f7e2150b-397a-47bf-bb27-a6971aca3cea
TNBS-specific T cells and TCR
Allergic contact dermatitis is a widespread T cell-mediated inflammatory skin disease, but in vitro monitoring of chemical-specific T cells remains challenging. We here introduce short-term CD154/CD137 upregulation to monitor human T cell responses to the experimental sensitizer 2,4,6-trinitrobenzenesulfonic acid (TNBS). Peripheral blood mononuclear cells (PBMC) from healthy donor buffy coats were TNBS-modified and incubated with unmodified PBMC. After 5 and 16 hours, we detected TNBS-specific activated CD154+CD4+ and CD137+CD8+ T cells by multi-parameter flow cytometry, respectively. Activated cells were sorted for restimulation and bulk T cell receptor (TCR) high-throughput sequencing (HTS). We confirmed suitable TNBS concentrations ranging between ~3 - 10 mM. Stimulation with TNBS-modified cells induced CD154 expression on 0.04% of CD4+ and CD137 expression on 0.60% of CD8+ memory T cells, respectively (means, n = 12 - 18 donors). CD69 co-expression argued for TCR-mediated activation, which was further supported by TNBS-specific restimulation of 10/13 CD154+CD4+ and 11/15 CD137+CD8+ T cell clones and lines. Major histocompatibility complex (MHC) blocking antibodies prevented activation, illustrating MHC restriction. The high frequencies of TNBS-specific T cells were associated with distinct common changes in the TCR ß-chain repertoire. We observed an overrepresentation of tryptophan and lysine in the complementarity determining regions 3 (CDR3) (n = 3 - 5 donors), indicating a preferential interaction of these amino acids with the TNBS-induced epitopes. In summary, the detection of chemical-specific T cells by CD154/CD137 upregulation is a fast, comprehensive and quantitative method. Combined with TCR HTS, the mechanisms of chemical allergen recognition that underlie unusually frequent T cell activation can be assessed. In the future, this approach may complement allergy diagnosis and chemical sensitizer prediction.
Allergic contact dermatitis is a widespread T cell-mediated inflammatory skin disease, but in vitro monitoring of chemical-specific T cells remains challenging. We here introduce short-term CD154/CD137 upregulation to monitor human T cell responses to the experimental sensitizer 2,4,6-trinitrobenzenesulfonic acid (TNBS). Peripheral blood mononuclear cells (PBMC) from healthy donor buffy coats were TNBS-modified and incubated with unmodified PBMC. After 5 and 16 hours, we detected TNBS-specific activated CD154+CD4+ and CD137+CD8+ T cells by multi-parameter flow cytometry, respectively. Activated cells were sorted for restimulation and bulk T cell receptor (TCR) high-throughput sequencing (HTS). We confirmed suitable TNBS concentrations ranging between ~3 - 10 mM. Stimulation with TNBS-modified cells induced CD154 expression on 0.04% of CD4+ and CD137 expression on 0.60% of CD8+ memory T cells, respectively (means, n = 12 - 18 donors). CD69 co-expression argued for TCR-mediated activation, which was further supported by TNBS-specific restimulation of 10/13 CD154+CD4+ and 11/15 CD137+CD8+ T cell clones and lines. Major histocompatibility complex (MHC) blocking antibodies prevented activation, illustrating MHC restriction. The high frequencies of TNBS-specific T cells were associated with distinct common changes in the TCR ß-chain repertoire. We observed an overrepresentation of tryptophan and lysine in the complementarity determining regions 3 (CDR3) (n = 3 - 5 donors), indicating a preferential interaction of these amino acids with the TNBS-induced epitopes. In summary, the detection of chemical-specific T cells by CD154/CD137 upregulation is a fast, comprehensive and quantitative method. Combined with TCR HTS, the mechanisms of chemical allergen recognition that underlie unusually frequent T cell activation can be assessed. In the future, this approach may complement allergy diagnosis and chemical sensitizer prediction.