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Chemotherapy and Inflammation Induced Damage of Intestinal Epithelium Is Associated with Increased T Cell Chemotaxis
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Abstracts / Biol Blood Marrow Transplant 26 (2020) S96 S255
Memorial Sloan Kettering Cancer Center, New York, NY; Department of Medicine, Weill Cornell Medical College, New York, NY; 6 Department of Medicine, Adult Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, New York, NY; 7 Princess Maxima Centre for Pediatric Oncology, Utrecht, Netherlands; 8 Pediatric Blood and Marrow Transplantation Program, University Medical Center Utrecht, Utrecht, Netherlands 5
Figure 1. ILCs recovery post allogeneic hematopoietic stem cell transplant. Circles represent mean with bars representing standard error of mean (SEM).
Figure 2. ILCs recovery difference between patients who developed GVHD versus patients who did not develop GVHD. Circles represent mean with bars representing SEM.
Figure 3. Type 1 ILC recovery difference between patients who developed GVHD versus patients who did not develop GVHD. Circles represent mean with bars representing SEM. **p = 0.0062.
Gastro-intestinal (GI) Graft-versus-Host Disease (GvHD) involves a complex interplay of conditioning-related injury and the pathologic immune response at the epithelial level. Enhanced tissue damage is a risk factor for GVHD. We demonstrated that donor T cell-derived IFNg causes intestinal stem cell damage in GvHD. Recent studies have proposed that donor T cells directly interact with GI epithelium in GvHD in vivo, but the precise mechanisms remain incompletely understood. Here, we evaluate the influence of both cytokine- and chemotherapy-induced intestinal epithelial damage on T cell migration and direct epithelial cell-T cell interactions using GI organoids as a model system. We developed a human intestinal organoid-T cell interaction co-culture system and studied the role of T cells in intestinal damage during GVHD. We observed that polyclonally activated T cells accumulate in close proximity to intestinal organoids (Fig. 1). In this co-culture system T cell-derived IFNgplays a key role in tissue damage. RNA-seq analyses of IFNgtreated intestinal organoids indicated significant upregulation of T cell chemotaxis associated genes including CXCL9, 10 and 11. Luminex analysis of conditioned media confirmed release of these chemokines in co-culture versus controls. In addition, stimulation of organoids with IFNg led to upregulation of HLAI and HLA-II at both the RNA and membrane protein expression level (Fig. 2A). These experiments place activated T cells at the cusp of intestinal damage during GVHD through the production of cytokines, the induction of T cell recruiting chemokines and mechanisms of antigen driven cytotoxicity. Next, we set out to characterize the effect of chemotherapy on epithelium by treating organoids with Busulfan (Bu), Fludarabine (Flu) or Clofarabine (Clo). Epithelial damage was confirmed by the presence of increasing levels of caspase activation (Fig. 3A), a decrease in the number of surviving organoids, a smaller organoid size and decreased proliferation. Bu, Flu, Clo treatment did not affect HLA-I-II mRNA expression, but a variable, organoid donor-dependent upregulation of membrane HLA-I upon exposure to Bu was observed (Fig. 2B). By using a migration assay, we established that medium that we obtained from chemotherapytreated organoids exerts increased T cell chemotaxis compared to control medium (Fig. 3B). In conclusion, both (T cell derived) IFNg- and chemotherapy induced intestinal epithelial damage can lead to increased T cell migration ex vivo, resulting in direct contact between allogeneic T cells and co-cultured human intestinal epithelium. Cytokine signaling and conditioning-related injury may contribute to GVHD pathogenesis in vivo by promoting recruitment to intestinal crypt epithelium.
240 Chemotherapy and Inflammation Induced Damage of Intestinal Epithelium Is Associated with Increased T Cell Chemotaxis Suze A. Jansen MD1, Marliek van Hoesel1, Michal Mokry2, Leire Saiz Sierra3, Shuichiro Takashima4, Edward E. Nieuwenhuis1, Alan M. Hanash MD, PhD5,6, Caroline A. Lindemans MD, PhD7,8. 1 Division of Pediatrics, University Medical Center Utrecht, Utrecht, Netherlands; 2 Department of Cardiology, University Medical Center Utrecht, Utrecht, Netherlands; 3 Division of Pediatrics, University Medical Center Utrecht, Utrrecht, Netherlands; 4 Department of Medicine,
Figure 1. Images of an intestinal organoid and CellTraceViolet-stained T-cells in co-culture (scale bar = 200 um, left: DAPI channel only, right: overlay).
