Abstract A039: FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1

Abstract

Abstract The outcome of cancer patients has recently been improved by utilizing novel immunotherapies that inhibit the regulatory immune system. Regulatory T-cells (Treg) contribute not only to suppression of excessive immune responses in the steady state but also to reduction of antitumor immune responses in cancer patients. We have shown that down-regulation of the Helios transcription factor undermines CD4 Treg stability inducing these cells to convert into effector cells that enhance the antitumor immune response. Helios is also the canonical transcription factor of CD8 Treg and essential for CD8 Treg function. CD8 Treg are Qa-1 (HLA-E in human)–restricted CD8 T-cells that mainly regulate Qa-1+ immune cells. The peptide FAM49B(p190-198) is a Qa-1 binding peptide derived from ubiquitously expressed FAM49B. Although FAM49B(p190-198) is regularly degenerated in the endoplasmic reticulum normal cells, we found that FAM49B(p190-198) is stably presented in some tumor cells. Here, we investigate the potential contribution of FAM49B(p190-198)/Qa-1 specific CD8 Treg to cancer.FAM49B(p190-198)/Qa-1 specific CD8 Treg were detected by FAM49B(p190-198)/Qa-1 tetramer after immunization with FAM49B(p190-198) peptide–loaded dendritic cells in CD8 Treg (i.e., CD44+CD122+Ly49+ CD8 T-cells). T-cell receptor cDNAs were amplified from the sorted single cells and inserted into retrovirus expression vectors for transduction in 58α–β– hybridoma cells and bone marrow cells. EL4-Qa-1 KO cells and EL4-FAM49B KO cells were generated using CRISPR/Cas9 technology. These tools allowed validation of the specificity of the TCRs and their Qa-1–restriction. For in vivo studies, TCR retrogenic mice were developed by transferring TCR-transduced Rag2–/– bone marrow hematopoietic stem cells into lethally irradiated CD45.1 mice. Retrogenic T-cells were prepared by CD8 enrichment of retrogenic mouse splenocytes and in vitro culture for 3 days in the presence of IL15c. For in vivo studies, we use an adoptive transfer model of retrogenic T-cells for EL4 tumor bearing mice. Rag2–/–γC–/– mice were inoculated with 0.5 x 10^6 EL4 WT-cells and 0.5 x 10^6 EL4 Qa-1 KO cells on each lateral flank, then intravenously injected with retrogenic T-cells (on day 13 and 17). FAM49B(p190-198)/Qa-1 specific CD8 T-cells preferentially use TCR Vα3.2 (TRAV9N-3) and Vβ5.1/5.2 (TRBV12-1/12-2) chains independent of their expression of Ly49+. We tested 17 pairs obtained from Ly49+ cells using the in vitro system and found that 11 pairs bound strongly to Fam49b(p190-198)/Qa-1 tetramer while 8 out of the 11 pairs exhibited antigen-specific activation. Interestingly, one TCR pair (No.8) responded to WT EL4 cells without antigen peptides, which was abolished in coculture wells with Qa-1 KO EL4 cells and FAM49B KO EL4 cells. This TCR hybridoma was also activated by stimulated WT B6 splenocytes but not by Qa-1 KO splenocytes suggesting its regulatory potential. Adoptive transfer of TCR No.8-expressing retrogenic T-cells delayed EL4 WT tumor growth but not EL4 Qa-1 KO tumor growth when inoculated in the same hosts. Tumor-infiltrating CD8 T-cells displayed a more activated phenotype in WT tumors compared to KO tumors, indicating that retrogenic T-cells recognize and target EL4 cells in a Qa-1-dependent manner. Citation Format: Hidetoshi Nakagawa, Hye-Jung Kim, Harvey Cantor. FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A039.