 IS - 2 PB - Amer. Assoc. Immunologists PY - 2003 SN - 0022-1767 ER - TY - JOUR AU - Schroeder, T. AU - Kohlhof, H. AU - Rieber, N. AU - Just, U. C1 - 22330 C2 - 21170 SP - 5538-5548 TI - Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression. JO - J. Immunol. VL - 170 IS - 11 PY - 2003 SN - 0022-1767 ER - TY - JOUR AB - Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs. AU - Weber, M.S. AU - Lange, C.M.* AU - Günther, W.H.H.* AU - Franz, M.* AU - Kremmer, E.* AU - Kolb, H.J.* C1 - 33222 C2 - 21048 SP - 5861-5868 TI - Minor histocompatibility antigens on canine hemopoietic progenitor cells. JO - J. Immunol. VL - 170 IS - 12 PY - 2003 SN - 0022-1767 ER - TY - JOUR AU - Ziegler-Heitbrock, L. AU - Lötzerich, M.* AU - Schaefer, A.* AU - Werner, T. AU - Frankenberger, M. AU - Benkhart, E.* C1 - 10077 C2 - 21256 SP - 285-290 TI - IFN-alpha Induces the Human IL-10 Gene by Recruiting Both IFN Regulatory Factor 1 and Stat3. JO - J. Immunol. VL - 171 PY - 2003 SN - 0022-1767 ER - TY - JOUR AB - In human blood two monocyte populations can be distinguished, i.e.,, the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmityloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-LVS4_ OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity, for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) ' monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14+CD16+ monocytes are major producers of TNF in human blood. AU - Belge, K.-U.* AU - Dayyani, F. AU - Horelt, A.* AU - Siedlar, M.* AU - Frankenberger, M. AU - Frankenberger, B. AU - Espevik, T.* AU - Ziegler-Heitbrock, L. C1 - 10073 C2 - 20259 SP - 3536-3542 TI - The Proinflammatory CD14+CD16+DR++ Monocytes Are a Major Source of TNF1. JO - J. Immunol. VL - 168 PB - American Assoc. of Immunologists PY - 2002 SN - 0022-1767 ER - TY - JOUR AB - A highly attractive approach to investigate the influence and hierarchical organization of viral proteins on cellular immune responses is to employ mutant viruses carrying deletions of various virus-encoded, immune-modulating genes. Here, we introduce a novel set of deletion mutants of the human CMV (HCMV) lacking the UL40 region either alone or on the background of a deletion mutant devoid of the entire US2–11 region. Deletion of UL40 had no significant effect on lysis of infected cells by NK cells, indicating that the expected enhancement of HLA-E expression by specific peptides derived from HCMV-encoded gpUL40 leader sequences was insufficient to confer target cell protection. Moreover, the kinetics of MHC class I down-regulation by US2–11 genes observed at early and late phases postinfection with wild-type virus correlated with increased susceptibility to NK lysis. Thus, the influence of HCMV genes on NK reactivity follows a hierarchy dominated by the US2–11 region, which encodes all viral genes capable of down-modulating expression of classical and non-classical MHC class I molecules. The insights gained from studies of such virus mutants may impact on future therapeutic strategies and vaccine development and incorporate NK cells in the line of defense mechanisms against HCMV infection. AU - Falk, C.S. AU - Mach, M.* AU - Schendel, D.J. AU - Weiss, E.H.* AU - Hilgert, I.* AU - Hahn, G.* C1 - 10074 C2 - 20297 SP - 3257-3266 TI - NK Cell Activity During Human Cytomegalovirus Infection Is Dominated by US2-11-Mediated HLA Class I Down-Regulation. JO - J. Immunol. VL - 169 PB - American Assoc. of Immunologists PY - 2002 SN - 0022-1767 ER - TY - JOUR AB - Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting. AU - Nößner, E. AU - Gastpar, R. AU - Milani, V. AU - Brandl, A. AU - Hutzler, P.J.S. AU - Kuppner, M.C.* AU - Roos, M. AU - Kremmer, E. AU - Asea, A.