Background: IL-33 is a pleotropic cytokine that can protect against cardiac transplant rejection by expanding regulatory T cells (Treg), particularly a Treg subset expressing the IL-33 receptor, Suppression of Tumorigenicity 2 (ST2). However, if IL-33-mediated signals directly impact on the ST2-expressing Treg expansion and alloimmune regulatory functions is poorly understood. The goals of this study was to precisely characterize IL-33-mediated Treg signaling and establish the importance of direct IL-33 stimulation of Treg for control of alloimmune responses.

Methods: To determine the importance of ST2 expression on Treg for regulation of alloimmune responses, we utilized a rodent model of graft-vs. host disease (GVHD) where C57BL/6 recipient mice were given lethal total body irradiation followed by a subsequent BALB/c bone marrow graft. Graft recipients also received wild-type (WT) BALB/c CD3⁺ effector T cells (Teff) alone, or with CD4⁺ CD25⁺ Treg from WT (st2^+/+) or st2^-/- mice (Treg:Teff=1:2). In related experiments, fluorescence activated cell sorting was used to obtain ST2^– or ST2⁺ populations of Foxp3⁺ cells from the splenocytes of Foxp3 Reporter mice (FOXP3-IRES-mRFP mice). The capacity of IL-33 to activate signaling downstream of ST2 in each population was quantitated by phosphoflow cytometry.

Results: Where as 90% of recipients receiving st2^+/+ CD4⁺ CD25⁺ were free of GVHD at Day 100 post-alloHCT, only 10% of mice that received st2^-/- Treg survived long-term (p=0.0017, st2^+/+ v. st2^-/-). Associated mechanistic studies revealed that Treg frequency was reduced in recipients receiving st2^–/- Treg cells compared to those receiving st2^+/+ Treg. Phosphoflow established that IL-33 signaling via ST2⁺ on Treg activates both NF-κB and p38 MAPK. Yet, only IL-33-stimulation of p38 MAPK, but not NF-kB, was required for ST2⁺ Treg expansion.

Conclusions: These studies make the novel observation that IL-33 activates p38 MAPK to drive selective ST2⁺ Treg expansion. We also establish that IL-33-mediated ST2 signaling is critical to the capacity of adoptively transferred Treg to prevent the alloimmune responses leading to GVHD. In total, our data reveal the importance of IL-33 stimulation of Treg for effective regulation of alloimmunity.

CITATION INFORMATION: Zhang X, Matta B, Reichenbach D, Blazar B, Turnquist H. Direct IL-33 Stimulation of T Regulatory Cells via Suppression of Tumorigenicity 2 (ST2) Is Required for Effective Control of Alloimmune Responses. Am J Transplant. 2016;16 (suppl 3).

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