Single cell analysis reveals similar functional competence of dominant and nondominant CD8 T-cell clonotypes

Abstract

Immune protection from infectious diseases and cancer is mediated by individual T cells of different clonal origin. Their functions are tightly regulated but not yet fully characterized. Understanding the contribution of each T cell will improve the prediction of immune protection based on laboratory assessment of T-cell responses. Here we developed techniques for simultaneous molecular and functional assessment of single CD8 T cells directly ex vivo. We studied two groups of patients with melanoma after vaccination with two closely related tumor antigenic peptides. Vaccination induced T cells with strong memory and effector functions, as found in virtually all T cells of the first patient group, and fractions of T cells in the second group. Interestingly, high functionality was not restricted to dominant clonotypes. Rather, dominant and nondominant clonotypes acquired equal functional competence. In parallel, this was also found for EBV-and CMV-specific T cells.

Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence

“…2B, left panel). Together, the functional heterogeneity present in our large clonal library yielded similar results to our previous ex vivo analyses based on the same cohorts of patients (22)(23)(24)31), thereby indicating that these clones provide a powerful model to study the impact of TCR/CD8:pMHC avidity on vaccine-induced tumorspecific T cell responses in a clinically relevant setting.…”

“…These results were based on various assays including ex vivo single cell gene expression profiling (23,24), ex vivo intracellular cytokine staining (22,23), ex vivo IFN-g ELISPOT (22,24), and 51 Cr-release cytotoxicity using T2-pulsed target (22,24) or melanoma cell lines (23). Overall, these data on polyclonal Melan-A-specific CD8 + T cell populations provided convincing results demonstrating the superior antitumoral immune response generated by vaccination with the low-affinity (native) peptide formulation.…”

“…Together, the data obtained from this large collection of vaccineinduced, tumor-specific CD8 T cell clones extended on our recent ex vivo analyses (23,24) by showing that the type of peptide vaccine (low or high affinity) qualitatively impacted the T cell cytotoxicity, the expression of effector molecules, to a lesser extent Ca 2+ signaling, and with significant differences observed in the frequencies of nonfunctional clones (nonkiller or non-Ca 2+ flux).…”

“…We have previously compared the sorting of Melan-A-specific CD8 T cells either with the analog-peptide or the native-peptide multimers. Compared with the native-specific multimer, the use of the analog-specific multimer does not introduce qualitative bias in the TCR Vb repertoires nor quantitative differences in multimer-specific T cell frequencies (22,23).…”

“…Few studies are based on quantifying the association between TCR/CD8:pMHC avidity and T cell responsiveness in naturally occurring TCR repertoires and in humans. Our group has thoroughly characterized a cohort of melanoma patients who received peptide-based vaccination in combination with CpG and IFA (21 (22)(23)(24). We will define hereafter the analog peptide as high affinity in comparison with the low-affinity native peptide as the analog Melan-A MART-1 26-35 (A27L) peptide has a 10-fold increased binding to HLA-A2 (25).…”