Abstracts / Biol Blood Marrow Transplant 26 (2020) S96 S255
Figure 2. A) Representative figures of membrane HLA-I (left) and II (right) expression of IFNg-treated organoids vs. control (IFN = rhIFNg 20 ng/ml 24h, Iso-1 = Isotype HLA-A,B,C-BV510, Iso 2 = Isotype HLA-DP,DQ,DR-PE/Cy7, n=4, from 2 healthy donors). B) Membrane HLA-I expression of Bu treated organoids vs. control (Bu 10 uM, n=5, from 4 healthy donors).
Figure 3. A) CaspaseGlo assay of organoids treated with chemotherapy for 48h (n=6, from 2 healthy human donors, Ordinary One-way ANOVA, *P<0,05, **P<40,01, ***P<0,001, ****P<0,0001, error bar = S.E.M.). B) Migration assay of polyclonally activated T-cells (Conditioned media (CM) after 48h Flu 5uM treatment, n=2, from 1 healthy donor, Ordinary One-way ANOVA, **P=0,0012, error bar = S.E.M.)
241 Pretransplant Short-Term Exposure of Donor Graft Cells to ITK Selective Inhibitor ONO-7790500 Prevents Acute Gvhd in Mouse BMT Model Takumi Kondo MD, Yasuhisa Sando MD, Yuichi Sumii MD, Shuntaro Ikegawa MD, Hiroyuki Sugiura M.D., Makoto Nakamura MD, Miki Iwamoto, Yusuke Meguri MD, Yoshinobu Maeda MD, PhD, Ken-ichi Matsuoka MD, PhD. Department of Hematology and Oncology, Okayama University Hospital, Okayama, Japan Introduction and Objectives: Graft-versus-host disease (GVHD) triggered by activated donor graft cells still remains a major complication and course of non-relapse mortality after HSCT. Interleukin-2-inducible T-cell kinase (ITK) is a TEC family cytoplasmic tyrosine kinase of crucial importance for T cell development and TCR signaling. Recent studies demonstrated that ITK-deficient CD4+ T cells easily differentiated into regulatory T cells (Tregs) and ITK/BTK inhibitor ibrutinib could improve chronic GVHD symptoms in mouse model and human clinical study, suggesting that ITK may have important roles in post-transplant immune tolerance. In this study, we examined the effects of ITK-selective inhibitor (ITKsi; ONO-7790500) on acute GVHD by using murine BMT model. Methods: Allogeneic response of ITKsi-treated CD4+ T cells from B6 was evaluated by in vitro system and murine BMT model. In BMT experiments, lethally irradiated 10-week old B6D2F1 recipient mice were transplanted with 5 £ 106 TCD BM together with 1.5 £ 106 CD4+ T cells from B6 donors. Survival and GVHD severity were clinically monitored, and T cell subset reconstitution and serum cytokine profile were also examined. Results: First, to examine the influence of ITKsi on alloreactive T cell to the corresponding antigens in vitro, CD4+ T cells isolated from B6 mice were exposed to ITKsi for 3 hours and then
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cultured with irradiated Balb/c stimulators. Proliferation and IFN-gamma production of ITKsi-treated T cells in response to allogeneic stimulators were significantly reduced in comparison to those from controls (P < 0.0001, < 0.0001, respectively). As expected, ITKsi-treatment did not interfere with Treg maintenance. Analysis of transcription factors revealed that the expressions of both T-bet and GATA3 were suppressed in ITKsi-treated T cells. We then performed experimental allogeneic BMT. In ITKsi-treated group, donor CD4+ T cells were cultured in the presence of ITKsi during 3 hours just before transplant. GVHD mortality was significantly less in recipients of ITKsi-treated donor graft than in those of a control graft (10% vs 64.5% on day 60, p < 0.01). Early proliferation of donor T cells and the production of proinflammatory cytokines including TNF-alpha and IL-2 in the first week after transplant were significantly reduced in ITKsi-treated group than control group (P = 0.04, 0.003, respectively). Th2 cytokines, IL-4 and IL-5, were also suppressed. Conclusion: Taken together, these data showed that pretreatment of CD4+ T cells with ITKsi efficiently regulated alloreactive T cell and improved the symptoms of acute GVHD. ITKsi inhibited both Th1 and Th2 response but spared the expansion of donor-derived Tregs. Our findings suggest that graft manipulation with short-term culture with ITKsi just before transplant could provide a simple but efficient strategy to modulate alloreactive T cell response and clinical GVHD.
