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Background Immunotherapies such as immune checkpoint blockade (ICB) harness the power of the adaptive immune system to control tumors. However, ICB efficacy varies widely across cancer types. Recently, Tertiary Lymphoid Structures (TLSs) have emerged as key biomarkers associated with better prognosis and increased ICB efficacy. TLSs are ectopic lymphoid aggregates comprising of T, B, and supporting myeloid cells, such as follicular dendritic cells. They are thought to act as niches for antigen drainage and to provide a pro-inflammatory microenvironment to enhance anti-tumor T cell responses. The factors that are thought to drive TLS formation have yet to be thoroughly interrogated. In particular, the importance of antigenic determinants of TLS formation is not well defined, and, whether T and B cells recognizing tumor antigens interact to form TLSs in tumors is unknown. Determining antigenic drivers of TLS formation is key to gaining a deeper understanding of their mechanisms and importance in tumor control, and to developing precision immunotherapies that specifically target TLSs.Methods We hypothesized that the interactions between tumor-antigen specific Tfh cells and tumor reactive B cells are critical for TLS formation, and these interactions lead better tumor control. To address the clonality and antigenic-specificity of Tfh and B cells, we used a B16-OVA model. To determine how the presence of antigen-specific T cells influences the TLSs, we performed intraperitoneal (ip) injections of B16 or B16-OVA cell lines into C57Bl/6 mice and then adoptively transferred naïve CD4 OTII T cells. 12 days post injection, tumors were removed and processed for imaging, scRNA-seq, flow cytometry and spatial transcriptomics.Results IP injections of our cell line create TLS-like structures. In our B16-OVA model, scRNA-seq reveals that clonally expanded CD4 cells are Tfh-like. In our adoptive transfer models, we saw a decrease in tumor burden and an increase in Tfh cells, when OTII T cells were present in our B16-OVA model. We also were able to map tumor-infiltrating T, B and myeloid cells using Slide-seq.Conclusions Presence of both Ag-specific T cells and the antigen on tumor led to increased TLS formation, differences in T, B and myeloid cells and a decrease in tumor burden. We are currently testing whether expanded Tfh TCR clones from tumors are tumor-reactive, and whether they can be mapped in a spatial manner. The completion of these studies will lead to a greater understanding of the T-B interactions that fuel TLS formation, allowing the development of antigen-specific therapies that enhance tumor clearance.Ethics Approval The studies discussed have been approved by the University of Pittsburgh IACUC and are be covered by IACUC protocol #20037102 (University of Pittsburgh).