* AU - Calderwood, S.K.* AU - Issels, R.D. C1 - 10072 C2 - 20426 SP - 5424-5432 TI - Tumor-Derived Heat Shock Protein 70 Peptide Complexes Are Cross-Presented by Human Dendritic Cells. JO - J. Immunol. VL - 169 PB - American Assoc. of Immunologists PY - 2002 SN - 0022-1767 ER - TY - JOUR AU - Stassen, M.* AU - Müller, Ch.* AU - Arnold, M.* AU - Hültner, L. AU - Klein-Hessling, S.* AU - Neudörfl, Ch.* AU - Reineke, T.* AU - Serfling, E.* AU - Schmitt, E.* C1 - 21977 C2 - 20501 SP - 4391-4398 TI - IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide : NF-kB is decisively involved in the expression of IL-9. JO - J. Immunol. VL - 166 PY - 2002 SN - 0022-1767 ER - TY - JOUR AU - Vogel, T.U.* AU - Horton, H.* AU - Fuller, D.H.* AU - Carter, D.K.* AU - Vielhuber, K.* AU - O'Connor, D.* AU - Shipley, T.* AU - Fuller, J.* AU - Sutter, G. AU - Erfle, V. AU - Wilson, N.* AU - Picker, L.J.* AU - Watkins, D.I.* C1 - 10075 C2 - 20562 SP - 4511-4521 TI - Differences Between T Cell Epitopes Recognized After Immunization and After Infection. JO - J. Immunol. VL - 169 PY - 2002 SN - 0022-1767 ER - TY - JOUR AB - Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-alpha. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-alpha from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets. AU - Bauer, M.* AU - Redecke, V.* AU - Ellwart, J.W. AU - Scherer, B.* AU - Kremer, J.-P. AU - Wagner, H.* AU - Lipford, G.B.* C1 - 24115 C2 - 31400 SP - 5000-5007 TI - Bacterial CpG-DNA triggers activation and maturation of human CD11c-, CD123+ dendritic cells. JO - J. Immunol. VL - 166 IS - 8 PB - Amer. Assoc. Immunologists PY - 2001 SN - 0022-1767 ER - TY - JOUR AU - Benkhart, E.M.* AU - Siedlar, M.* AU - Wedel, A.* AU - Werner, T. AU - Ziegler-Heitbrock, L. C1 - 1545 C2 - 22800 SP - 1612-1617 TI - Role of Stat3 in lipopolysaccharide-induced Il-10 gene expression. JO - J. Immunol. VL - 165 PB - American Assoc. of Immunologists PY - 2000 SN - 0022-1767 ER - TY - JOUR AU - Hültner, L. AU - Kölsch, S.* AU - Stassen, M.* AU - Kaspers, U. AU - Kremer, J.-P. AU - Mailhammer, R. AU - Moeller, J.* AU - Broszeit, H.* AU - Schmitt, E.* C1 - 21391 C2 - 19509 SP - 5556-5563 TI - In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9. JO - J. Immunol. VL - 164 PY - 2000 SN - 0022-1767 ER - TY - JOUR AU - Sgadari, C.* AU - Toschi, E.* AU - Palladino, C.* AU - Barillari, G.* AU - Carlei, D.* AU - Cereseto, A.* AU - Ciccolella, C.* AU - Yarchoan, R.* AU - Monini, P.* AU - Stürzl, M. AU - Ensoli, B.* C1 - 21397 C2 - 19515 SP - 509-517 TI - Mechanism of Paclitaxel Activity in Kaposi's Sarcoma. JO - J. Immunol. VL - 165 PY - 2000 SN - 0022-1767 ER - TY - JOUR AU - Stassen, M.* AU - Arnold, M.* AU - Hültner, L. AU - Müller, C.* AU - Neudörfl, C.* AU - Reineke, T.* AU - Schmitt, E.* C1 - 21292 C2 - 19407 SP - 5549-5555 TI - Murine bone marrow-derived mast cells as potent producers of IL-9: Costimulatory function of IL- 10 and kit ligand in the presence of IL-1. JO - J. Immunol. VL - 164 PY - 2000 SN - 0022-1767 ER - TY - JOUR AU - Ulbrecht, M.* AU - Martinozzi, S.* AU - Grzeschik, M.* AU - Hengel, H.* AU - Ellwart, J.W. AU - Pla, M.* AU - Weiss, E.H.* C1 - 21293 C2 - 19408 SP - 5019-5022 TI - Cutting Edge: The Human Cytomegalovirus UL40 Gene Product Contains a Ligand for HLA-E and Prevents NK Cell-Mediated Lysis. JO - J. Immunol. VL - 164 PY - 2000 SN - 0022-1767 ER - TY - JOUR AU - Sparwasser, T.* AU - Hültner, L. AU - Koch, E.S.* AU - Luz, A. AU - Lipford, G.* AU - Wagner, H.