Exploring the pro-tumor role of IL17 producing γδT (Tγδ17) cells in gallbladder cancer

Rushikesh Patil^1,3, Sagar Shah¹, Shailesh Shrikhande^2,3, Mahesh Goel^2,3, Rajesh Dikshit^2,3, and Shubhada Chiplunkar^1,3

¹Chiplunkar Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, 410210, India.

²Tata Memorial Hospital, Dr. E. Borges Road, Parel, Mumbai, Maharashtra, 400012, India

³Homi Bhabha National Institute, Anushaktinagar, Mumbai -400094

Introduction: Gall bladder cancer (GBC) is a highly malignant cancer known for its poor clinical outcome. Prognostic biomarkers and effective immunotherapy for GBC are unavailable. T cells expressing γδ-TCR exhibit potent anti-tumour activity and have gained significant importance in cell based therapies. However, the role of γδT cells in cancer promoting inflammation is not well explored. The present prospective study aimed at investigating the contribution of pro and anti-inflammatory immune cells in pathogenesis of GBC.

Methodology: Multicolor flowcytometry was used for immunophenotyping. Serum cytokines were examined by cytometric bead array. Suppressive potential of Tregs was evaluated by CFSE dilution. Cell migration and angiogenesis was investigated using transwell assay and angiogenesis array respectively.

Results: Immunophenotyping of GBC patients revealed that IL17 producing inflammatory subtypes of γδ (Tγδ17), CD4 (Th17) and CD8 (Tc17) T cells were increased in peripheral blood (PBL) and tumor infiltrating lymphocytes (TIL) of GBC patients compared to healthy individuals (HI). Regulatory T cells (Tregs) were decreased in PBLs and increased in TILs of GBC patients but their suppressive potential was comparable to HI. The ratios of Th17/Treg, Tγδ17/Treg and Tc17/Treg were increased but CD8⁺IFNg⁺ and γδ⁺IFNg⁺ cells were decreased in TILs. Serum cytokines profile of GBC patients showed elevated levels of cytokines (IL6, IL23 and IL1β) required for differentiation of IL17 producing cells. We demonstrated that Tγδ17 cells migrate towards tumour bed through CXCL9-CXCR3 axis. IL17 secreted by Tγδ17 induced VEGF production and other angiogenesis related factors in GBC cells. Tgd17 cells promote vasculogenesis as studied by chick chorioallantoic membrane assay. Addition of IL17 to GBC cell lines (OCUG1 and NOZ) induced proliferation, migration, matrigel invasion and VEGF production in concentration dependent manner. Tγδ17, Th17, and Treg were associated with poor survival of GBC patients.

Discussion and conclusion: Our data establishes Tgd17 as a pro-tumorigenic subtype of gdT cells, which correlates with the negative clinical outcome in GBC patients.

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