Background Immune cells play an integral role in the prevention as well as the progression of cancers. Within the T cell compartment, regulatory T cells (T_regs) suppress the anti-tumor T cell response through use of direct and indirect mechanisms.^1,2 However, under some circumstances T_regs can lose their suppressive activity while retaining expression of their master transcription factor, Foxp3, a phenomenon our lab has termed T_reg fragility.³ A hallmark of T_reg fragility is production of IFNg, a pleiotropic cytokine necessary for response to cancer immunotherapies such as anti-PD1.⁴ However, it is unknown the exact mechanism that leads to T_reg fragility as well as the consequences of T_reg-produced IFNg. By using a novel transgenic mouse with a conditional deletion of IL-12Rb2 (Il12rb2.Thy1.1 ^L/LhNGFR Foxp3 ^Cre.YFP) in T_regs we were able to test the effect of IL-12 signaling on T_reg function and response to different immunotherapies during tumor progression.

Methods We inoculated wildtype mice with MC-38 adenocarcinoma or B16 melanoma and determined the amount of IL-12 and IFNg induced by different immunotherapies. We also inoculated mice with a T_reg-specific IL12R deletion with MC-38 or B16 and characterized the effect of IL-12R loss on T_reg function after immunotherapy treatment by measuring tumor growth and flow cytometric analyses of intratumoral T_regs.

Results We have found that anti-PD1 therapy is able to cause modest increases in IFNg and IL-12 levels within the tumor. In contrast, when using an IL-12 inducing therapy (anti-CD40 agonism) there was a larger amount of IL-12 intratumorally along with very large increases of IFNg levels in both tumors and serum of wildtype mice. However, when mice with IL-12R deficient T_regs were treated with either of these therapies, tumor growth was not affected. Additionally, flow cytometric analyses of these mice after anti-CD40 agonist treatment suggests that these T_regs are still sensitive to fragility induction.

Conclusions These data suggest that IL-12 is not necessary for the induction of T_reg fragility. More specifically, these data may indicate that the increase in IFNg that is induced by immunotherapy may circumvent the IL-12 signaling loss in our mouse model and induce T_reg fragility. While the mechanism of how IL-12 is effecting these changes still unknown, this evidence suggests that IL-12 can indirectly induce fragility in T_regs and therefore augment the TME and the response to immunotherapy.

References

1. Scott EN, Gocher AM, Workman CJ, Vignali DAA. Regulatory T cells: barriers of immune infiltration into the tumor microenvironment. Front Immunol 2021;12:702726.

2. Vignali DAA, Collison LW, Workman CJ. How regulatory T cells work. Nat Rev Immunol. 2008;8(7):523–32.

3. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, et al. Interferon-? Drives Treg Fragility to Promote Anti-tumor Immunity. Cell 2017;169(6):1130–1141.e11.

4. Gocher AM, Workman CJ, Vignali DAA. Interferon-?: teammate or opponent in the tumour microenvironment? Nat Rev Immunol 2022 Mar;22(3):158–72.

Ethics Approval All mice were co-housed in the University of Pittsburgh Animal Facility under SPF conditions. All experiments strictly adhered to the University of Pittsburgh Animal Care and Use Committee requirements.