Visualizing B Cell Infiltration and Aggregation Within the Tumor Microenvironment

Project Summary Emerging clinical evidence shows that activated immune cells, especially B and T lymphocytes, enhance the response to cancer immunotherapies but are often excluded from tumors. High endothelial venules (HEVs) may facilitate immune cell entry and tertiary lymphoid structure (TLS) formation within tumors, as observed in histological samples. Dr. Komatsu’s lab has developed an immunostimulatory treatment to induce HEV and TLS formation in previously HEV/TLS-free tumors. The combination of lymphotoxin beta receptor (LTβR) and STING agonists reduces tumor size and promotes robust anti-tumor immunity. This study uses transgenic mice with a dorsal skin window chamber (DSWC) tumor model undergoing intravital microscopy (IVM) to visualize B cell infiltration through HEVs and immunity development via TLS formation. Bulk sequencing and immunofluorescence analysis suggest that HEVs are the main route for B cell recruitment into tumors; intravital imaging shows B cells roll more slowly on the endothelium of HEVs, indicating HEVs facilitate B cell entry into tumors. Transgenic mice with fluorescent red B cells and fluorescent green blood vessels in DSWCs will visualize and analyze B cell recruitment, rolling speeds, transmigration through tumor-associated HEVs, and real-time TLS formation in agonist-treated KPC tumors using repeated IVM. These results will demonstrate for the first time that HEVs are critical gateways for B cell entry and TLS formation within tumors, providing foundational insights for targeted immunotherapies to enhance anti-tumor immunity. TLS formation in tumors may require continuous B cell recruitment via chemokine signaling. This study investigates whether TLS are sustained by ongoing B cell recruitment from circulation or clonal expansion within the tumor. Adoptive transfer experiments suggest that TLS recruit B cells from the circulation, evident by the upregulation of chemokine genes known for B cell recruitment following LTβR/STING agonist combination therapy that induces TLS formation. Maintenance may involve both initial B cell infiltration and subsequent clonal expansion, marked by germinal center gene upregulation. The anticipated findings will reveal that continuous recruitment of naïve B cells is essential for TLS maintenance, offering new insights into tumor immunity mechanisms and paving the way for novel therapeutic strategies that exploit B cell recruitment and clonal expansion to improve cancer treatment outcomes. The role of chemotaxis in B cell recruitment to TLS will be tested by inhibiting this process with pertussis toxin-treated naïve B cells, determining whether LTβR/STING agonist combination therapy promotes clonal expansion of B cell-comprised TLS and anti-tumor immunity development. Results are expected to show that continuous recruitment of naïve B cells and clonal expansion are essential for TLS formation and maintenance, providing insights for novel targeted cancer immunotherapies. This research will produce novel insights into immune cell infiltration and aggregation into intratumoral TLS, demonstrating experimental models needed for continued onco-immunology research.