Abstract PR-12: Single-cell profiling of tumor-infiltrating B cells reveals autoantibody repertoires and potential cross-talk with T cells

Abstract A growing body of evidence suggests that tumor-infiltrating B lymphocytes (TIBs) play important roles in anti-tumor immunity. However, their functions and characteristics—particularly the antigens they recognize—remain poorly understood. In this study, we aimed to comprehensively characterize TIBs, including their immunoglobulin repertoire, antigen specificity, and phenotypic diversity. We performed single-cell profiling of both the transcriptome and immunoglobulin repertoire of TIBs isolated from 11 gastric adenocarcinoma tissues. Using single-cell immunoglobulin repertoire sequencing (scRepertoire-seq), we obtained thousands of paired heavy- and light-chain sequences per case. We observed hallmarks of antigen-specific stimulation—including class switching to IgG, somatic hypermutation, clonal expansion, and differentiation into plasma cells—underscoring the importance of antigen identification for understanding TIB biology. Based on the scRepertoire-seq data, we reconstructed human monoclonal antibodies and explored their target antigens. To focus on cases with strong humoral response, we selected three tumors with high plasma cell infiltration and generated 6–10 immunoglobulins per case from dominant TIB clones. We screened these antibodies against human cell line lysates via immunoprecipitation and mass spectrometry, complemented by ELISA and human protein arrays, and identified 10 autoantigens (2–5 per case). The identified autoantigens displayed diverse functions and subcellular localizations, while several were notably linked to nucleic acid–related proteins. Notably, whole-genome sequencing revealed that several identified autoantigens harbored somatic missense mutations in the same tumors where the corresponding autoantibodies were detected. In these tumors, multiple distinct TIB clones recognized the same antigen, suggesting high immunogenicity of the mutated proteins. However, the mutations do not appear to be essential for antibody recognition, as the antigen sources in our experiments were generic human cell lines lacking these mutations. These findings raise the possibility that oligoclonal expansion of TIBs toward mutated proteins is driven by neoantigen-specific T cell responses. Such coordination may involve linked recognition, where T cell–mediated B cell activation leads to both B and T cells recognizing the same or physically associated antigens, potentially at different epitopes. To investigate this, we assessed the MHC binding affinity of the mutated peptides using bioinformatic analysis. Collectively, our findings provide new insights into TIB-related autoimmunity in cancer, suggesting that these responses are not merely non-specific reactions to abundant self-proteins, but may reflect coordinated, antigen-specific interactions with neoantigen-driven T cell immunity. These insights may inform the development of more effective strategies to harness tumor immunity. Citation Format: Mikiya Takata, Hiroto Katoh, Daisuke Komura, Miwako Kakiuchi, Shumpei Ishikawa. Single-cell profiling of tumor-infiltrating B cells reveals autoantibody repertoires and potential cross-talk with T cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr PR-12.