242 Dimethyl Fumarate Ameliorates Disease Severity in a Mouse Model of Chronic Graft-Versus-Host Disease. Julian C. Assmann PhD, Kathrynne Warrick BS, Christopher Johns BS, Don E. Farthing PhD, Ronald E. Gress MD, Nataliya P. Buxbaum MD. Experimental Transplantation and Immunology Branch, National Cancer Institute, Bethesda, MD Introduction: Chronic graft-versus host disease (cGvHD) is a major complication of allogeneic hematopoietic stem cell transplantation. Standard treatment mainly consists of corticosteroids but is limited by a high percentage of steroid-refractory cases. Alloreactive T cells are a major driver of the disease, infiltrating target organs such as the liver. However, global ablation of T cells interferes with the graft-versus-leukemia effect and bears the risk of increased infections. An increased use of glycolysis has been shown to drive T cell activation and its inhibition can blunt T cell effector functions. Dimethyl fumarate (DMF), an immunomodulatory drug already approved for the treatment of multiple sclerosis and psoriasis, has been shown to inhibit the activity of glyceraldehyde-3phosphate dehydrogenase (GAPDH), a key glycolytic enzyme. Objective: We hypothesized that metabolic targeting of T cells could specifically inhibit activated T cells while preserving other T cell populations in cGvHD. Methods: Chronic GvHD was induced using the B10.D2 into BALB/c minor-mismatch mouse model. Daily oral treatment with 100 mg/kg DMF or vehicle was initiated on the day of the transplant and continued for a 42-day period. Weight and cGvHD severity were scored twice weekly and liver and spleen tissue was analyzed via flow cytometry on day 42. Results: DMF treatment reduced weight loss and cGvHD severity over a 42-day period. In addition, DMF decreased the frequency of activated CD4/CD8 T cells in the liver while simultaneously increasing the frequency of naïve T cells. Importantly, the frequency of regulatory T cells was not affected by the treatment. Conclusion: DMF has already been shown to be safe and effective in autoimmune diseases. Our studies indicate that DMF
Abstracts / Biol Blood Marrow Transplant 26 (2020) S96 S255
Memorial Sloan Kettering Cancer Center, New York, NY; Department of Medicine, Weill Cornell Medical College, New York, NY; 6 Department of Medicine, Adult Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, New York, NY; 7 Princess Maxima Centre for Pediatric Oncology, Utrecht, Netherlands; 8 Pediatric Blood and Marrow Transplantation Program, University Medical Center Utrecht, Utrecht, Netherlands 5
Figure 1. ILCs recovery post allogeneic hematopoietic stem cell transplant. Circles represent mean with bars representing standard error of mean (SEM).
Figure 2. ILCs recovery difference between patients who developed GVHD versus patients who did not develop GVHD. Circles represent mean with bars representing SEM.
Figure 3. Type 1 ILC recovery difference between patients who developed GVHD versus patients who did not develop GVHD. Circles represent mean with bars representing SEM. **p = 0.0062.
