PuSH - Publication Server of Helmholtz Zentrum München: Identification and characterization of antigen-specific CD8<sup>+</sup> T cells using surface-trapped TNF-α and single-cell sequencing.

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Abdulhaqq, S.* ; Ventura, A.B.* ; Reed, J.S.* ; Bashirova, A.A.* ; Bateman, K.B.* ; McDonald, E.* ; Wu, H.L.* ; Greene, J.M.* ; Schell, J.B.* ; Morrow, D.* ; Wisskirchen, K. ; Martin, J.N.* ; Deeks, S.G.* ; Carrington, M.N.* ; Protzer, U. ; Früh, K.* ; Hansen, S.G.* ; Picker, L.J.* ; Sacha, J.B.* ; Bimber, B.N.*

Identification and characterization of antigen-specific CD8⁺ T cells using surface-trapped TNF-α and single-cell sequencing.

J. Immunol. 207, 2913-2921 (2021)

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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.

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PuSH - Publication Server of Helmholtz Zentrum München

Identification and characterization of antigen-specific CD8+ T cells using surface-trapped TNF-α and single-cell sequencing.

Identification and characterization of antigen-specific CD8⁺ T cells using surface-trapped TNF-α and single-cell sequencing.