Lymph nodes (LNs) function as immunological hubs whereby antigen and naïve lymphocytes meet to facilitate an immune response. In cancers, the tumour-draining lymph node (TDLN) is where the initial anti-tumour immune response occurs. Previous studies have demonstrated that TDLNs undergo dramatic reprogramming in response to tumour drainage. However, the precise nature of these alterations and their contribution to tumour-induced immune escape requires further exploration.

Utilising the B16-F10 melanoma and E0771 breast cancer (BC) model I identified significant changes in the T cell landscape of TDLNs. While the CD8-mediated immune response is effectively suppressed in TDLNs, the CD4 compartment undergoes dramatic alterations. Most notably, regulatory T cells (Tregs) expand and become more active, upregulating suppressive markers, including TGF-B, CTLA-4 and FasL. Concurrently, CD4 and CD8 T cells show diminished secretion of IL-2 and IFNy, common markers of T cell activation. Localisation of Tregs also differed in TDLNs, displaying closer contacts with stromal fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs). Supporting these observations, in vitro studies demonstrated that tumour-conditioned FRCs display an enhanced capacity to recruit and adhere T cells. These findings support a model whereby tumour drainage reprograms the stromal and immune populations in TDLNs to create an immunosuppressive environment, thereby hindering the anti-tumour immune response.

Immune checkpoint inhibitors (ICIs) have emerged as a novel strategy to reinvigorate the immune system promoting tumour clearance; however, therapeutic success is often followed by resistance and tumour resurgence. To date most research has focused on the impact of ICIs at the tumour site, and the TDLN remains largely ignored. In this regard, I also examined the T cell response of ND- and TDLNs following a-PD-1 or a-CTLA-4 monotherapy. Immunotherapy bolstered the anti-tumour immune response in TDLNs, driving the differentiation of both CD4 and CD8+ T cells into effector and CM subsets, which circulated to mediate tumour cell clearance. Interestingly, checkpoint blockade also induced Treg expansion in TDLNs, with cells adopting a more immunosuppressive phenotype, as confirmed by in vitro and ex vivo suppression assays. Correspondingly, tumour cell death was reduced in LN isolates from therapy versus control treated LNs, demonstrating that the inhibitory niche is sufficient to overcome the immunostimulatory effects afforded by checkpoint blockade. Consequently, we have devised the “Homeostatic Hypothesis” to explain this phenomena. Immunotherapy effective bolsters the anti-tumour immune response within the TDLN, enhancing cancer clearance. Tregs subsequently expand in TDLNs to quell the immune response and prevent overt activation, resulting from checkpoint blockade. Complete Treg depletion within TDLNs therefore holds promise to maximize therapeutic effect, following checkpoint blockade, and mitigate resistance.