Gastro-intestinal (GI) Graft-versus-Host Disease (GvHD) involves a complex interplay of conditioning-related injury and the pathologic immune response at the epithelial level. Enhanced tissue damage is a risk factor for GVHD. We demonstrated that donor T cell-derived IFNg causes intestinal stem cell damage in GvHD. Recent studies have proposed that donor T cells directly interact with GI epithelium in GvHD in vivo, but the precise mechanisms remain incompletely understood. Here, we evaluate the influence of both cytokine- and chemotherapy-induced intestinal epithelial damage on T cell migration and direct epithelial cell-T cell interactions using GI organoids as a model system. We developed a human intestinal organoid-T cell interaction co-culture system and studied the role of T cells in intestinal damage during GVHD. We observed that polyclonally activated T cells accumulate in close proximity to intestinal organoids (Fig. 1). In this co-culture system T cell-derived IFNgplays a key role in tissue damage. RNA-seq analyses of IFNgtreated intestinal organoids indicated significant upregulation of T cell chemotaxis associated genes including CXCL9, 10 and 11. Luminex analysis of conditioned media confirmed release of these chemokines in co-culture versus controls. In addition, stimulation of organoids with IFNg led to upregulation of HLAI and HLA-II at both the RNA and membrane protein expression level (Fig. 2A). These experiments place activated T cells at the cusp of intestinal damage during GVHD through the production of cytokines, the induction of T cell recruiting chemokines and mechanisms of antigen driven cytotoxicity. Next, we set out to characterize the effect of chemotherapy on epithelium by treating organoids with Busulfan (Bu), Fludarabine (Flu) or Clofarabine (Clo). Epithelial damage was confirmed by the presence of increasing levels of caspase activation (Fig. 3A), a decrease in the number of surviving organoids, a smaller organoid size and decreased proliferation. Bu, Flu, Clo treatment did not affect HLA-I-II mRNA expression, but a variable, organoid donor-dependent upregulation of membrane HLA-I upon exposure to Bu was observed (Fig. 2B). By using a migration assay, we established that medium that we obtained from chemotherapytreated organoids exerts increased T cell chemotaxis compared to control medium (Fig. 3B). In conclusion, both (T cell derived) IFNg- and chemotherapy induced intestinal epithelial damage can lead to increased T cell migration ex vivo, resulting in direct contact between allogeneic T cells and co-cultured human intestinal epithelium. Cytokine signaling and conditioning-related injury may contribute to GVHD pathogenesis in vivo by promoting recruitment to intestinal crypt epithelium.
240 Chemotherapy and Inflammation Induced Damage of Intestinal Epithelium Is Associated with Increased T Cell Chemotaxis Suze A. Jansen MD1, Marliek van Hoesel1, Michal Mokry2, Leire Saiz Sierra3, Shuichiro Takashima4, Edward E. Nieuwenhuis1, Alan M. Hanash MD, PhD5,6, Caroline A. Lindemans MD, PhD7,8. 1 Division of Pediatrics, University Medical Center Utrecht, Utrecht, Netherlands; 2 Department of Cardiology, University Medical Center Utrecht, Utrecht, Netherlands; 3 Division of Pediatrics, University Medical Center Utrecht, Utrrecht, Netherlands; 4 Department of Medicine,
Figure 1. Images of an intestinal organoid and CellTraceViolet-stained T-cells in co-culture (scale bar = 200 um, left: DAPI channel only, right: overlay).
Abstracts / Biol Blood Marrow Transplant 26 (2020) S96 S255
Figure 2. A) Representative figures of membrane HLA-I (left) and II (right) expression of IFNg-treated organoids vs. control (IFN = rhIFNg 20 ng/ml 24h, Iso-1 = Isotype HLA-A,B,C-BV510, Iso 2 = Isotype HLA-DP,DQ,DR-PE/Cy7, n=4, from 2 healthy donors). B) Membrane HLA-I expression of Bu treated organoids vs. control (Bu 10 uM, n=5, from 4 healthy donors).
Figure 3. A) CaspaseGlo assay of organoids treated with chemotherapy for 48h (n=6, from 2 healthy human donors, Ordinary One-way ANOVA, *P<0,05, **P<40,01, ***P<0,001, ****P<0,0001, error bar = S.E.M.). B) Migration assay of polyclonally activated T-cells (Conditioned media (CM) after 48h Flu 5uM treatment, n=2, from 1 healthy donor, Ordinary One-way ANOVA, **P=0,0012, error bar = S.E.M.)
