Febrile temperature change modulates CD4 T cell differentiation via a TRPV channel-regulated Notch-dependent pathway.

Fever is a conserved and prominent response to infection. Yet, the issue of how CD4 T cell responses are modulated if they occur at fever temperatures remains poorly addressed. We have examined the priming of naive CD4 T cells in vitro at fever temperatures, and we report notable fever-mediated modulation of their cytokine commitment. When naive CD4 T cells were primed by plate-bound anti-CD3 and anti-CD28 monoclonal antibodies at moderate fever temperature (39 °C), they enhanced commitment to IL4/5/13 (Th2) and away from IFNg (Th1). This was accompanied by up-regulation of the Th2-relevant transcription factor GATA3 and reduction in the Th1-relevant transcription factor Tbet. Fever sensing by CD4 T cells involved transient receptor potential vanilloid cation channels (TRPVs) since TRPV1/TRPV4 antagonism blocked the febrile Th2 switch, while TRPV1 agonists mediated a Th2 switch at 37 °C. The febrile Th2 switch was IL4 independent, but a γ-secretase inhibitor abrogated it, and it was not found in Notch1-null CD4 T cells, identifying the Notch pathway as a major mediator. However, when naive CD4 T cells were primed via antigen and dendritic cells (DCs) at fever temperatures, the Th2 switch was abrogated via increased production of IL12 from DCs at fever temperatures. Thus, immune cells directly sense fever temperatures with likely complex physiological consequences.

Intraperitoneal activation of myeloid cells clears ascites and reveals IL27-dependent regression of metastatic ovarian cancer.

Patients with metastatic ovarian cancer (OvCa) have a 5-year survival rate of less than 30% due to persisting dissemination of chemoresistant cells in the peritoneal fluid and the immunosuppressive microenvironment in the peritoneal cavity. Here, we report that intraperitoneal administration of ß-glucan and IFNγ (BI) induced robust tumor regression in clinically relevant models of metastatic OvCa. BI induced tumor regression by controlling fluid tumor burden and activating localized antitumor immunity. ß-glucan alone cleared ascites and eliminated fluid tumor cells by inducing intraperitoneal clotting in the fluid and Dectin-1-Syk-dependent NETosis in the omentum. In omentum tumors, BI expanded a novel subset of immunostimulatory IL27+ macrophages and neutralizing IL27 impaired BI efficacy in vivo. Moreover, BI directly induced IL27 secretion in macrophages where single agent treatment did not. Finally, BI extended mouse survival in a chemoresistant model and significantly improved chemotherapy response in a chemo-sensitive model. In summary, we propose a new therapeutic strategy for the treatment of metastatic OvCa.

The African-centric P47S Variant of TP53 Confers Immune Dysregulation and Impaired Response to Immune Checkpoint Inhibition.

The tumor suppressor TP53 is the most frequently mutated gene in cancer and is mutationally inactivated in 50% of sporadic tumors. Inactivating mutations in TP53 also occur in Li Fraumeni syndrome (LFS). In addition to germline mutations in TP53 in LFS that completely inactivate this protein, there are many more germline mutant forms of TP53 in human populations that partially inactivate this protein: we call these partially inactivating mutations "hypomorphs." One of these hypomorphs is a SNP that exists in 6%-10% of Africans and 1%-2% of African Americans, which changes proline at amino acid 47 to serine (Pro47Ser; P47S). We previously showed that the P47S variant of p53 is intrinsically impaired for tumor suppressor function, and that this SNP is associated with increased cancer risk in mice and humans. Here we show that this SNP also influences the tumor microenvironment, and the immune microenvironment profile in P47S mice is more protumorigenic. At basal levels, P47S mice show impaired memory T-cell formation and function, along with increased anti-inflammatory (so-called "M2") macrophages. We show that in tumor-bearing P47S mice, there is an increase in immunosuppressive myeloid-derived suppressor cells and decreased numbers of activated dendritic cells, macrophages, and B cells, along with evidence for increased T-cell exhaustion in the tumor microenvironment. Finally, we show that P47S mice demonstrate an incomplete response to anti-PD-L1 therapy. Our combined data suggest that the African-centric P47S variant leads to both intrinsic and extrinsic defects in tumor suppression. Significance: Findings presented here show that the P47S variant of TP53 influences the immune microenvironment, and the immune response to cancer. This is the first time that a naturally occurring genetic variant of TP53 has been shown to negatively impact the immune microenvironment and the response to immunotherapy.

Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer.

