Abstract A010: B cell infiltration and clonal expansion in tertiary lymphoid structures: Insights from intravital microscopy

Abstract Emerging clinical evidence highlights the importance of tumor infiltration of activated immune cells, particularly T and B lymphocytes, for the efficacy of immune-based cancer therapies. However, many patients show limited immune cell presence in the tumor microenvironment. High endothelial venules (HEVs) are specialized blood vessels that enable lymphocyte trafficking from circulation into lymph nodes or tumors. Histological analyses of clinical tumor samples indicate that HEVs are positioned near dense aggregates of B and T lymphocytes, known as tertiary lymphoid structures (TLS), underscoring their role in immune cell recruitment and anti-tumor immunity. The abundant development of TLS and HEVs is associated with clinical response to immune checkpoint inhibition therapy and cancer survival. Recently, we found that simultaneous activation of innate immune effectors, STING and lymphotoxin-β receptor (LTβR) using their agonists induces the formation of TLS and HEVs in TLS/HEV-free tumors. In the current study, mouse pancreatic ductal adenocarcinoma (KPC) tumors were grown in the dorsal skin fold window chamber (DSWC) and received STING/LTβR agonist combination therapy to monitor the real-time TLS/HEV development and dynamic tumor microenvironment in intravital microscopy (IVM). Transgenic mice with fluorescent red B cells and green fluorescent CD31+ blood vessels were infused with MECA-79 antibody (HEV marker) to visualize B cell trafficking through HEVs. Four-color confetti mice crossed with B cell-specific Cre mice, allowed us to analyze the clonal expansion of B cells expressing one of four colors, with daughter cells retaining their parent color. Our findings demonstrated that tumoral HEVs closely resemble those found in lymph nodes, characterized by fewer branches, longer segment lengths, and increased diameters compared to non-HEV tumor blood vessels, which typically exhibit chaotic patterns. Circulating B cells traveling through tumoral HEVs displayed significantly slower rolling velocities than non-HEV tumor vessels, likely due to enhanced integrin and L-selectin expression induced by the combination therapy. By repeated intravital imaging, we were also able to observe a quadruple increase in B cell number in TLS during the day 4-7 treatment time frame. Following adoptive transfer of splenic leukocytes from naïve donor mice, we found that these leukocytes preferentially homed to TLS-rich areas, with B cells constituting the majority of the recruited cells. Importantly, analyses using the confetti mouse model indicate that tumor infiltrating B cells underwent clonal expansion in TLS, with different TLS having different colors. Overall, our study demonstrates that HEVs are the gateways for B cell entry to tumors, and the intratumoral TLS formation is driven by localized clonal expansions of these B cells, resembling lymph node germinal center activities, which drive the development of humoral immunity against tumors. Citation Format: Gabrielle Rowe-Brown, Masanobu Komatsu. B cell infiltration and clonal expansion in tertiary lymphoid structures: Insights from intravital microscopy [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 A010.