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Background Loss of MHC-I expression, particularly due to β2-microglobulin (B2M) deficiency, is a well-known mechanism of immune evasion in cancer, limiting both T cell-mediated immune surveillance and the efficacy of immunotherapy. We previously demonstrated that intratumoral (i.t.) injection of murine cytomegalovirus (mCMV)-derived and vaccine-derived MHC-I and MHC-II restricted peptides activates bystander antiviral and anti-vaccine T cells in situ, resulting in tumor control, tumor microenvironment (TME) remodeling, and epitope spreading. 1 2 Here, we investigated whether this strategy could overcome resistance in MHC-I-deficient tumors.Methods We generated B2M knockout (KO) TC1 tumor cells using CRISPR/Cas9 and validated MHC-I loss in vitro and in vivo. Parental and B2M KO tumors were implanted in syngeneic mice. Tumor growth, immune infiltrates, and immune activation were assessed following systemic vaccination with HPV16E7 peptide or i.t. injection of mCMV-derived MHC-I-restricted peptides. Multiparametric flow cytometry, proteomics, and RNA sequencing were used to analyze the TME and systemic immune responses.Results B2M KO tumors grew at similar rates to parental TC1 tumors, suggesting no compensatory NK cell-mediated rejection. Systemic vaccination with the E7 tumor-associated peptide failed to control B2M KO tumors but was effective against parental TC1. In contrast, i.t. injection of mCMV-derived peptides delayed growth of both tumor types. Expansion of antiviral CD8+ T cells within the tumor was comparable in B2M KO and parental tumors, indicating that peptide presentation occurred primarily via non-tumor cells. Recruitment of NK cells and neutrophils was observed in both tumor types. Despite the absence of MHC-I, B2M KO tumors upregulated IFN-γ-induced molecules (PD-L1, FAS) and immunogenic cell death markers (calreticulin). Proteomic analysis showed similar induction of proinflammatory cytokines and chemokines (IFN-γ, TNF-α, CCL2, GM-CSF, CXCL9, CXCL10) in both models. RNA-seq confirmed a ‘cold’ immune signature in untreated B2M KO tumors. Following i.t. treatment, both models exhibited gene signatures consistent with enhanced antigen presentation, IFN signaling, T cell activation, cytotoxicity, extracellular matrix remodeling, and reduced fibroblast content.Conclusions Intratumoral injection of virus-derived peptides activates bystander T cells via non-tumor antigen-presenting cells and can eliminate MHC-I-deficient tumor cells. These findings have important implications for vaccine design and support intratumoral delivery as a strategy to bypass tumor-intrinsic immune escape mechanisms.References Çuburu N, et al. Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors. Proc Natl Acad Sci U S A. 2022 Jun 28;119(26).Sethi SK, et al. Repurposing anti-viral subunit and mRNA vaccines T cell immunity for intratumoral immunotherapy against solid tumors. NPJ Vaccines. 2025 Apr 25;10(1):84.