Upon acquisition of large cell samples comprising more than 1×106

Upon acquisition of large cell samples comprising more than 1×106 PBMC, a small but detectable level of HLA/peptide tetramer-reactivity was found in these donors. Importantly, this reactivity remained unaffected by SPV-T3b pretreatment (Fig. 4). In Crizotinib mouse parallel analyses, the MFI of the population of HLA-A2/flu tetramer-reactive T cells in these donors decreased by SPV-T3b pretreatment, indicating that SPV-T3b pretreatment efficiently discriminates true TCR/CD3-mediated

binding from background HLA/peptide tetramer-reactivity. Furthermore, these data show that the low-level reactivity of HLA/peptide tetramers with CD8+ T cell populations that lack the relevant antigen-specificity is not mediated through binding to the TCR/CD3 complex. Analyses of the immune response to vaccination are mostly performed based on the reactivity of the CD8+ T cells using HLA class I tetramers. However, successful activation of an effector

CD8+ T cell response frequently involves the activation of CD4+ T cells. An increasing number of epitopes recognized by CD4+ T cells has been identified over the last years, and for which class II tetramers may be generated [3], [5], [14], [30], [43] and [44]. As the background reactivity of HLA-class Venetoclax cell line II tetramer may vary [13], extra testing for TCR/CD3-mediated binding is useful to improve reliable detection of antigen-specific CD4+ T cells. We have tested

our SPV-T3b pretreatment method on HLA class II tetramers, composed of the influenza virus hemagglutinin peptide (HA307-319) bound to HLA-DRA1⁎0101/DRB1⁎0401 molecules (HLA-DR4/flu tetramers). As a source of PBMC containing antigen-specific T cells, we used PBMC of a DRA1⁎0101/DRB1⁎0401-positive donor stimulated with the influenza virus hemagglutinin peptide (HA307-319), which resulted in an increase in HLA-DR4/flu tetramer-reactive T cells [22]. When the PBMC were labeled 3-mercaptopyruvate sulfurtransferase with CFSE prior to peptide stimulation, the decreased CFSE intensity of the HLA-DR4/flu tetramer-positive CD4+ T cells indicated that these cells had proliferated following peptide stimulation (not shown). SPV-T3b pretreatment decreased the MFI of HLA-DR4/flu tetramer-reactive cells, indicating that mAb SPV-T3b also interfered with binding of HLA class II tetramers (Fig. 5A). As was observed for the HLA class I tetramers, pretreatment with mAb T10B9 did not inhibit HLA-class II tetramer-binding. To study the inhibition of tetramer-binding at the level of a single TCR specificity, we generated T cell clones from the HLA-DR4/flu tetramer-reactive T cell population. As shown in Fig. 5B, binding intensity of HLA-DR4/flu tetramers to T cell clones is almost ten-fold decreased by SPV-T3b pretreatment, indicating that HLA-DR4/flu tetramer bound to the T cells by interaction with the TCR/CD3 complex.

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