Allogeneic immune cells, particularly T cells in donor grafts, recognize and eliminate leukemic cells via graft-versus-leukemia (GVL) reactivity, and transfer of these cells is often used for high-risk hematological malignancies, including acute myeloid leukemia. Unfortunately, these cells also attack host normal tissues through the often fatal graft-versus-host disease (GVHD). Full separation of GVL activity from GVHD has yet to be achieved. Here, we show that, in mice and humans, a population of interleukin-9 (IL-9)–producing T cells activated via the ST2–IL-33 pathway (T9IL-33 cells) increases GVL while decreasing GVHD through two opposing mechanisms: protection from fatal immunity by amphiregulin expression and augmentation of antileukemic activity compared with T9, T1, and unmanipulated T cells through CD8α expression. Thus, adoptive transfer of allogeneic T9IL-33 cells offers an attractive approach for separating GVL activity from GVHD.
T-cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self-antigen. We previously showed in C57BL/6 mice that part of the CD4(+) T-cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 also recognizes the neuronal antigen neurofilament medium (NF-M) 15-35. Such bi-specific CD4(+) T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self-antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF-M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG35-55 -reactive CD4(+) T cells were increased in MOG-deficient but not in NF-M-deficient mice. We found that presentation of NF-M15-35 by I-A(b) on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC-anchoring residue into NF-M15-35 (NF-M15-35 T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi-specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self-antigens.
Soluble stimulation-2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2+ Tregs were thymus derived and predominantly localized to the intestine. ST2-/- Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2-/- versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type 1 and increased IL-4/IL-10-producing type 2 T cells. Cotransfer of ST2+ Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33-stimulated Tregs (TregIL-33) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that TregIL-33 has potential as a cellular therapy avenue for preventing GVHD.
Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble membrane-bound ST2 (ST2) are elevated in human and murine aGVHD and correlated to type 1 T cells response. ST2 signals through the adapter protein MyD88. The role of MyD88 in T cells during aGVHD has yet to be elucidated. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of IL-1R and TLR4 signaling in two murine HCT models. This protection was entirely driven by MyD88-/- CD4 T cells. Transplanting donor MyD88-/- conventional T cells (Tcons) with wild-type (WT) or MyD88-/- regulatory T cells (Tregs) lowered aGVHD severity and mortality. Transcriptome analysis of sorted MyD88-/- CD4 T cells from the intestine 10 d post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, Batf, and Jak2 Transplanting donor ST2-/- Tcons with WT or ST2-/- Tregs showed a similar phenotype with what we observed when using donor MyD88-/- Tcons. Decreased ST2 was confirmed at the protein level with less secretion of soluble ST2 and more expression of ST2 compared with WT T cells. Our data suggest that Treg suppression from lack of MyD88 signaling in donor Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways, and ST2 represents a potential aGVHD therapeutic target sparing Tregs.
Restricted accessAbstractFirst published online March 1, 2016Il-33/St2 Triggering of Il-9-Secreting T Cells: From Proteomics to TherapeuticsA Ramadan, J Zhang, […], M Abu Zaid, L Taylor, H O'Leary, R Kapur, H Hanenberg, HE Broxmeyer, MH Kaplan, and S Paczesny+7-7View all authors and affiliationsVolume 64, Issue 4https://doi.org/10.1136/jim-2016-000120.73
Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, as illustrated by soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis and graft-versus-host disease (GVHD). sST2 contains only the extra-cellular domain (ECD) of ST2 and binds its unique ligand, interleukin-33, via an extensive interaction interface ─ a challenging target for drug development. Here, we report the discovery of small molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, three compounds were selected for evaluation in two experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our results support the feasibility of targeting the ECD of ST2 with small molecule inhibitors in an inflammatory disease mediated by sST2.
Graft-versus-host disease (GVHD) remains the major barrier to the success of allogeneic stem cell transplantation (SCT). We have reported that soluble suppression of tumoriginicity 2 (ST2) measured in plasma of post-SCT patients is a marker for risk of therapy-resistant GVHD and death. ST2 is a member of the IL-1 receptor family whose sole known ligand is IL-33. Soluble ST2 acts as a decoy receptor for IL-33. Thus, we hypothesized that blockade of soluble ST2 will increase the amount of free IL-33 that will be able to bind to membrane ST2 on CD4+ T cells, driving them toward a Th2 phenotype, which will alleviate GVHD. First, we found that similar to plasma ST2 course in SCT patients, plasma ST2 was markedly increased prior to and at the onset of GVHD symptoms in multiple clinically relevant GVHD murine models (B6 to C3H.SW shown, Fig. 1A). Based on this observation, anti-ST2 antibody given with a prophylactic schedule (one dose before SCT and one dose at day+1 post-SCT) significantly reduced GVHD severity and mortality (Fig. 1B). Pathology analysis indicated that anti-ST2 treated recipients showed lower histopathologic score in liver and intestine (Fig. 1C). Strikingly, anti-ST2 significantly increased plasma IL-33 (Fig. 1D). Whole transcriptome analysis of mesenteric lymph node T cells showed that anti-ST2 modulated gene expression of helper T cell (Th1/Th2) related cytokines (Fig. 1E), suggesting that ST2 blockade may affect the helper T cell compartment. To further assess the effects of ST2 blockade on helper T cells, we examined the Th1/Th2 balance by flow cytometry. Administration of anti-ST2 shifted Th1/Th2 balance toward a Th2 phenotype (Fig. 2). In addition, ST2 blockade also induced expansion of regulatory T cells (Tregs) (Fig. 2). Given that anti-ST2 administration also up-regulated expression of genes, such as Granzyme A, that can mediate graft vs. leukemia (GVL) responses (Fig. 1E), we postulated that ST2 blockade would not affect therapeutic GVL activity. To confirm this, we developed a clinically relevant model of leukemia, in which C3H.SW recipients were challenged with syngeneic GFP+ MLL-AF9 induced acute myeloid leukemia cells. Our results indicated that administration of anti-ST2 preserved substantial GVL activity and resulted in significantly improved leukemia-free survival (Fig. 3), suggesting that anti-ST2 ameliorated GVHD and maintained GVL response. In summary, we found that prophylaxis with anti-ST2 antibody could alleviate GVHD severity and mortality while preserving GVL effect. ST2 blockade increased the level of plasma IL-33, skewed the Th1/Th2 balance toward a Th2 phenotype, and induced Foxp3+Tregs (with preserved expression of membrane ST2 [not shown]). Our findings suggest that ST2 is a novel therapeutic target to ameliorate GVHD.Figure 2Blockade of ST2 shifts the Th1/Th2 balance toward Th2 phenotype and increases CD4+ regulatory T cells. Spleen T cells were collected for intracellular staining at day l0 post-transplantation. ∗p<0.05, ∗∗p<0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Anti-ST2 mAb treatment preserves substantial GVL activity. (A) Survival cure. (B) GVHD score (bars) and percent of tumor relapse (lines). ∗∗p<0.01, IgG vs Anti-ST2View Large Image Figure ViewerDownload Hi-res image Download (PPT)
