LTBR-mediated immunomodulation of the tumor microenvironment promotes anti-tumor responses

The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis in many cancer types and has been associated with better treatment responses to immune-checkpoint blockade (ICB). However, the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses remain unclear. Lymphotoxin beta receptor (LTBR) signaling is a critical regulator of lymph node organogenesis and when combined with antiangiogenic and ICB treatment can augment tumor-associated HEV formation. Here we demonstrate that LTBR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTBR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTBR agonism on DC activation and maturation and associated DC- mediated T cell activation. Single agent LTBR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner; and the LTBR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies, respectively. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTBR agonism and lymphotoxin alpha (LT?) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTBR signaling in modulating the tumor microenvironment and inform future HEV/TLS- based strategies for cancer treatments. Overall design: The GFP-expressing tumor cells were subcutaneously implanted in BALB/c mice. The tumor-bearing mice were treated with 3 doses of isotype control or LTBR agonist antibody at 2 mg/kg, every other day. There are 4 treatment groups: "Isotype control mAb treated", "LTBR agonist mAb treated", "Isotype control mAb treated + LTa expression", "LTBR agonist mAb treated + LTa expression". At the end of study, 5 tumors per treatment group (N=5) were harvested, dissociated, stained by endothelial cell marker CD31 and immune cell marker CD45, and sorted by flow cytometry to collect the immune cells (CD45+CD31-GFP-), endothelial cells (CD31+CD45-GFP-) and stromal cells (CD45-CD31-GFP-) for scRNA sequencing.