241 Pretransplant Short-Term Exposure of Donor Graft Cells to ITK Selective Inhibitor ONO-7790500 Prevents Acute Gvhd in Mouse BMT Model Takumi Kondo MD, Yasuhisa Sando MD, Yuichi Sumii MD, Shuntaro Ikegawa MD, Hiroyuki Sugiura M.D., Makoto Nakamura MD, Miki Iwamoto, Yusuke Meguri MD, Yoshinobu Maeda MD, PhD, Ken-ichi Matsuoka MD, PhD. Department of Hematology and Oncology, Okayama University Hospital, Okayama, Japan Introduction and Objectives: Graft-versus-host disease (GVHD) triggered by activated donor graft cells still remains a major complication and course of non-relapse mortality after HSCT. Interleukin-2-inducible T-cell kinase (ITK) is a TEC family cytoplasmic tyrosine kinase of crucial importance for T cell development and TCR signaling. Recent studies demonstrated that ITK-deficient CD4+ T cells easily differentiated into regulatory T cells (Tregs) and ITK/BTK inhibitor ibrutinib could improve chronic GVHD symptoms in mouse model and human clinical study, suggesting that ITK may have important roles in post-transplant immune tolerance. In this study, we examined the effects of ITK-selective inhibitor (ITKsi; ONO-7790500) on acute GVHD by using murine BMT model. Methods: Allogeneic response of ITKsi-treated CD4+ T cells from B6 was evaluated by in vitro system and murine BMT model. In BMT experiments, lethally irradiated 10-week old B6D2F1 recipient mice were transplanted with 5 £ 106 TCD BM together with 1.5 £ 106 CD4+ T cells from B6 donors. Survival and GVHD severity were clinically monitored, and T cell subset reconstitution and serum cytokine profile were also examined. Results: First, to examine the influence of ITKsi on alloreactive T cell to the corresponding antigens in vitro, CD4+ T cells isolated from B6 mice were exposed to ITKsi for 3 hours and then
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cultured with irradiated Balb/c stimulators. Proliferation and IFN-gamma production of ITKsi-treated T cells in response to allogeneic stimulators were significantly reduced in comparison to those from controls (P < 0.0001, < 0.0001, respectively). As expected, ITKsi-treatment did not interfere with Treg maintenance. Analysis of transcription factors revealed that the expressions of both T-bet and GATA3 were suppressed in ITKsi-treated T cells. We then performed experimental allogeneic BMT. In ITKsi-treated group, donor CD4+ T cells were cultured in the presence of ITKsi during 3 hours just before transplant. GVHD mortality was significantly less in recipients of ITKsi-treated donor graft than in those of a control graft (10% vs 64.5% on day 60, p < 0.01). Early proliferation of donor T cells and the production of proinflammatory cytokines including TNF-alpha and IL-2 in the first week after transplant were significantly reduced in ITKsi-treated group than control group (P = 0.04, 0.003, respectively). Th2 cytokines, IL-4 and IL-5, were also suppressed. Conclusion: Taken together, these data showed that pretreatment of CD4+ T cells with ITKsi efficiently regulated alloreactive T cell and improved the symptoms of acute GVHD. ITKsi inhibited both Th1 and Th2 response but spared the expansion of donor-derived Tregs. Our findings suggest that graft manipulation with short-term culture with ITKsi just before transplant could provide a simple but efficient strategy to modulate alloreactive T cell response and clinical GVHD.
242 Dimethyl Fumarate Ameliorates Disease Severity in a Mouse Model of Chronic Graft-Versus-Host Disease. Julian C. Assmann PhD, Kathrynne Warrick BS, Christopher Johns BS, Don E. Farthing PhD, Ronald E. Gress MD, Nataliya P. Buxbaum MD. Experimental Transplantation and Immunology Branch, National Cancer Institute, Bethesda, MD Introduction: Chronic graft-versus host disease (cGvHD) is a major complication of allogeneic hematopoietic stem cell transplantation. Standard treatment mainly consists of corticosteroids but is limited by a high percentage of steroid-refractory cases. Alloreactive T cells are a major driver of the disease, infiltrating target organs such as the liver. However, global ablation of T cells interferes with the graft-versus-leukemia effect and bears the risk of increased infections. An increased use of glycolysis has been shown to drive T cell activation and its inhibition can blunt T cell effector functions. Dimethyl fumarate (DMF), an immunomodulatory drug already approved for the treatment of multiple sclerosis and psoriasis, has been shown to inhibit the activity of glyceraldehyde-3phosphate dehydrogenase (GAPDH), a key glycolytic enzyme. Objective: We hypothesized that metabolic targeting of T cells could specifically inhibit activated T cells while preserving other T cell populations in cGvHD. Methods: Chronic GvHD was induced using the B10.D2 into BALB/c minor-mismatch mouse model. Daily oral treatment with 100 mg/kg DMF or vehicle was initiated on the day of the transplant and continued for a 42-day period. Weight and cGvHD severity were scored twice weekly and liver and spleen tissue was analyzed via flow cytometry on day 42. Results: DMF treatment reduced weight loss and cGvHD severity over a 42-day period. In addition, DMF decreased the frequency of activated CD4/CD8 T cells in the liver while simultaneously increasing the frequency of naïve T cells. Importantly, the frequency of regulatory T cells was not affected by the treatment. Conclusion: DMF has already been shown to be safe and effective in autoimmune diseases. Our studies indicate that DMF