Immunological ignorance is an enabling feature of the oligo-clonal T cell response to melanoma neoantigens

Abstract

The impact of intratumoral heterogeneity (ITH) and the resultant neoantigen landscape on T cell immunity are poorly understood. ITH is a widely recognized feature of solid tumors and poses distinct challenges related to the development of effective therapeutic strategies, including cancer neoantigen vaccines. Here, we performed deep targeted DNA sequencing of multiple metastases from melanoma patients and observed ubiquitous sharing of clonal and subclonal single nucleotide variants (SNVs) encoding putative HLA class I-restricted neoantigen epitopes. However, spontaneous antitumor CD8+ T cell immunity in peripheral blood and tumors was restricted to a few clonal neoantigens featuring an oligo-/monoclonal T cell-receptor (TCR) repertoire. Moreover, in various tumors of the 4 patients examined, no neoantigen-specific TCR clonotypes were identified despite clonal neoantigen expression. Mature dendritic cell (mDC) vaccination with tumor-encoded amino acid-substituted (AAS) peptides revealed diverse neoantigen-specific CD8+ T responses, each composed of multiple TCR clonotypes. Isolation of T cell clones by limiting dilution from tumor-infiltrating lymphocytes (TILs) permitted functional validation regarding neoantigen specificity. Gene transfer of TCRαβ heterodimers specific for clonal neoantigens confirmed correct TCR clonotype assignments based on high-throughput TCRBV CDR3 sequencing. Our findings implicate immunological ignorance of clonal neoantigens as the basis for ineffective T cell immunity to melanoma and support the concept that therapeutic vaccination, as an adjunct to checkpoint inhibitor treatment, is required to increase the breadth and diversity of neoantigen-specific CD8+ T cells.

Keywords: CD8+ T cells; cancer vaccine; dendritic cells; melanoma; neoantigen.

Fig. 1.

Neoantigen landscape of patient MEL66 tumors. Clonal neoantigen architecture of patient MEL66 tumors. Primers were designed to 375 MMs encoding putative neoantigens, and sequencing was performed using the Ion Torrent platform. A total of 350 of 375 targets yield sequencing results with an average depth of 1,776×. (A) Derived variant allelic fraction (VAF) values were used to infer clonal architecture of tumors using the SciClone algorithm, which revealed 7 neoantigen clusters as represented in the 2-dimensional analysis of SQ1 vs. SQ4. Each cluster is identified by a distinct color. (B, Bottom) Two-dimensional analysis representing distribution of neoantigen clusters across the 6 tumors obtained from patient MEL66. (B, Top) Two-dimensional analysis representing cluster distribution of 15 vaccine- and TIL-reactive neoantigens across MEL66 tumors. Identities of these neoantigens are detailed in SI Appendix, Fig. S1D.

Fig. 2.

Neoantigen-specific T cell responses in patient MEL66. Neoantigen T cell reactivities in prevaccine PBMCs (Pre-Vacc) and TILs and postvaccine PBMCs (Post-Vacc). Pre- and postvaccine PBMCs were cultured in vitro in the presence of peptide and IL-2 for 10 d, followed by p-HLA multimer staining. Prevaccine TILs were cultured in vitro in the presence of mDC/peptide and IL-2 for 10 d, followed by restimulation with artificial APCs pulsed with peptide for 10 d and staining with p-HLA multimers. Numbers in dot plot represent percentage of neoantigen-specific T cells in lymph/CD8+ gated cells. TIL reactivity to neoantigen EXT2 F350I could not be assessed due to insufficient cells.

Fig. 3.

Antigen specificity of vaccine-induced T cell responses in patient MEL66. (A) Vaccine-induced CD8+ T cell recognition of AAS- (solid circle) and WT- (semicircle) peptide-pulsed HLA-A*02:01-expressing K562 line in a 4-h ⁵¹Cr-release assay. (B) Vaccine-induced CD8+ T cell recognition of AAS- (solid circle) and WT- (semicircle) TMC in HLA-A*02:01-expressing K562 line in a 4-h ⁵¹Cr-release assay. Open circle represents lysis obtained with parental K562 cell line. Percent specific lysis of triplicates (mean ± SD) is shown for each data point; spontaneous lysis <5%. Representative experiment of 2 to 3 independent evaluations is shown.

Fig. 4.

MEL66 neoantigen TCRVB CDR3 repertoires in blood and tumors. (A) Frequency of individual neoantigen-specific TCRVB clonotypes identified in tumors (SQ1–4, RM, and PM) and pre- and postvaccine peripheral blood CD8+ T cells as determined using neoantigen-specific TCRVB_CDR3 reference libraries. Each colored dot represents a unique neoantigen TCRVB clonotype. White dots represent neoantigen-specific TCRVB clonotypes found only in blood samples. For clarity, only TCR clonotypes with tumor frequencies >0.00001 are shown (Dataset S6 provides further details). TCRα/β sequencing of T cell clones isolated from (B) AKAP9 L947F- and (C) PORCN H346Y-specific stimulated TILs (SI Appendix, Fig. S4) yielded uniquely expressed TCRVB/TCRVA pairs (SI Appendix, Fig. S5). Lentiviral vector expression of these TCRα/β pairs into Jurkat 76 T cells revealed reactivity with HLA-A*02:01/AKAP9 L947F and HLA-A*02:01/PORCN H346Y multimers, respectively. Functionality of TCR was assessed upon stimulation of TCR+ Jurkat 76 T cells with K562 HLA-A2+ cells pulsed with 10 µg/mL WT or AAS peptide for 16 h. Geometric mean fluorescent intensity of NFAT-eGFP reporter is shown for a representative experiment.

Fig. 5.

Immunological ignorance is a feature in melanoma. (A) Using variant allelic fraction (VAF) values for SNV encoding neoantigens and the SciClone algorithm, the tumor architecture of these antigens was inferred in MEL21, MEL38, and MEL69 tumors. Two-dimensional analyses are shown, with each cluster defined by a distinct color. ID of mutated genes encoding neoantigens are indicated in figures. (B–D) Distribution of neoantigen-specific TCR clonotypes in tumors and blood. Tumors and peripheral blood pre- and postvaccine CD8+ T cells from patients (B) MEL21, (C) MEL38, and (D) MEL69 were characterized for neoantigen TCRVB repertoires using TCRVB_CDR3 reference libraries. Each colored dot represents a unique neoantigen TCR clonotype identified in tumors. White dots represent unique neoantigen TCR clonotypes found only in blood-derived samples. Identities of neoantigen-specific TCR clonotypes are listed in Dataset S10. For clarity, only TCR clonotypes with tumor frequencies >0.00001 are shown.