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity.

The microbiome-derived metabolite TMAO drives immune activation and boosts responses to immune checkpoint blockade in pancreatic cancer.

The composition of the gut microbiome can control innate and adaptive immunity and has emerged as a key regulator of tumor growth, especially in the context of immune checkpoint blockade (ICB) therapy. However, the underlying mechanisms for how the microbiome affects tumor growth remain unclear. Pancreatic ductal adenocarcinoma (PDAC) tends to be refractory to therapy, including ICB. Using a nontargeted, liquid chromatography-tandem mass spectrometry-based metabolomic screen, we identified the gut microbe-derived metabolite trimethylamine N-oxide (TMAO), which enhanced antitumor immunity to PDAC. Delivery of TMAO intraperitoneally or via a dietary choline supplement to orthotopic PDAC-bearing mice reduced tumor growth, associated with an immunostimulatory tumor-associated macrophage (TAM) phenotype, and activated effector T cell response in the tumor microenvironment. Mechanistically, TMAO potentiated the type I interferon (IFN) pathway and conferred antitumor effects in a type I IFN-dependent manner. Delivering TMAO-primed macrophages intravenously produced similar antitumor effects. Combining TMAO with ICB (anti-PD1 and/or anti-Tim3) in a mouse model of PDAC significantly reduced tumor burden and improved survival beyond TMAO or ICB alone. Last, the levels of bacteria containing CutC (an enzyme that generates trimethylamine, the TMAO precursor) correlated with long-term survival in patients with PDAC and improved response to anti-PD1 in patients with melanoma. Together, our study identifies the gut microbial metabolite TMAO as a driver of antitumor immunity and lays the groundwork for potential therapeutic strategies targeting TMAO.

Immunophenotype and function define TCRγδ + T-ALL as a distinct subgroup from TCRαß + T-ALL patients.

We recently demonstrated TCRγδ + T-ALL as a distinct subgroup from TCRαß + T-ALL at genomic level. TCRγδ + T-ALL subgroup possess significant survival advantage compared to TCRαß + T-ALL. In the present study, functional level differences in these two subgroups of T-ALL were studied to understand the immune scenario contributing to survival benefit of TCRγδ + T-ALL subgroup. TCRγδ clonal T-ALL patients showed significantly high levels of γδ T cells compared to TCRαß clonal T-ALL patients. TCRγδ + T-ALL patients expressed significantly high central memory and terminally differentiated (TemRA) Vδ1 and Vδ2 T cells. TCR γδ clonal leukemic blasts stimulated increased number of Vδ2 T cells from healthy lymphocytes. TCR γδ clonal leukemic blasts were able to form efficient immune synapse with effector γδ T cells. The differences in immunophenotype, cytotoxicity and immune synapse formations corroborate TCRγδ + T-ALL as a distinct subgroup from TCRαß + T-ALL patients and explain the survival benefit of TCRγδ + T-ALL patients.

Risk stratification of T-cell Acute Lymphoblastic Leukemia patients based on gene expression, mutations and copy number variation.

Gene expression, copy number variations (CNV), mutations and survival were studied to delineate TCRγδ+T-cell acute lymphoblastic leukemia (T-ALL) as a distinct subgroup from TCRαß+T-ALL. Gene Ontology analysis showed that differential regulation of genes involved in pathways for leukemogenesis, apoptosis, cytokine-cytokine receptor interaction and antigen processing/presentation may offer a survival benefit to TCRγδ+T-ALL patients. Genes involved in disease biology and having equal expression in both the subgroups, were further analysed for mutations and CNV using droplet digital PCR. TCRγδ+T-ALL patients exhibited differential level of mutations for NOTCH1 and IKZF3; however BRAF mutations were detected at equal levels in both the subgroups. Although TCRγδ+T-ALL patients with these mutations demonstrated improved disease-free survival (DFS) as compared TCRαß+T-ALL patients, it was not statistically significant. Patients with homozygous deletion of CDKN2A/CDKN2B showed poor DFS in each subgroup. TCRγδ+T-ALL patients with wild type/heterozygous deletion of CDKN2A/CDKN2B possess significantly better DFS over TCRαß+T-ALL patients (p=0.017 and 0.045, respectively). Thus, the present study has for the first time demonstrated TCRγδ clonality and CDKN2A/CDKN2B CNV together as potential prognostic markers in management of T-ALL. Further understanding the functional significance of differentially regulated genes in T-ALL patients would aid in designing risk based treatment strategies in subset specific manner.