antigenic specificities
In the last few decades, immunotherapy has played a leading role in innovative treatments against cancers such as melanoma, or viral infections such as CMV. In most immunotherapy- related strategies, cytotoxic CD8 T-cells play a central role, due to their capacity to kill tumor or infected cells. Therefore, extensive research has been undertaken to investigate which properties of these cells are essential to generate protective and durable immune responses. In that regard, high T cell functional avidity, which is the antigen sensitivity measured by functional readouts, has been associated with better control of viral infections in both animal models [71-73] and humans [74-78], as well as better tumor control in mice [79, 80] and melanoma patients [81, 82]. However, ex vivo functional avidity and polyfunctional assessments remain laborious and time consuming, and are often not possible because relatively large number of cell numbers must be withdrawn from patients. Furthermore, the TCR-pMHC binding avidity remains often neglected, mainly due to technical limitations. In fact, TCR-ligand avidity may offer a better biometric by which the quality of the antigen-specific T cell response can be directly evaluated, since it controls numerous aspects of T cell biology such as T-cell activation, differentiation, and functional efficacy (reviewed in [255]) as well as Listeria
Objectives
1.1. Strong relationships between TCR-pMHC binding avidity and T cell
functional potency
To better characterize the relationship between the physical interaction of the TCR-pMHC complex and the ensuing T cell activation, we used the recently developed NTAmer technology. This novel flow cytometry-based approach allows for the quantification of monomeric TCR-pMHC dissociation kinetics (koff) directly on living antigen-specific CD8 T
cells. Using this technology, our group documented robust correlations between NTAmer kinetic values (koff) and those obtained by SPR [108]. Moreover, we showed that NTAmer-
based koff measurements strongly correlate with the killing capacity of TCR-engineered CD8 T
cells [108] as well as following therapeutic vaccination [109]. In the latter study, we found differences in TCR-pMHC binding avidity depending on the type of Melan-A26-35 peptide used
for vaccination [109]. To elucidate the precise causality between TCR-pMHC binding avidity and the overall CD8 T cell functional profile, NTAmer-based koff measurements of antigen-
specific CD8 T cell clones were combined with multiple functional assays including killing capacity, CD107a degranulation, cytokine production, proliferation, costimulatory and inhibitory receptor expression, as well as the ability of tumor-specific CD8 T cells to control tumor growth in vivo. This part of the study was performed on large libraries of HLA-A*0201– restricted effector-memory CD8 T cell clones specific for self/tumor antigen (i.e. Melan-A26-35
and NY-ESO-1157-165) and viral antigen (i.e. CMV/pp65495-504 and EBV/BMFL1259-267). Results
obtained from these experiments show that TCR-pMHC off-rate is a major determinant controlling the functions of CD8 T cells in vitro and in vivo. These observations further enhance our understanding of the impact of TCR avidity on CD8 T cell activation and function.
1.2. Variations of TCR-pMHC avidity according to the antigenic specificity of
CD8 T cells
Only limited information is available on the overall quality of TCR-pMHC binding avidity of self/tumor–specific versus non–self/pathogen–specific CD8 T cell repertoires [54, 55]. The most detailed study investigating this question was carried out by Aleksic et al. [55] who compared the TCR affinities of 14 tumor-specific TCRs directed against various tumor antigens, versus 10 TCRs that bind to different viral antigens. Using the SPR approach, they observed that TCRs that bind viral antigens fall within a higher affinity range than those that bind cancer-related antigens [55]. However, one of the major caveats of SPR analysis is that it
Objectives
ignores the contribution of CD8 coreceptor and/or other molecules present in the vicinity of the TCR to the overall TCR-pMHC avidity. In addition, this study focused on only one TCR sequence per antigenic specificity due to technical limitations (i.e. the laborious and expensive production of soluble TCRs and their cognate pMHC), thus introducing a strong selection bias, as it is known that TCRs directed against the same epitope can cover a large range of TCR affinities [256]. To better address the question of whether or not TCR avidity depends on the antigenic origin (i.e. self/tumor versus non-self/viral), we performed a comprehensive analysis of TCR-pMHC off-rates using the novel NTAmer technology on large panels of effector- memory CD8 T cell clones (n = 414) specific for (i) the differentiation antigen A2/Melan-A26- 35, (ii) the cancer testis antigen A2/NY-ESO-1157-165, (iii) the viral A2-CMV/pp65495-504 antigen
and (iv) the viral A2-EBV/BMFL1259-267 antigen isolated from five melanoma patients and two
healthy donors. In addition, we investigated potential differences in terms of TCR binding avidity between the type of tumor antigen (i.e. Melan-A versus NY-ESO-1) as well as following peptide vaccination in combination with CpG oligodeoxynucleotides (CpG ODN) and incomplete Freund’s adjuvant (IFA) (i.e. Melan-A) or from patients with naturally occurring anti-tumoral T cell responses (i.e. NY-ESO-1). Our data highlight superior TCR-ligand binding avidities of virus-specific T cell repertoires compared with self/tumor-specific T cell ones. Moreover, higher avidity T cells were found in melanoma patients with natural responses against NY-ESO-1 tumor antigen than following therapeutic vaccination with the Melan-A peptide. Nevertheless, several clones with enhanced TCR binding avidity could still be detected, indicating the presence of rare self/Melan-A-specific CD8 T cells that are selected upon vaccination, emphasizing the relevance of therapeutic vaccination approaches in enhancing the quality of a tumor-specific repertoire.
1.3. Stability and robustness of TCR-pMHC off-rates
T cell functional avidity can greatly vary depending on the chosen functional readouts and the laboratory protocols used to assess them, the activation state of the T cells, their differentiation status or the expression of inhibitory and costimulatory receptors [70]. In this regard, the TCR- pMHC off-rate may provide a more reliable biophysical parameter to assess T cell potency. To investigate this question, we assessed the reproducibility of NTAmer-derived TCR-pMHC off- rate measurements in parallel with T cell functional capacities, in separate experiments. In addition, we investigated the impact of the T cell activation state on TCR-pMHC off-rate and on T cell functional avidity. Our results revealed that the TCR-pMHC off-rate is a more stable
Objectives
and robust biomarker of CD8 T cell potency than the frequently used functional assays/metrics, that depend on T cell activation state, and therefore show major intra- and inter-experimental variability. The identification of novel T cell-based parameters able to overcome some of the limitations associated with functional assays, and thus to resolve the lack of universal standards of T cell potency assessment, is of great interest. Indeed, the efficient and reproducible identification and isolation of high avidity T cells is of significant value to improve the therapeutic potential of T cells for immunotherapy. In this line, TCR- pMHC off-rate represents an interesting candidate as a biomarker of T cell therapeutic efficacy.