Abdulraouf Ramadan

ST2 blockade reduces sST2-producing T cells while maintaining protective mST2-expressing T cells during graft-versus-host disease

Blocking sST2 before transplant with a neutralizing monoclonal antibody reduced GVHD severity and mortality. Blocking graft-versus-host disease Bone marrow transplantation replaces unhealthy bone marrow with bone marrow from a healthy donor. However, the donor-derived immune cells may recognize the transplant recipient as foreign and attack, resulting in graft-versus-host disease (GVHD). Now, Zhang et al. report that blocking soluble suppression of tumorigenicity 2 (sST2), a plasma marker for GVHD, with a neutralizing antibody can reduce GVHD severity and mortality. The blockade decreased the production of proinflammatory cytokines and increased the frequency of anti-inflammatory molecules and cells while maintaining graft-versus-leukemia activity. These data suggest that targeting sST2 may help decrease GVHD after bone marrow transplantation. Graft-versus-host disease (GVHD) remains a devastating complication after allogeneic hematopoietic cell transplantation (HCT). We previously identified high plasma soluble suppression of tumorigenicity 2 (sST2) as a biomarker of the development of GVHD and death. sST2 sequesters interleukin-33 (IL-33), limiting its availability to T cells expressing membrane-bound ST2 (mST2) [T helper 2 (TH2) cells and ST2+FoxP3+ regulatory T cells]. We report that blockade of sST2 in the peritransplant period with a neutralizing monoclonal antibody (anti-ST2 mAb) reduced GVHD severity and mortality. We identified intestinal stromal cells and T cells as major sources of sST2 during GVHD. ST2 blockade decreased systemic interferon-γ, IL-17, and IL-23 but increased IL-10 and IL-33 plasma levels. ST2 blockade also reduced sST2 production by IL-17–producing T cells while maintaining protective mST2-expressing T cells, increasing the frequency of intestinal myeloid–derived suppressor cells, and decreasing the frequency of intestinal CD103 dendritic cells. Finally, ST2 blockade preserved graft-versus-leukemia activity in a model of green fluorescent protein (GFP)–positive MLL-AF9 acute myeloid leukemia. Our findings suggest that ST2 is a therapeutic target for severe GVHD and that the ST2/IL-33 pathway could be investigated in other T cell–mediated immune disorders with loss of tolerance.

Various Forms of Tissue Damage and Danger Signals Following Hematopoietic Stem-Cell Transplantation

Hematopoietic stem-cell transplantation (HSCT) is the most potent curative therapy for many malignant and non-malignant disorders. Unfortunately, a major complication of HSCT is graft-versus-host disease (GVHD), which is mediated by tissue damage resulting from the conditioning regimens before the transplantation and the alloreaction of dual immune components (activated donor T-cells and recipient’s antigen-presenting cells). This tissue damage leads to the release of alarmins and the triggering of pathogen-recognition receptors that activate the innate immune system and subsequently the adaptive immune system. Alarmins, which are of endogenous origin, together with the exogenous pathogen-associated molecular patterns (PAMPs) elicit similar responses of danger signals and represent the group of damage-associated molecular patterns (DAMPs). Effector cells of innate and adaptive immunity that are activated by PAMPs or alarmins can secrete other alarmins and amplify the immune responses. These complex interactions and loops between alarmins and PAMPs are particularly potent at inducing and then aggravating the GVHD reaction. In this review, we highlight the role of these tissue damaging molecules and their signaling pathways. Interestingly, some DAMPs and PAMPs are organ specific and GVHD-induced and have been shown to be interesting biomarkers. Some of these molecules may represent potential targets for novel therapeutic approaches.

Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA-transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA- transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT.

Specifically differentiated T cell subset promotes tumor immunity over fatal immunity

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 CD8&agr; expression. Thus, adoptive transfer of allogeneic T9IL-33 cells offers an attractive approach for separating GVL activity from GVHD.

From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

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, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. 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, 3 compounds were selected for evaluation in 2 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 data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.

Editorial: Danger signals triggering immune response and inflammation

1 Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, United States, 2 Department of Microbiology Immunology, Indiana University School of Medicine, Indianapolis, IN, United States, 3 Melvin and Bren Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN, United States, 4 German Academy of Transplantation Medicine, Munich, Germany, 5 Molecular ImmunoRheumatology, INSERM UMR_S1109, Laboratory of Excellence Transplantex, Faculty of Medicine, University of Strasbourg, Strasbourg, France

