Radiation combines with immune checkpoint blockade to enhance T cell priming in a murine model of poorly immunogenic pancreatic cancer

Radiation has been a pillar of cancer therapy for decades. The effects of radiation on the anti-tumour immune response are variable across studies and have not been explicitly defined in poorly immunogenic tumour types. Here, we employed combination checkpoint blockade immunotherapy with stereotactic body radiation therapy and examined the effect on tumour growth and immune infiltrates in subcutaneous and orthotopic mouse models of pancreatic cancer. Although immune checkpoint blockade and radiation were ineffective alone, their combination produced a modest growth delay in both irradiated and non-irradiated tumours that corresponded with significant increases in CD8+ T cells, CD4+ T cells and tumour-specific T cells as identified by IFNγ ELISpot. We conclude that radiation enhances priming of tumour-specific T cells in poorly immunogenic tumours and that the frequency of these T cells can be further increased by combination with immune checkpoint blockade.

ACKR4 in Tumor Cells Regulates Dendritic Cell Migration to Tumor-Draining Lymph Nodes and T-Cell Priming

Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but have failed in mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is implicated in regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and anti-tumor immunoregulation are not known. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Moreover, tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockade. Finally, we identified that microRNA miR-552 negatively regulates ACKR4 expression in human CRC. Taken together, our studies identified a novel and crucial mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC.

P08.02 CCR2/CCR5 dual-antagonist ‘licenses’ the radiation-induced effector T-cell infiltration in the anti-PD-1 antibody-treated pancreatic adenocarcinoma

BackgroundThe resistance of pancreatic ductal adenocarcinoma(PDAC) to immune checkpoint inhibitors(ICIs) is mainly attributed to the immune-quiescent nature of its tumor microenvironment(TME). Radiotherapy(RT) activates innate responses including the RAGE and TLR2/4 pathways and subsequently modifies the TME by promoting the release of chemokines that recruit inflammatory cells into the TME. In this preclinical study, we examined the PDAC vaccine or RT as a T-cell priming mechanism together with BMS-687681, a small molecule dual-antagonist of CCR2 and CCR5(CCR2/5i) as an immunosuppressive TME-targeting agent, in combination with the anti-PD-1 antibody(αPD-1) as a new treatment.Materials and MethodsThe hemi-spleen and Orthotopic mice model were used to investigate both GVAX and RT as T-cell priming agents in combination regimens that included αPD-1 and CCR2/5i. Dissected orthotopic pancreatic tumors were collected for analysis of tumor-infiltrating immune cells by flow cytometry. RNA from tumor-infiltrating immune cell pellets and whole-exome RNA sequencing was performed for further mechanism research.ResultsCCR2 and CCR5 are associated with the immunosuppressive TME of PDAC patients and their expression were induced after treatment with GVAX+nivolumab. Using a mouse model of PDAC, we demonstrated that the addition of GVAX to CCR2/5i+αPD-1 combination therapy did not significantly improve antitumor activity. However, RT followed by αPD-1 and prolonged treatment with CCR2/5i conferred significantly better antitumor efficacy compared to the other combination treatments we studied. The combination of RT, αPD-1, and CCR2/5i enhanced intratumoral effector and memory T-cell infiltration. This combination suppressed Treg, M2-like TAM, and M-MDSC infiltration, but not M1-like TAM and PMN-MDSC infiltration. Finally, RNA sequencing showed that CCR2/5i partially inhibited RT-induced TLR2/4&RAGE signaling, which would have otherwise led to the release of immunosuppressive cytokines including CCL2 and CCL5. The inhibition of TLR2/4&RAGE signaling permitted the expression of effector T-cell chemokines such as CCL17 and CCL22.ConclusionsThis study thus supports the clinical development of CCR2/5i in combination with RT and ICIs for PDAC treatment.Disclosure InformationJ. Wang: None. M. Tun Saung: None. K. Fujiwara: None. N. Niu: None. A. Narang: None. J. He: None. L. Zheng: None.

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8+ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8+ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8+ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E2 (PGE2) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE2 production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE2 production early during infection whereas vacuole-constrained strains fail to induce PGE2 over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2+ or CD11c+ cells produce less PGE2, suggesting these cell subsets contribute to PGE2 levels in vivo, while depletion of phagocytes with clodronate abolishes PGE2 production completely. Taken together, this work demonstrates that optimal PGE2 production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8+ T-cell responses may be to facilitate production of PGE2.

Loss of ACKR4 in tumor cells dysregulates dendritic cell migration to tumor-draining lymph nodes and T-cell priming

Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but failed in the mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is known for regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and immunoregulation are unclear. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockades. Finally, we identified that microRNA-552 negatively regulates ACKR4 expression in human CRC. Taken together, our work identifies a critical mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC tumors.