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1048 Interplay of IL-12 and IFNg to induce Treg fragility within the tumor microenvironment
  1. Ellen Scott,
  2. Angela Gocher,
  3. Creg Workman and
  4. Dario Vignali
  1. University of Pittsburgh, Pittsburgh, PA, USA


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 (Tregs) suppress the anti-tumor T cell response through use of direct and indirect mechanisms.1,2 However, under some circumstances Tregs can lose their suppressive activity while retaining expression of their master transcription factor, Foxp3, a phenomenon our lab has termed Treg fragility.3 A hallmark of Treg 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 Treg fragility as well as the consequences of Treg-produced IFNg. By using a novel transgenic mouse with a conditional deletion of IL-12Rb2 (Il12rb2.Thy1.1 L/LhNGFR Foxp3 Cre.YFP) in Tregs we were able to test the effect of IL-12 signaling on Treg 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 Treg-specific IL12R deletion with MC-38 or B16 and characterized the effect of IL-12R loss on Treg function after immunotherapy treatment by measuring tumor growth and flow cytometric analyses of intratumoral Tregs.

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 Tregs 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 Tregs are still sensitive to fragility induction.

Conclusions These data suggest that IL-12 is not necessary for the induction of Treg 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 Treg 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 Tregs and therefore augment the TME and the response to immunotherapy.


  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.

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