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Response to mechanical signals is an important mechanism by which T lymphocytes drive inflammation. A potential contributor in this process is Piezo1, a mechanosensitive cation channel which is abundantly expressed in T lymphocytes. Previously, our lab has shown that Piezo1 colocalizes with high affinity LFA-1 on activated human T lymphocytes during chemokine-activated crawling, suggesting a role for Piezo1 in the adhesion cascade. However, the mechanism by which Piezo1 contributes to the regulation of the adhesion cascade is unknown. We hypothesize that Piezo1 mediates T lymphocyte migration through direct contact with integrin to regulate its function. We investigate the role of Piezo1 in the expression, binding affinity, and turnover of LFA-1 and VLA-4. Co-immunoprecipitation revealed a preferential association of Piezo1 with the alpha subunit of integrins, specifically LFA-1, MAC-1, and VLA-4 in the active forms. Moreover, microfluidic devices were used to study the contribution of Piezo1 on T lymphocytes under laminar flow-induced shear stress. We found that activated T lymphocytes with CRISPR/Cas9-mediated Piezo1 ablation resulted in a reduced chemokine-induced crawling speed that correlates to the efficiency of Piezo1 gene ablation. Finally, in studies of lymph node homing where fluorescently labeled T lymphocytes were tracked in vivo, Piezo1 deficient cells exhibit increased ingress and decreased egress through the lymph node, suggesting an intricate role of Piezo1 on T lymphocyte migration. Taken together, our data support the hypothesis that Piezo1 modulates inflammatory processes through complex integrin interactions and mediates T lymphocyte migration from the lymph node to the site of inflammation. Case Postbaccalaureate Research Education Program grant NIH/NIGMS 5R25GM075207-15; St. Baldrick’s Foundation; Hyundai Hope-on-Wheels Program; The I’m Not Done Yet Foundation; and Pediatric Cancer Research Foundation

PD-L1 blockade has shown great success in the rejection of specific PD-L1+ tumor-types within various tissues. Blocking PD-L1 interactions with PD-1 molecules expressed on effector T cells allow them to engage their targets unimpeded and enhances tumor rejections. However, most pediatric tumors and some adult tumors express low to no surface PD-L1. Interestingly, some reports have demonstrated the rejection of PD-L1 negative tumors after treatment with anti-PD-L1 antibodies (Abs). When we administered anti-PD-L1 Abs 7 days after injection of CRISPR-negative PD-L1 (crPD-L1neg) medulloblastoma (MM1) tumors, all tumors were rejected. However, treating with anti-PD-L1 Abs at the time of tumor inoculation (day 0), resulted in a greatly diminished response to further treatment and a loss of protection. In addition, analysis of day 7 treated tumors in comparison to isotype and day 0 treated tumors, revealed an ~4- and ~2-fold increase in PD-1+ CD8+ T cells by day 21, respectively. These data suggest that the efficacy of anti-PD-L1 Abs on PD-L1 negative tumors is dependent on the timing and the microenvironment in which the T cell priming occurs. Our lab is investigating this phenomenon by interrogating the interaction of DC-T cell priming in the tumor draining lymph nodes (TDLNs) during the early stages of tumor growth. These studies may lead to improved efficacy of immune checkpoint blockade in PD-L1 negative tumors.

CCL3 is important in orchestrating cellular contacts in vaccinated lymph nodes (LNs) and enhancing memory T cell generation. Furthermore, dendritic cells (DCs) have been shown to mediate both tumor-tolerance and antitumor immunity in some tumor models. We hypothesize that incorporating CCL3 into tumor vaccines can enhance antitumor immunity. We injected into the footpad of naïve recipients, wild-type tumors (WTTUs) or CCL3-secreting (L3TUs) colon tumors (CT26). Immunocompetent mice injected with L3TUs resulted in a suppression of tumor growth compared to the WTTU group in a CD8+ T cell dependent manner. 1- and 5- days after injection with WTTUs or L3TUs, there was a superior accumulation of endogenous DCs and activated T cells in the draining lymph node (DLN) of the L3TU group. Concurrently bone marrow dendritic cells (BMDC)-tumor or -T cell co-culturing experiments were conducted to assess the effects the tumor and its microenvironment (+/− CCL3) has on DC function. In vitro experiments using BMDCs cultured with CCL3 enhanced their expression of MHC-I, CD86, and CCR7. BMDCs cultured with CCL3, then pulsed with SIINFEKL peptide, also showed an enhance capacity to proliferate OT-I (CD8+) T cells. In vivo analysis reveal that BMDCs injected directly into mice bearing L3TUs showed an enhanced ability to home to DLNs. Moreover, ElISpot and intracellular analysis of isolated T cells from LNs revealed an enhanced ability for T cells to upregulate IFNγ post-tumor injection with L3TUs. Together these data reveal that CCL3 in the tumor microenvironment leads to a reduction in tumor growth and improved T cell responses in the DLN, which may be a result of an enhanced capacity for DCs to home to the DLN and activate antitumor T cells.