Single cell analysis of the CD8+ T cell response to therapeutic Melan-A peptide vaccination over time

Background Therapeutic vaccines are currently being optimized to enhance melanoma patients' own immune defenses against cancer cells. The Melan-AMART-1 tumor antigen is frequently expressed in melanoma and has been widely used in clinical immunotherapy studies. We previously reported that vaccination with the native unmodified Melan-A (defined as EAA hereafter), but not the analog peptide (defined as ELA), despite its stronger binding to HLA-A2, elicited a polyfunctional phenotype (i.e. co-expressing genes associated with memory and effector attributes) by tumor-specific T cells, together with increased tumor cell killing capacities by these cells. Objective The present study aims at characterizing the timing of acquisition of memory/effector properties among individual tumor-reactive CD8 T cell clones in melanoma patients following vaccination with the native/EAA peptide, compared to responses induced with the analog/ELA peptide. Moreover, we assessed the evolution of the tumor-specific T cell clonotype repertoire across time following vaccination in both cohorts of patients and its specific selection at early time points as compared to the specific responses pre-existing before the start of immunotherapy. Methods We generated a large library of cDNAs (n = 1500) isolated from single Melan-A-specific effector- memory (EM) CD28+ (memory-like; EM28+) and CD28- (effector-like; EM28-) T cells at different time points before and after peptide vaccination. Our highly sensitive single cell gene expression approach allowed the direct ex vivo characterization of individual tumor-specific T cells in melanoma patients who received either the native/EAA (n = 4) or the analog/ELA (n = 3) peptide vaccine. PCRs specific for the TCRβ-chain variable region were further performed to determine the TRBV/CDR3-based clonotype of each individual tumor-specific T cells across time after vaccination. Results When compared to pre-vaccine T cell phenotype, tumor-specific T cells underwent a drastic differentiation change into effector-memory following CpG vaccination, regardless of the type of peptide vaccine used (native/EAA or analog/ELA). Acquisition of the EM28+ phenotype shortly after vaccination was specifically associated with the significant increase in effector gene expression and concomitant reduction of a memory gene expression pattern (IL-7R CXCR3- CCR5-). Dominant tumor-specific TCR clonotypes were expanded during this process and many of them were maintained stable over time, as they also were found present at late time points after immunotherapy. Using the advantage of the single cell resolution, a difference in CD27 gene expression could be identified between EM T cells with dominant or non-dominant TCR clonotypes. Conclusions Melan-A peptide/CpG vaccination induced the strong T cell differentiation of tumor-reactive T cells, which was accompanied by increased effector gene expression and expansion of dominant clonotypes, in both native/EAA and analog/ELA based vaccine. Moreover, our data further suggest that tumor-specific T cells may undergo a maturation process during the course of vaccination, which may account for the differences observed in the co-expression of memory and effector genes within native/EAA EM28+ T cells at later time points. Finally, once established, the clonal composition of tumor-specific T cell responses was kept stable along immunotherapy. Collectively, such analyses provide important insights on the in vivo impact of natural over analog peptide vaccination on T cell polyfunctionality and clonotype selection induced by each type of vaccination