Distinct T Cell Interactions with HLA Class II Tetramers Characterize a Spectrum of TCR Affinities in the Human Antigen-Specific T Cell Response

Abstract

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16369–379-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16369–379 peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16369–379 tetramer at either 23 or 37°C. Weak staining was also observed at 4°C. Clone 5 showed weaker staining compared with clone 48 at 37°C, and no staining was observed at 23°C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16369–379 complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

The CD4+ T‐cell epitope‐binding register is a critical parameter when generating functional HLA‐DR tetramers with promiscuous peptides

“…3A, two different p57/58-specific T-cell clones, but not an irrelevant T-cell line, were stained by HLA-DR Ã 1101 tetramers loaded with p57/58C3AL peptide. Consistent with published data [8][9][10], the tetramers did not stain the two T-cell clones with the same intensity, in agreement with the different TCR avidity exhibited by the two T-cell clones for the p57/58 peptide (data not shown); 6.22, the T-cell clone with a higher avidity of recognition was stained more brightly by the HLA-DR Ã 1101 loaded with the p57/58C3AL peptide than clone 2E5.53.HLA-DR*1101 tetramers loaded with anchor-tailored p58 peptide stain MAGE-A3-specific CD4 1 T-cell clonesIn the p58 peptide, we maintained the single putative anchor residue Met 290 and replaced the two additional putative Val 286 and Leu 287 anchor residues with two Ala [39]. The resulting p58AA analogue was tested for its ability to form functional HLA-DR Ã 1101 tetramers.…”

“…HLA-DR tetramers loaded with MAGE-A3 anchor-tailored analogues, however, were unable to bind and sort low-affinity CD4 1 T cells from the polyclonal T-cell line, which remained in the negatively sorted fraction. This observation is consistent with the poor efficiency of HLA-DR tetramers in binding T cells bearing low-affinity TCR [10,12], which include CD4 1 T-cell responses specific for peptides derived from non-mutated TAA, such as the cancer-testis family [50,51].A possible drawback of using MHC class II tetramers loaded with anchor-tailored peptide analogues is that they can narrow the TCR clonal representation of the bound T cells. It has been shown that residues flanking the core peptide might be involved in the interaction between the TCR and the MHC class II-peptide complex [36,52].…”

“…The identification of primary antigen-specific CD4 1 T cells by peptide-pulsed MHC class II tetramers seems, however, less straightforward than that of CD8 1 T cells by peptide-loaded MHC class I tetramers [7,8]. Several biological and structural characteristics of the CD4 1 T-cell response that differ from those displayed by the CD8 1 one seem to affect the capacity of MHC class II tetramers to optimally stain specific CD4 1 T cells ex vivo: (i) the lower frequency of peptide-specific CD4 1 T cells, (ii) their apparent lower affinity for the peptide-MHC class II complexes [9][10][11][12][13], and (iii) the requirement for an activation-induced TCR reorganization to bind with sufficient avidity cognate peptide-MHC class II tetramers [12,14,15].Furthermore, the open structure of the MHC class II molecules allows peptides as long as 20 aa to extend out of the binding groove at both ends [16][17][18]. It is thus possible that a peptide, which contains multiple predicted anchor residues, might slide forward and backward in the MHC class II groove, assuming different binding registers, only one of which is optimal for the recognition by a specific TCR.…”

Qualitative difference between the cytotoxic T lymphocyte responses to melanocyte antigens in melanoma and vitiligo

“…TCR affinity was defined through tetramer binding analyses, as described [19][20][21][22]. Tetramer-binding kinetic analysis indicated that similar staining levels were reached after 30 min by the melanoma cell clones versus only 5 min in the case of vitiligo clones, suggesting a difference in the TCR affinity of T cell clones established from the two diseases (Fig.…”

“…Tetramer binding assays were performed essentially as described [19][20][21], with minor modifications [22]. Briefly, to investigate the tetramer binding kinetics, 4 Â 10 5 cells were stained with a subsaturating concentration of tetramer (0.015 lg/mL) for 30 min on ice.…”