1289 Activation of bystander anti-viral T cells by in situ peptide vaccination in MHC-I deficient syngeneic tumor models

<h3>Background</h3> Intrinsic tumor defect in MHC-I presentation pathway, notably, beta-2-microglobulin (B2M) loss, can limit the activity of immune surveillance by anti-tumor T cells or response to immunotherapy. We have previously reported that intratumoral injection of mouse cytomegalovirus (mCMV)-derived peptides restricted to MHC-I and MHC-II can activate infection-induced bystander anti mCMV T cells in situ in both immunologically ‘hot’ and ‘cold’ syngeneic murine tumor models (PMID: 35749366). This led to tumor killing, tumor microenvironment reprogramming and epitope spreading against tumor-associated antigens. <h3>Methods</h3> We generated a B2M knockout cell line (B2M KO) using CRISPR/CAS9 in the TC1 syngeneic model driven by HPV16E6E7 oncogenes. We confirmed MHC-I loss in B2M KO cells in vitro and in vivo. B2M KO tumors grew at similar rate in vivo than parental TC1 tumors suggesting that Natural Killer cells (NK) cells were not activated by the absence of MHC-I. <h3>Results</h3> Systemic vaccination with a tumor-associated long peptide (E7) failed to control B2M KO in contrast to the TC1 parental line. However, in situ activation of bystander anti-viral CD8+ T cells by intratumoral injection of MHC-I restricted peptides delayed tumor growth. Local T cell expansion of intratumoral bystander anti-viral CD8+ T cells was similar in B2M KO compared to TC1 parental tumors suggesting that direct presentation by tumor cells contributed minimally to local T cell activation. NK cells and neutrophils recruitment was increased in B2M KO as well as in parental TC1 tumors. In TME analysis experiments, we found that local activation of bystander T cells led to tumor cell death independently of MHC-I expression and upregulation of IFN-gamma associated molecules (PD-L1, FAS) and immunogenic cell death markers (Calreticulin) by tumor cells. Proteomic analysis showed simultaneous induction of proinflammatory cytokines and chemokines such as IFN-gamma, TNF-alpha, CCL2, GM-CSF, CXCL9 and CXCL10 in both B2M KO and parental TC1 tumors. Finally, RNAseq analysis revealed a ‘cold’ signature in the B2M KO TME compared to parental TC1. Upon intratumoral activation of bystander T-cells, both B2M KO and TC1 parental tumors displayed gene expression signatures in antigen presentation, IFN-gamma, T-cell activation, cellular cytotoxicity, ECM remodeling and reduction in cancer-associated fibroblasts. <h3>Conclusions</h3> These results suggest that MHC-I restricted peptides injected intratumoral are largely presented by non-tumor cells. Local activation of bystander T-cells leads to eradication of MHC-deficient tumor cells. These results have implication for the development of anti-tumor vaccines and on the choice of optimal route of delivery of such vaccines.