Abstract: Objective To investigate the effects of TLR7/8 agonist Resiquimod (R848) on the generation and immunosuppressive function of γδ Treg induced by TGF-β1, and explore the underlying molecular mechanisms. Methods γδ T cells were expanded with anti-human γδ TCR antibodies in the presence of different concentrations of TGF-β1, and the expression of transcription factor forkhead box protein P3 (FoxP3) in γδ T cells was subsequently analyzed by flow cytometry. Then Vδ1 and Vδ2 γδ T cells were amplified by anti-human Vδ1 TCR anti- body and anti-human γδ TCR antibody respectively, and R848 was added to the induction system to examine its effect on the proportion of FoxP3⁺γδ Treg. Induced Vδ1 and Vδ2 γδ T cells were isolated by magnetic-activated cell sorting, and the effect of R848 on their suppressive function against CD4⁺ T cell proliferation was assessed by a CFSE dilution assay. Flow cytometry was used to detect the expression of perforin and granzyme B. Western blot was performed to measure the expression level of granzyme K. ELISA was used to determine the concentration of TGF-β1 in the supernatant, and RNA sequencing was performed to identify differentially expressed genes after R848 treatment with validation using Western blot. Results 3 ng/mL TGF-β1 significantly increased the expression of FoxP3 in γδ T cells. R848 had no significant effect on the generation of TGF-β1-induced FoxP3⁺ Vδ1 γδ Treg, but significantly reduced the proportion of FoxP3⁺ Vδ2 γδ Treg. R848 treatment reversed the suppressive effect of Vδ2 γδ Treg on CD4⁺ T cell proliferation, whereas no such effect was observed in Vδ1 γδ Treg. R848 partially reversed the suppressive function of Vδ2 γδ Treg, promoted granzyme B expression, and significantly decreased their secretion of TGF-β1.RNA-seq analysis and Western blot validation demonstrated that R848 reversed the suppressive function of Vδ2 γδ Treg by downregulating the expression of SOCS1 and CISH, two key negative regulators of the JAK/STAT signaling pathway and inhibiting STAT3 phosphorylation. Conclusions The TLR7/8 agonist R848 reverses the generation and suppressive function of TGF-β1-induced Vδ2 γδ Treg. These findings provide experimental support for the development of a combinational immunotherapeutic strategy involving γδ T cells and R848.

Key words: γδ T cells, TLR agonist, R848, TGF-β1