* C1 - 20817 C2 - 18884 SP - 2368-2374 TI - Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis. JO - J. Immunol. VL - 162 PY - 1999 SN - 0022-1767 ER - TY - JOUR AB - Bispecific Abs (bsAb) are promising immunological tools for the elimination of tumor cells in minimal residual disease situations. In principle, they target an Ag on tumor cells and recruit one class of effector cell. Because immune reactions in vivo are more complex and are mediated by different classes of effector cell, we argue that conventional bsAb might not yield optimal immune responses at the tumor site. We therefore constructed a bsAb that combines the two potent effector subclasses mouse IgG2a and rat IgG2b. This bispecific molecule not only recruits T cells via its one binding arm, but simultaneously activates FcgammaR+ accessory cells via its Fc region. We demonstrate here that the activation of both T lymphocytes and accessory cells leads to production of immunomodulating cytokines like IL-1beta, IL-2, IL-6, IL-12, and DC-CK1. Thus this new class of bsAb elicits excellent antitumor activity in vitro even without the addition of exogenous IL-2, and therefore represents a totally self-supporting system. AU - Zeidler, R.* AU - Reisbach, G. AU - Wollenberg, B.* AU - Lang, S.* AU - Chaubal, S.* AU - Schmitt, B.* AU - Lindhofer, H.* C1 - 27636 C2 - 32778 SP - 1246-1252 TI - Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing. JO - J. Immunol. VL - 163 IS - 3 PY - 1999 SN - 0022-1767 ER - TY - JOUR AU - Forster, R.* AU - Kremmer, E. AU - Schubel, A.* AU - Breitfeld, D.* AU - Kleinschmidt, A. AU - Nerl, C.* AU - Bernhardt, G.* AU - Lipp, M.* C1 - 21168 C2 - 19214 SP - 152-1531 TI - Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusion on various leukocyte subset: Rapid internalization and recycling upon activation. JO - J. Immunol. VL - 160 PY - 1998 SN - 0022-1767 ER - TY - JOUR AB - Conventional mouse/mouse or rat/rat hybrid-hybridoma supernatants contain up to 10 different IgG molecules consisting of various combinations of heavy and light chains. Hence, the yield of functional bispecific Ab is low, and purification is often complicated, hampering a general preclinical evaluation of, e.g., bispecific Ab-mediated tumor immunotherapy in animal models. In experiments to overcome this drawback we found that fusion of rat with mouse hybridomas opens the possibility of large scale production of bispecific Ab due to the increased incidence of correctly paired Ab and facilitated purification. In essence, rat/mouse quadroma-derived bispecific Ab have the following advantages: 1) enrichment of functional bispecific Ab because of preferential species-restricted heavy/light chain pairing (observed in four of four rat-mouse quadromas) in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, and 2) a possible one-step purification of the quadroma supernatant with protein A. This simple chromatography step does not bind unwanted variants with parental rat/rat heavy chain configuration, and the desired rat/mouse bispecific Ab are retained, which can then easily be separated from parental mouse Ab by sequential pH elution. AU - Lindhofer, H. AU - Mocikat, R. AU - Steipe, B. AU - Thierfelder, S. C1 - 58137 C2 - 0 SP - 219-25 TI - Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies. JO - J. Immunol. VL - 155 IS - 1 PY - 1995 SN - 0022-1767 ER - TY - JOUR AB - IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. Recently it has been shown to influence several functions of keratinocytes, including HLA-DR expression, chemotaxis, and proliferation by binding to a specific receptor. Because psoriasis vulgaris is characterized by epidermal hyperproliferation and infiltration of inflammatory cells, we investigated the expression of IL-8 and its receptor in normal and psoriatic epidermis using semiquantitative reverse-transcriptase-polymerase chain reaction. In addition the mRNA levels