Kinetics of Neoantigen Specific T Cells in Post-Transplant Relapse

Abstract

Introduction: In allogeneic stem cell transplantation (alloSCT), donor-derived T cells mediate the graft-versus-leukemia effect (GVL) by recognizing antigens expressed by leukemia cells. These antigens may include either major- or minor-histocompatibility alloantigens, leukemia over-expressed antigens (leukemia-associated antigens), or neoantigens derived from leukemic somatic mutations. In solid tumors, higher somatic mutation burdens are associated with better response to checkpoint inhibitors, which underscores the importance of neoantigen-specific T-cells in cancer immunotherapy. However, mutation burdens are relatively low in acute leukemia, and the role of neoantigens in GVL remains unknown. Here, we screened potential neoantigens and identified neoantigen-reactive T cells in the patients who developed post-transplant relapse. Materials and methods: Leukemic blasts from patients in relapse were FACS-isolated from bone marrow aspirate or peripheral blood samples. Recipient T cells or saliva were isolated from pre-transplant peripheral blood as a germline control, and donor monocyte or CD34-positive cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) at 100X coverage, paired with RNAseq at 40M reads per sample. Somatic mutations were called between recipient blasts and germline controls with mutect and mpileup, followed by annotation with SnpEff. Somatic mutations with variant allele frequency >20% were subjected to pVAC-seq for prediction of neoantigen likely presented on recipient MHC (IC50 < 500nM). 73 SNPs loci associated with publicly known minor histocompatibility antigens (miHAs) were also genotyped to find the miHA disparities between donors and recipients. Serial peripheral blood mononuclear cell samples (PBMCs) were obtained before relapse if available, at the time of relapse, and after treatment for relapse. For HLA-A*02:01-restricted neoantigens, PBMCs were stained with HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65 (control). Purified CD3 cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: Eight patients with relapsed acute leukemia after allo-SCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their transplant donors (4 matched siblings, 1 matched unrelated, 3 haploidentical) were screened for neoantigens. We analyzed samples from 7 subjects with available WES/RNAseq and detected an average of 273 somatic mutations per patient (range 108- 609) on average. Targeted next-generation sequencing was used to detect 2 somatic mutations in one subject. Among these mutations, potential neoantigen candidates were identified in seven out of eight subjects (87.5%), with a median number of 9 candidate neoantigens per subject. In contrast, only 2 subjects (25%) were found with donor:receipient miHAs at tested loci. CD8 T cells derived from UPN8, a responder to DLI, showed dominant CMVpp65-specific T cells at the time of relapse and persistent low frequencyTP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse by HLA-A*02:01 tetramer staining (Figure A). HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these T cell clones during GVHD (Figure B). In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells reactive to neoantigens were detectable by IFN-γ/CD107a production specific to mutant peptides ( SDE2.pK123X and TP53.pR248W respectively). Discussion: Our in-silico analysis suggested the possibility of somatic mutations in acute leukemia to serve as putative neoantigens in alloSCT. We were able to detect neoantigen-specific T cells by tetramer staining and cytokine production assays from a subject who achieved a durable remission after DLI. Although the sample size is very small, we demonstrated the feasibility of studying neoantigen-specific T cells in post-transplant specimens. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with post-relapse outcomes, especially for response to DLI.