508. IL-33/ST2 Triggering of IL-9-Secreting T Cells: From Proteomics to Therapeutics

As one of the most validated immunotherapies to date, allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative option for high-risk hematological malignancies, particularly acute myeloid leukemia (AML). The immunotherapeutic activity of allo-HCT is known as the graft-vs-leukemia (GVL) activity. However, GVL activity is often accompanied by T-cell reactivity to allo-antigens in normal host tissues, which leads to graft-versus-host disease (GVHD), another major cause of death after HCT. Therefore, there is a great unmet need to improve the current process of allo-HCT through increasing the GVL activity and decreasing GVHD. We have shown that an elevated plasma level of soluble (s)ST2 in HCT patients is a risk factor for severe GVHD. ST2 blockade reduces sST2-producing T cells while maintaining protective membrane (m)ST2-expressing T cells such as type 2 T cells and regulatory T cells during aGVHD. A novel IL-9 producing T helper subset, Th9, expresses mST2. Furthermore, Th9 cells and IL-9 producing CD8 cytotoxic (Tc9) cells have higher antitumor activity than Th1 and Tc1 cells in melanoma models. Interestingly, we found that the addition of IL-33 during T9 differentiation (T9IL-33) increased expression of mST2 and PU.1, a transcription factor that promotes IL-9 production in both CD4 and CD8 T cells. Adoptive transfer of T9IL-33 cells with bone marrow cells in a murine model of HCT resulted in less severe GVHD compared to transfer of T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Furthermore, cytolytic molecules implicated in anti-leukemic activity (granzyme B and perforin) were upregulated in WT T9IL-33 cells while ST2−/− T9IL-33 cells did not. WT T9IL-33 cells also exhibited higher anti-leukemic activity when cultured with a retrovirally transduced MLL-AF9 leukemic cells in comparison to ST2−/− T9IL-33 in in vitro cytolytic assays. In vivo GVL experiments with MLL-AF9 AML and adoptive transfer of T9IL-33 cells resulted in increased survival compared to syngeneic mice, allo-HCT mice transferred with T1 cells, or T9 cells or T9IL-33 cells generated from ST2−/− or IL-9−/− T cells (Figure 1Figure 1). Human T9 cells are poorly studied. Here we demonstrate that IL-33 has the same impact on human T cells through enhancing IL-9 and Granzyme B production compared to T9 cells as well as demonstrated higher in vitro anti-leukemic cytolytic activity when incubated with MOLM14, an aggressive AML tumor cell line expressing FLT3/ITD mutations. Importantly, CD8α expression was upregulated in WT T9IL-33 (both CD4 and CD8) cells in comparison to ST2−/− T9IL-33 cells, and CD8α blockade with neutralizing antibody during allogeneic specific T9IL-33 differentiation reduced cytotoxicity of both murine T9IL-33, and human T9IL-33 cells as compared to the cell blocked with isotype control, suggesting that CD8α was associated with MHC-restricted cytolytic activity in T9IL-33 cells. Altogether, our observations demonstrated that adoptive transfer of T9IL-33 cells represents a promising cellular therapy following HCT.View Large Image | Download PowerPoint Slide

ID: 95: IL-33/ST2 TRIGGERING OF IL-9–SECRETING T CELLS: FROM PROTEOMICS TO THERAPEUTICS

As one of the most validated immunotherapies to date, allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative option for high-risk hematological malignancies, particularly acute myeloid leukemia (AML). The immunotherapeutic activity of allo-HCT is known as the graft-vs-leukemia (GVL) activity. However, GVL activity is often accompanied by T-cell reactivity to allo-antigens in normal host tissues, which leads to graft-versus-host disease (GVHD), another major cause of death after HCT. Therefore, there is a great unmet need to improve the current process of allo-HCT through increasing the GVL activity and decreasing GVHD. We have shown that an elevated plasma level of soluble (s)ST2 in HCT patients is a risk factor for severe GVHD. ST2 blockade reduces sST2-producing T cells while maintaining protective membrane (m)ST2-expressing T cells such as type 2 T cells and regulatory T cells during aGVHD. A novel IL-9 producing T helper subset, Th9, expresses mST2. Furthermore, Th9 cells and IL-9 producing CD8 cytotoxic (Tc9) cells have higher antitumor activity than Th1 and Tc1 cells in melanoma models. Interestingly, we found that the addition of IL-33 during T9 differentiation (T9IL-33) increased expression of mST2 and PU.1, a transcription factor that promotes IL-9 production in both CD4 and CD8 T cells. Adoptive transfer of T9IL-33 cells with bone marrow cells in a murine model of HCT resulted in less severe GVHD compared to transfer of T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Furthermore, cytolytic molecules implicated in anti-leukemic activity (granzyme B and perforin) were upregulated in WT T9IL-33 cells while ST2−/− T9IL-33 cells did not. WT T9IL-33 cells also exhibited higher anti-leukemic activity when cultured with a retrovirally transduced MLL-AF9 leukemic cells in comparison to ST2−/− T9IL-33 in in vitro cytolytic assays. In vivo GVL experiments with MLL-AF9 AML and adoptive transfer of T9IL-33 cells resulted in increased survival compared to syngeneic mice, allo-HCT mice transferred with T1 cells, or T9 cells or T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Human T9 cells are poorly studied. Here we demonstrate that IL-33 has the same impact on human T cells through enhancing IL-9 and Granzyme B production compared to T9 cells as well as demonstrated higher in vitro anti-leukemic cytolytic activity when incubated with MOLM14, an aggressive AML tumor cell line expressing FLT3/ITD mutations. Importantly, CD8α expression was upregulated in WT T9IL-33 (both CD4 and CD8) cells in comparison to ST2−/− T9IL-33 cells, and CD8α blockade with neutralizing antibody during allogeneic specific T9IL-33 differentiation reduced cytotoxicity of both murine T9IL-33, and human T9IL-33 cells as compared to the cell blocked with isotype control, suggesting that CD8α was associated with MHC-restricted cytolytic activity in T9IL-33 cells. Altogether, our observations demonstrated that adoptive transfer of T9IL-33 cells represents a promising cellular therapy following HCT.