of the proto-oncogenes c-ras, c-raf, c-myc, and HER-2 were also investigated as potential growth-promoting stimuli in psoriatic epidermis. IL-8 mRNA was only detected in lesional psoriatic epidermis, and IL-8R-specific mRNA was found to be 10 times increased in lesional psoriatic epidermis. There was no significant difference in the proto-oncogene mRNA levels. In order to test the relevance of the massively increased IL-8R levels in psoriatic epidermis, we investigated the effect of the antipsoriatic drug FK-506 on specific IL-8 and IL-8R mRNA expression. FK-506 dose dependently inhibited IL-8R expression and function. Our data suggest that in psoriatic skin, elevated IL-8 levels and markedly increased IL-8R expression may act in concert to induce the cardinal signs of psoriasis - epidermal hyperproliferation and leukocyte infiltration. IL-8R may prove a molecular target for antipsoriatic drugs such as FK-506. AU - Schulz, B.S.* AU - Michel, G.* AU - Wagner, S.A. AU - Süß, R.* AU - Beetz, A.* AU - Peter, R.U.P.* AU - Kemény, L.V.* AU - Ruzicka, T.* C1 - 40442 C2 - 40100 SP - 4399-4406 TI - Increased expression of epidermal IL-8 receptor in psoriasis: Down-regulation by FK-506 in vitro. JO - J. Immunol. VL - 151 IS - 8 PY - 1993 SN - 0022-1767 ER - TY - JOUR AU - Schendel, D.J. AU - Reinhardt, C. AU - Nelson, P.N. AU - Maget, B. AU - Pullen, L. AU - Bornkamm, G.W. AU - Steinle, A. C1 - 20500 C2 - 13709 SP - 2406-2414 TI - Cytotoxic T Lymphocytes Show HLA-C-restricted Recognition of EBV-Bearing Cells and Allorecognition of HLA Class I Molecules Presenting Self-Peptides. JO - J. Immunol. VL - 149 PY - 1992 SN - 0022-1767 ER - TY - JOUR AU - Eulitz, M. AU - Murphy, C. AU - Weiss, D.T. AU - Solomon, A. C1 - 19601 C2 - 12710 SP - 3091-3096 TI - Serologic and Chemical Differentiation of Human LambdaIII Light Chain Variable Regions. JO - J. Immunol. VL - 146 PY - 1991 SN - 0022-1767 ER - TY - JOUR AB - A novel growth factor for bone marrow derived murine mucosal type mast cells has been isolated from the conditioned medium of the Mlsa-reactive mouse Th cell line MLS-4.2. In proliferation assays this growth factor synergizes, like IL-4, with IL-3 on established mast cell lines and was therefore termed MEA: mast cell growth enhancing activity. MEA was characterized as a glycoprotein with a Mr range between 37,000 and 43,000. Apparent homogeneity was obtained by using a four-step purification scheme including cation exchange chromatography, Procion red affinity chromatography, IEF, and gel filtration. Inasmuch as MEA was N-terminally blocked during automated Edman-degradation, peptide fragments after digestion with trypsin were used for partial amino acid sequence determination. All evaluable MEA peptide fragments showed complete sequence homology to a recently purified and cloned novel T cell growth factor (P40/TCGF III), the mouse homologue of human IL-9. AU - Moeller, J. AU - Hültner, L.* AU - Schmitt, E.* AU - Breuer, M.* AU - Dörmér, P.G.* C1 - 42594 C2 - 40234 SP - 4231-4234 TI - Purification of MEA, a mast cell growth-enhancing activity, to apparent homogeneity and its partial amino acid sequencing. JO - J. Immunol. VL - 144 IS - 11 PY - 1990 SN - 0022-1767 ER - TY - JOUR AB - A series of permanent IL-3-dependent cell lines have been established from normal BALB/c or C3H bone marrow using alpha-thioglycerol-supplemented culture medium and PWM-stimulated spleen cell-conditioned medium as a source of IL-3. The cell lines and derivatives cloned in agar resembled "mucosal type" mast cells with respect to phenotypic and functional properties. In this report we demonstrate that in vitro growth of these mast cell lines was not only dependent on IL-3 and