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

Abstract

Experimental models demonstrated that therapeutic induction of CD8 T cell responses may offer protection against tumors or infectious diseases providing that T cells have sufficiently high TCR/CD8:pMHC avidity for efficient Ag recognition and consequently strong immune functions. However, comprehensive characterization of TCR/CD8:pMHC avidity in clinically relevant situations has remained elusive. In this study, using the novel NTA-His tag–containing multimer technology, we quantified the TCR:pMHC dissociation rates (koff) of tumor-specific vaccine-induced CD8 T cell clones (n = 139) derived from seven melanoma patients vaccinated with IFA, CpG, and the native/EAA or analog/ELA Melan-AMART-126–35 peptide, binding with low or high affinity to MHC, respectively. We observed substantial correlations between koff and Ca2+ mobilization (p = 0.016) and target cell recognition (p < 0.0001), with the latter independently of the T cell differentiation state. Our strategy was successful in demonstrating that the type of peptide impacted on TCR/CD8:pMHC avidity, as tumor-reactive T cell clones derived from patients vaccinated with the low-affinity (native) peptide expressed slower koff rates than those derived from patients vaccinated with the high-affinity (analog) peptide (p < 0.0001). Furthermore, we observed that the low-affinity peptide promoted the selective differentiation of tumor-specific T cells bearing TCRs with high TCR/CD8:pMHC avidity (p < 0.0001). Altogether, TCR:pMHC interaction kinetics correlated strongly with T cell functions. Our study demonstrates the feasibility and usefulness of TCR/CD8:pMHC avidity assessment by NTA-His tag–containing multimers of naturally occurring polyclonal T cell responses, which represents a strong asset for the development of immunotherapy.

Relevance of the T cell Receptor-Ligand Avidity for Immunity to Infection

“…In the meantime, others have also developed TCR-pMHC k off -rate assays based on the same principles as our Streptamer based assay [25]. Their methodologies have found a clear correlation between the TCR-pMHC k off -rate and the functionality of the analysed cells [26], supporting our previous findings.…”

TCR-ligand dissociation rate is a robust and stable biomarker of CD8+ T cell potency

“…To precisely address the relationship between the TCR-pMHC off-rate and the overall CD8 + T cell functional profile, we generated large libraries of HLA-A*0201-restricted CD8 + T cell clones, by direct ex vivo sorting and cloning of self/tumor-specific (i.e., Melan-A [26][27][28][29][30][31][32][33][34][35] and NY-ESO-1 157-165 ) and virus-specific (i.e., cytomegalovirus CMV/pp65 495-504 and EpsteinBarr virus EBV/BMFL1 259-267 ) effector memory (EM) T cells (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.92570DS1). We analyzed all clones for TCR-pMHC dissociation rates using NTAmers loaded with the native Melan-A, NY-ESO-1, EBV/BMFL1, or CMV/pp65 peptide because they provided a more physiological assessment of the TCR-pMHC recognition efficacy as opposed to the corresponding analog peptides (as detailed in Methods).…”

“…However, owing to the faster decay of the multimeric complex onto monomeric pMHC when compared with Streptamers, NTAmers offer an increased sensitivity to detect T cells with low-avidity TCRs (26), such as those typically found in self/tumor-specific CD8 + T cell repertoires. Consequently, we recently showed that NTAmer-based k off strongly correlated with the killing capacity of TCR-engineered and natural tumor-specific human CD8 + T cells (28,29). With the aim to thoroughly evaluate possible correlations between T cell function and TCR-pMHC binding kinetics, we here undertook a large-scale analysis of combined multiple functions (i.e., killing, CD107a degranulation, cytokine production, proliferation, surface expression of activating/inhibitory receptors, and tumor control) and optimized off-rate measurements using NTAmers to characterize large libraries of tumorand virus-specific CD8 + T cell clones isolated from melanoma patients and healthy donors.…”

Flow cytometry‐based TCR‐ligand Koff‐rate assay for fast avidity screening of even very small antigen‐specific T cell populations ex vivo

“…For the measurement of relatively homogeneous populations, like T cell clones or TCR-transgenic T cells, flow cytometrybased assessment of TCR binding kinetics can be performed quite straight forward, as demonstrated in this report and by others (14). However, when aiming for direct ex vivo analysis of antigen-specific T cell populations, one typically has to deal with a small population within a plethora of additional leukocytes.…”

“…This is the main reason why we initially set up the microscopy-based platform, where single cell tracking overcomes this limitation. Gannon et al proposed to generate T cell clones from ex vivo T cell populations to get access to TCR ligand binding kinetics (14). In vitro culture can substantially bias the repertoire of expanding T cells and therefore obtained results might not reflect the composition of the original T cell population.…”