Abstract B038: Toll like receptor 4 (TLR4) agonism by lipopolysaccharide (LPS) in pancreatic cancer model stimulates anti-tumor immunity in CD4+T cell dependent fashion
Authors: Vaish Utpreksha , Ferrantella Anthony R. , Jain Tejeshwar , Sharma Prateek , Iyer Srikanth , Dudeja Vikas 
Published: November 2022

Abstract
Introduction: Pancreatic cancer, one of the most lethal malignancies, has a 5-year overall survival of approximately 10%. It is an immunologically cold tumor and is poorly responsive to immunotherapy. Globally, researchers are in quest of immunomodulatory modalities to improve the therapeutic response. lipopolysaccharide (LPS), a known toll like receptor 4 (TLR4) agonist is known to activate immune response. Here, we evaluate the effect of LPS mediated TLR4 agonism on the anti-tumor immune machinery of pancreatic cancer. Methods: We implanted KPC (Kras LSL-G12D; Trp53 LSL-R172H/+; Pdx1Cre/+) pancreatic cancer cells in C57BL/6 mice subcutaneously. The animals were treated intraperitoneally with LPS (1mg/kg) or saline (control) at day 10 and 17. Tumor samples were isolated at endpoint and were analyzed using flow cytometry and real time polymerase chain reaction (RT-PCR) for infiltration of immunocytes and expression of immune-associated genes respectively. Functional response of CD4+ T-cells to tumor antigen re-challenge was assessed by measuring interferon-γ (IFN-γ) release in-vitro. In another experiment, LPS or saline treatment was administered along with depletion of CD4+ or CD8+ T-cells employing monoclonal antibodies. CD4+ T-cells extracted from spleens of tumor bearing control or LPS treated mice were subjected to Calcein AM based cytotoxicity assay. Cytotoxicity assay was also performed by blocking major histo-compatibility II (MHCII) on KPC cells. Results: LPS administration in tumor bearing mice significantly reduced the tumor burden as compared to control group. Flow cytometry on tumor samples revealed increased infiltration of CD4+ T-cells in LPS group whereas CD8+ T-cell repertoire was unaffected. Tumor samples from the LPS group showed higher expression of genes for anti-tumor cytokines such as IFN-γ, granzymes and perforin. Depletion of CD4+ T cells, but not CD8+ T cells attenuated the effect of LPS on tumor growth. When the splenic CD4+ T-cells from both groups were pulsed with KPC cell lysate, LPS treated mice derived CD4+ T-cells demonstrated a greater ability to secrete IFN-γ. Splenic CD4+ T-cells from LPS treated mice showed significantly higher cytotoxicity against KPC cells as compared to control CD4+ T-cells. However, blocking MHCII molecules on KPC cells reduced the cytotoxicity in CD4+ T-cells derived from LPS treated mice. This suggests that CD4 cytotoxicity ensues in an MHCII dependent manner. Conclusions: LPS treatment reduces tumor burden in the subcutaneous tumor model of pancreatic cancer by upregulating the infiltration of CD4+ T-cells and translating them to a cytotoxic phenotype. It also stimulates the upregulation of anti-tumor immunity related cytokines in the tumor microenvironment. We have also found that the cytotoxic CD4+ T cells terminate the pancreatic cancer cells in an MHCII dependent manner. Therefore, our findings provide a novel insight into how “TLR4 agonism can stimulate CD4+ T-cell mediated antitumor immunity”, which can be harnessed as a potential source for pancreatic cancer immunotherapy.
Citation Format: Utpreksha Vaish, Anthony R. Ferrantella, Tejeshwar Jain, Prateek Sharma, Srikanth Iyer, Vikas Dudeja. Toll like receptor 4 (TLR4) agonism by lipopolysaccharide (LPS) in pancreatic cancer model stimulates anti-tumor immunity in CD4+T cell dependent fashion [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B038.