Introduction

^​​​​​​T cells, are a major regulator and source of pro-inflammatory cytokines, which may promote colitis, but be protective in some cancers^1,2. Nevertheless, limited efforts have been made to explore factors which determine the phenotype of T cells and their impact on the above-mentioned phenomena. The adhesion G protein-coupled receptor F5 (ADGRF5) belongs to seven-transmembrane receptor subfamily and may be related to development of some cancer types³.

Aims & Methods

The aim of the study was to assess the significance of ADGRF5 for T cell function and its impact on colitis and colon cancer progression. WT and ADGRF5 knockout CD4⁺ T cells were examined for cytokine genes and proteins using real-time PCR, Western blot and multiplex analysis. Tumor killing assay and flow cytometry analysis were performed to evaluate tumor killing properties of T cells co-cultured with MC38 colon cancer cells. ADGRF5 WT and KO mice were utilized to examine the impact of ADGRF5 on colitis, colitis-associated cancer development and colon cancer growth. Adoptive transfer of WT and ADGRF5 knockout CD4⁺ and CD8⁺ T cells were examined in tumor models to assess their role in disease progression.

Results

In murine model of colon cancer, animals with ADGRF5 knockout developed significantly smaller tumors which was accompanied by enhanced CD3 expression and hyperproduction of cytokines related to T cell action compared to wild type mice. Ex vivo analysis of two main population of T cells obtained from spleen of wild type and ADGRF5 knockout mice revealed that activated CD4⁺ but not CD8⁺ T cells with ADGRF5 were manifested by increased production of major anti-cancer molecules such as interferon γ (IFN- γ), perforin and granzyme B, and were characterized by secretion profile of cytokines related to mixed phenotype of T helper (T_h) 1 and T_h2 cells. Moreover, we documented higher proportion of apoptotic cells in co-culture of MC38 colon cancer cells with ADGRF5 knockout but not wild type CD4⁺ T cells. Finally, in the murine model of colon cancer adoptive transfer of CD4⁺ T cells with ADGRF5 knockout led to a significant reduction of tumor volume which was reversed by administration of antibody against IFN-γ. Nevertheless, ADGRF5 knockout in mice with colitis-associated colorectal cancer and colitis was related to significantly higher number of colon tumors and worse disease activity index compared to wild type animals, respectively. These results together with the analysis of gene expression signature in inflamed colons suggest opposite role for ADGRF5 in colitis than in the allograft colon cancer.

Conclusion

ADGRF5 is a critical regulator of T cell function and specific knockout of ADGRF5 reprograms CD4⁺ T cells into the pro-inflammatory phenotype. Our in vitro and in vivo studies indicated that ADGRF5 knockout in T cells promote pro-inflammatory cytokines, which prevent gastrointestinal cancer progression while boost both colitis and colitis-associated colorectal cancer development.

References

1. Jacenik D., Karagiannidis I., Beswick E.J. 2022. T_h2 cells inhibit growth of colon and pancreas cancers by promoting anti-tumorigenic responses from macrophages and eosinophils. Br J Cancer. 128: 387-397. doi: 10.1038/s41416-022-02056-2.
2. Shale M., Schiering C., Powrie F. 2013. CD4⁺ T cell subsets in intestinal inflammation. Immunol Rev. 252: 164-82. doi: 10.1111/imr.12039.
3. Jacenik D., Hikisz P., Beswick E.J., Fichna J. 2023. The clinical relevance of the adhesion G protein-coupled receptor F5 for human diseases and cancers. Biochim Biophys Acta Mol Basis Dis. 1869: 166683 doi: 10.1016/j.bbadis.2023.166683.

Disclosure

The work was supported by the National Science Centre, Poland under research project no 2019/32/C/NZ5/00064 (DJ).