THE “TYPE 2” CYTOKINE, INTERLEUKIN-33, GENERATES POTENT CD4+ST2+TBET+ TH1 RESPONSES INDEPENDENTLY OF IL-12 UNDER LYMPHOPENIC CONDITIONS

GaelenK. Dwyer ⁰; Heth R. Turnquist ^0,0; LisaR. Mathews ⁰; Anna Lucas ⁰; BruceR. Blazar ⁰

⁰Medical Scientist Training Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; ¹Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; ²Department of Pediatrics, University of Minnesota, Minneapolis, PA, United States; ³Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Introduction: The stromal-derived cytokine interleukin (IL)-33 is described as a promoter of CD4⁺ T helper type 2 (Th2) and regulatory T cells (Treg) responses. Yet, recipient conditioning before allogeneic hematopoietic cell transplantation (alloHCT) releases IL-33 to promote type 1 alloimmunity and graft-versus-host disease (GVHD). Conditioning causes lymphopenia and releases self and bacteria materials that promote myeloid cell secretion of IL-12, a type 1 immune response driver. IL-12 drives T cell expression of Tbet, which induces the IL-33 receptor, ST2, on T cells. While it is known that IL-33-mediated GVHD immunopathology involves direct stimulation of donor T cells, -the precise mechanisms mediating IL-33 support of GVHD are unclear. Herein we tested the hypothesis that neutralizing IL-12 post alloHCT would block IL-33-support of CD4⁺ T helper type 1 (Th1) response and limit GVHD by favoring Th2 or Treg responses. 

Methods: B6 Rag2^-/-γc^-/- mice were used to test the impact of IL-33 on CD4⁺ T cells in lymphopenic conditions.  These mice were infused with 5x10⁴ CD90.1⁺CD4⁺ST2⁺Treg and 4.5x10⁵ CD90.2⁺CD4⁺Foxp3^-CD44^loST2^lo T cells and recieved PBS  or IL-33 on day (d)1-8. Splenocytes were assessed by flow cytometry on d9. To establish the role of IL-12 in IL-33-mediated GVHD responses, irradiated BALB/c recipients were given 1x10⁷ B6 alloHCT with 2x10⁶ B6 T cells. Some cohorts also received IL-12p40 neutralizing Ab or control Ab alone, or with IL-33 (d3-7). Survival was determined. Splenocytes from additional mice were assessed at d7.

Results: Therapeutic doses of anti-IL-12p40 did not rescue mice from IL-33-mediated acceleration of GVHD (Fig. 1A). Post-alloHCT, IL-33 worked with IL-12 to expand ST2⁺Tbet⁺ Th1 cells (Fig. 1B). Surprisingly, only neutralization of IL-12, but not delivery of IL-33, increased Gata-3⁺ Th2 cells (Fig. 1C) or Treg. During lymphopenia, proliferating Foxp3^-CD4⁺ T cells display ST2 and IL-33 favored the expansion of these non-Treg cells over ST2⁺ Treg (Fig 2). 

Conclusion: We provide insights into how IL-33 contributes to GVHD. Specifically, donor CD4⁺ T cells transferred into the lymphopenic environments rapidly upregulate ST2 and are expanded by IL-33. Our data also suggest that after alloHCT, IL-33 does not facilitate ST2⁺ Treg- or ST2⁺Gata-3⁺ Th2 expansion, but works with IL-12 to support a ST2⁺Tbet⁺ Th1 response.
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