IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease Following Bone Marrow Transplantation,

Abstract 4037

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for many hematologic malignancies such as leukemia, lymphoma and myelodysplastic syndromes. A potentially fatal complication of HCT is graft-versus-host disease (GVHD), where conventional T cells of alloreactive-specificities (CD4⁺ and CD8⁺ T cells; T_con) infiltrate and destroy target organs such as the liver, gut and skin. While immunomodulation is necessary to control the adverse GVHD reactions, an effective immune response is required for successful tumor eradication, named graft-versus-tumor effect (GVT).

Following allogeneic bone marrow transplantation, our and other laboratories have shown that the transfer of highly purified regulatory T cells (T_reg), a key immunoregulatory cell population involved in the maintenance of immune tolerance, can prevent lethal GVHD induced by the donor-derived T_con in multiple models across both major and minor histocompatibility barriers. Notably, when transferred into recipient mice with established leukemia or lymphoma, T_reg cells effectively suppress T_con cell proliferation and prevent lethal GVHD, while preserving GVT activity. We have recently adapted our pre-clinical model to an ongoing clinical trial, and are currently assessing the efficacy of highly purified human T_reg cells in a clinical setting. However, recent evidence suggest that, under the influence of intense inflammatory signals, the T_regsuppressive phenotype can become unstable, a phenomenon that can culminate in T_regconversion into IL-17-producing pro-inflammatory cells. IL-17 is a proinflammatory cytokine that has been reported to promote GVHD in different models for human HCT. We hypothesized that under the influence of the intense proinflammatory signals, such as those released during an ongoing alloreaction, a fraction of the transferred T_reg might be redirected to the Th17 cell fate, thereby losing immunosuppressive potential and acquiring proinflammatory capacity. Our preliminary in vitro studies demonstrated that T_reg cells do indeed upregulate Il17 gene expression following activation by allogeneic antigen presenting cells (APCs). We, therefore, sought to evaluate the impact of Il17 gene ablation on T_reg stability and immunosuppressive capacity in a major MHC mismatch model, in order to ultimately assess the possible therapeutic potential of IL-17 targeting for the enhancement of T_reg-mediated GVHD suppressive ability following HCT.

On the basis of our preliminary studies we anticipated that T_reg cells purified from Il17 knockout (KO) donor mice would have enhanced immunomodulatory capacity compared to wild-type (WT) T_reg, given their inability to produce IL-17. To monitor ongoing GVHD our laboratory has developed an imaging system that allows for the in vivo visualization of T cell homing and proliferation over time, without the necessity to sacrifice the experimental animals. Specifically, the allogeneic T_con cells are isolated from donor mice where the gene coding for the enzyme luciferase (luc) is expressed by all hematopoietic cells. Importantly, the proliferation of the transferred luc⁺ T_con can be directly assessed by the number of photons detected by the bioluminescent imaging system (BLI), which gives a measure of ongoing GVHD. T_reg were purified from both WT and IL-17 KO animals and their GVHD-suppressive ability was directly compared in vivo following the transfer of luc+ T_con. IL-17 KO T_regcells were as proficient as WT T_reg in suppressing GVHD. Similarly, the GVHD-inhibitory capacity of T_reg cells isolated from IL-1R KO mice, that were previously shown to be resistant to Th17 conversion, was comparable to that of WT T_reg. Furthermore, upon ex vivo re-isolation of T_regthat were previously transferred into transplanted recipients, we could detect little or no IL-17 production by intracellular cytokine staining. Thus, we conclude that freshly isolated and highly purified T_reg do not convert into IL-17 producing cells in our model, suggesting that IL-17 production is not a factor affecting T_regstability during ongoing GVHD, and excluding IL-17 targeting as a valuable strategy to improve T_reg immunotherapy following bone marrow transplantation.