ID: 91: ST2 BLOCKADE REDUCES SST2-PRODUCING T CELLS WHILE MAINTAINING PROTECTIVE MST2-EXPRESSING T CELLS DURING GRAFT-VERSUS-HOST DISEASE

Allogeneic hematopoietic stem cell transplantation is an important curative therapy for hematological malignancies and other blood disorders, but graft-versus-host disease (GVHD) remains a devastating complication. We previously identified high plasma soluble suppression of tumorigenicity 2 (ST2) as a biomarker of the development of GVHD and death. ST2, also known as the interleukin (IL)-33 receptor, is one of the newest members of the IL-1 receptor family, and its only known ligand is IL-33. Due to alternative splicing, ST2 has two main isoforms: a membrane-bound form (mST2), expressed on hematopoietic cells that promote type 2 helper T (Th2) cell immune responses, and a soluble form (sST2), secreted by non-hematopoietic cells. sST2 acts as a decoy receptor for IL-33, limiting its availability to mST2-expressing cells. Thus, we hypothesized that blockade of the sST2 will free IL-33 for signaling in mST2-expressing T cells [Th2 cells and ST2+FoxP3+ regulatory T cells (Tregs)], which will attenuate GVHD. First, we found the onset of GVHD symptoms was correlated with a marked increase of plasma ST2 in multiple clinically relevant GVHD murine models, and blockade of sST2 in the peri-transplant period with a neutralizing monoclonal antibody (anti-ST2 mAb) reduced GVHD clinical and histopathological severity and mortality. We identified intestinal stromal cells and T cells as major sources of sST2 during GVHD. ST2 blockade decreased systemic interferon-γ, IL-17, and IL-23 but increased IL-10 and IL-33 plasma levels. ST2 blockade also reduced sST2 production by IL-17–producing T cells while maintaining protective mST2-expressing Th2 cells and Tregs. In addition, ST2 blockade increased the frequency of intestinal myeloid-derived suppressor cells (MDSCs) and decreased the frequency of intestinal CD103 dendritic cells (DCs). Finally, whole transcriptome analysis of T cells comparing anti-ST2 mAb-treated mice versus IgG control-treated mice showed that anti-ST2 mAb administration up-regulated gene expression of cytokines and cytolytic molecules that have been implicated in antitumoral or graft-vs-leukemia (GVL) activity, such as IL-27, IL-18, IL-9, type I interferons, and granzyme A. We therefore postulated that ST2 blockade would not affect therapeutic GVL activity. To confirm this in a clinically relevant GVL model, we developed primary retrovirally induced MLL-AF9 eGFP+ leukemic cells on the C3H.SW background. The phenotype of the leukemic cells in this model is eGFP+, CD3−, B220− and Mac-1hiGr-1hi. Our results indicated that administration of anti-ST2 mAb or ST2−/− donor T cells preserved substantial GVL activity and resulted in significantly improved leukemia-free survival. In summary, our findings identify intestinal alloreactive T cells as an important source of the decoy receptor for IL-33 that can be blocked with two doses of anti-ST2 mAb in the peri-transplant period without inhibiting the beneficial mST2 expression on Th2 cells and Tregs or the GVL activity. In addition, ST2 blockade induced expansion of tolerogenic MDSCs while inhibiting immunogenic CD103 DCs during GVHD. This study offers new perspectives on the translation of drug-targetable biomarkers for selectively and safely treating GVHD and other T cell-mediated human disorders.