synergistically enhanced by IL-4, but in addition regulated by alpha-thioglycerol which could be replaced by 2-ME or cysteamine. We show that these thiol-sensitive mast cell lines respond to a mast cell growth enhancing activity (MEA) present in spleen cell-conditioned medium and acting in concert with IL-3. Partially purified MEA was not able to stimulate the growth of IL-3-dependent 32Dcl.23 cells, IL-2-dependent CTLL-2 cells or the mouse T cell line F4/4K.6 (L3T4+) adapted to grow in purified IL-4. Moreover, 11B11 hybridoma-derived anti-IL-4 mAb specifically neutralizing mouse Il-4 were unable to abolish the bioactivity of MEA. PWM, CSF-1, GM-CSF, IL-1, IL-2, IL-5, IL-6, IL-7, IFN-gamma, TGF-alpha, TNF-alpha, NGF, or EPO did not substitute for MEA in our standard proliferation assay. AU - Hültner, L. AU - Moeller, J. AU - Schmitt, E. AU - Jäger, G. AU - Reisbach, G. AU - Ring, J. AU - Dörnmer, P. C1 - 17837 C2 - 10746 SP - 3440-3446 TI - Thiol-Sensitive Mast Cell Lines Derived from Mouse Bone Marrow Respond to a Mast Cell Growth-Enhancing Activity Different from Both IL-3 and IL-4. JO - J. Immunol. VL - 142 PY - 1989 SN - 0022-1767 ER - TY - JOUR AB - A growth factor acting synergistically with IL-3 on thiol-sensitive "mucosal type" bone marrow-derived mast cell lines, and therefore termed mast cell growth enhancing activity, is present in PWM stimulated spleen cell conditioned medium. Mast cell growth enhancing activity can be partially purified and completely separated from IL-3, IL-4, and IL-5, and for the most part from IL-6 and GM-CSF using strong cation exchange and Procion red affinity chromatography. Mast cell growth enhancing activity binds to Con A-Sepharose and can be digested with trypsin and chymotrypsin. It shows a Mr ranging from 37 to 43 kDa under nonreducing SDS-PAGE and a main isoelectric point ranging from 6.2 to 7.3. AU - Moeller, J. AU - Hültner, L. AU - Schmitt, E. AU - Dörmer, P. C1 - 17834 C2 - 10743 SP - 3447-3451 TI - Partial Purification of a Mast Cell Growth-Enhancing Activity and its Separation from IL-3 and IL-4. JO - J. Immunol. VL - 142 PY - 1989 SN - 0022-1767 ER - TY - JOUR AB - There is considerable interest in the use of monoclonal anti-T cell antibodies for immunosuppression during organ transplantation. However, the in vitro cytotoxic titers of these monoclonal reagents do not correlate with their immunosuppressive potency when injected in vivo. A relationship nevertheless seems to exist between immunosuppression and the isotype of anti-mouse Thy-1 antibodies, because among several anti-Thy-1 antibodies of mouse and rat origin, the only two found to cause immunosuppression in vivo belonged to the rat IgG2b and mouse IgG2a isotype. We show here that a quantitative positive correlation exists between an antibody-induced humoral effector mechanism and immunosuppression. We measured the uptake of the C1q complement subunit by polyclonal rabbit and rat anti-thymocyte globulin and also seven monoclonal anti-Thy-1 antibodies in an immunohistochemical assay or a radioimmunoassay. Immunosuppression was studied in a murine graft-vs-host and skin allograft model. Our results suggest strongly that a stable association between the C1 protein and a potential binding antibody is an essential prerequisite of antibody-dependent cell inhibition in vivo that suppresses the immunoresponse against strongly incompatible transplantation antigens. AU - Kummer, U. AU - Thierfelder, S.S. AU - Hoffmann-Fezer, G. AU - Schuh, R. C1 - 41958 C2 - 36228 SP - 4069-4074 TI - In vivo immunosuppression by pan-T cell antibodies relates to their isotype and to their C1q uptake. JO - J. Immunol. VL - 138 IS - 12 PY - 1987 SN - 0022-1767 ER -