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Neutrophil Granulocytes Mediate Cellular Communication Between the Damaged Intestinal Tract and Mesenteric Lymph Nodes at the Onset of Intestinal Graft-Versus-Host Disease
S398
Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
Our analysis highlights HDCTX as a compelling option for GVHD prophylaxis, but with nuances. Rates of ANC and Platelet recovery slightly lag behind Tac/MTX but HDCTX is associated with lower rates of severe GVHD, improved TRM and decreased admissions and inpatient hospital days. This is more impressive when taking into account that the HDCTX included more high risk patients (the haplo transplants) compared to the Tax/MTX group. Thus, HDCTX may have a significant positive impact on transplant outcomes and the long-term costs of allogeneic transplants. Longer follow-up and analysis of a larger pool of patients will be required to further expand on the observations made in our study.
598 Neutrophil Granulocytes Mediate Cellular Communication Between the Damaged Intestinal Tract and Mesenteric Lymph Nodes at the Onset of Intestinal Graft-Versus-Host Disease Jan Hülsdünker1, 2, 3, Gabriele Prinz1, Katja Ottmüller4, 5, Heide Dierbach1, Marie Follo 1, Susanne Kirschnek 6, Andreas Beilhack 4, 7, 8, Georg Häcker 6, Robert Zeiser 1. 1 Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Albert Ludwigs University (ALU) Freiburg, Freiburg, Germany; 2 Faculty of Biology, University Freiburg, Freiburg, Germany; 3 Spemann Graduate School of Biology and Medicine (SGBM), University Freiburg, Freiburg, Germany; 4 Interdisciplinary Center for Clinical Research (IZKF), Würzburg, Germany; 5 University of Würzburg Graduate School of Life Sciences, Würzburg, Germany; 6 Institute of Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany; 7 Else-Kröner-Forschungskolleg for Interdisciplinary Translational Immunology, Würzburg, Germany; 8 Department of Medicine II, Würzburg University Clinics, Würzburg, Germany Multiple studies have shown the critical role of conditioninginduced damage of the intestinal tract and the intestinal immune system in the onset of graft-versus-host disease (GVHD). However, it is unclear how and where the tissue damage signals are transmitted from the peri-intestinal tissue into the mesenteric lymph nodes (mLNs), where the adaptive T cell response leading to GVHD is very likely initiated. We have recently described a critical role for neutrophil granulocytes (neutrophils) in the initiation of GvHD. We now report that neutrophils are not normally found in mLN, but that neutrophils of recipient origin accumulate in mLN upon conditioning of mice by total body irradiation. This was in parallel to the appearance of bacterial 16S RNA in these LN, supporting the concept of an influx of bacteria or their components via the lymph system, possibly within myeloid cells. Using 3D light sheet fluorescence microcopy (LSFM) of the terminal ileum and the mLN we found neutrophil-containing clusters in the terminal ileum in untreated mice, which enlarged upon irradiation, together with the appearance of infiltrates in the marginal zone of the mLN. Reduction of the bacterial load in the intestinal tract reduced neutrophil recruitment by 90% in the terminal ileum and 70% in the mLN. We then analyzed candidate molecules that could be responsible for the selective recruitment of neutrophils into the ileum/mLN. Using gene-targeted mice and pharmacological inhibitors we could exclude a role for the following molecules on the recipient side: TNF, CCL2 and P2Y2 as well as TLR4 signal transduction, sphingosin-1phosphate and JAK1/2 signaling. Importantly, we found increased levels of MHC class II and the integrin CD11b on neutrophils residing in the ileum and mLN compared to
blood-derived neutrophils. This indicates a different biological function for these LN-residing neutrophils compared to their blood-derived counterparts. One possible function is the direct, MHC II-dependent antigen presentation by neutrophils towards donor T cells. In summary, our data support the concept that neutrophil granulocytes mediate cellular communication between the damaged intestinal tract and mesenteric lymph nodes, where donor T cells are being primed during the early phase of intestinal GVHD.
599 TIM-1 Blockade of the Donor Graft Provides Protection Against Lethal Gvhd Bettina P. Iliopoulou 1, Katie Hsu 1, Antonio Pierini 1, Gordon J. Freeman 2, Rosemarie H. Dekruyff 3, Everett Meyer 1. 1 Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, Stanford, CA; 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; 3 Division of Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA TIM-1 blockade of the donor graft provides protection against lethal GVHD Bettina P. Iliopoulou1, Katie Hsu1, Antonio Pierini1, Gordon J. Freeman2, Rosemarie H. Dekruyff1, Everett H. Meyer1 1 Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA, USA 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. Graft-versus-host disease (GVHD) continues to be a common cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) and new therapeutic approaches are needed to prevent or treat this disease. The T cell immunoglobulin and mucin (TIM) proteins represent a family of molecules that act in concert with Tecell receptor and costimulatory signals to regulate expansion, differentiation and effector function of T and NKT cells. More recently it has been shown that TIM-1 protein is also expressed on regulatory B cells as well plasmacytoid dendritic cells. TIM-1, a member of the TIM gene family, plays a critical role in regulating immune responses including allergy, asthma, autoimmunity and solid organ transplant tolerance. TIM-1 binds to phosphytidylserine, exposed on the surface of apoptotic cells. Given the immunoregulatory role of TIM-1 and the proapoptotic state inherent to HSCT, we explored how TIM-1 influences GVHD in a murine model of allogeneic HSCT. Here
Figure. Antagonistic anti-TIM-1 mAb protects from lethal GVHD. Survival was assessed 70 days post BMT of BALB/c recipient mice treated with lethal body irradiation before receiving allogeneic bone marrow and T cells (Tcon) from C57BL/6 mice. Mice that in addition received five doses (days -1, 3, 7, 11, 15; 400 mg) of the antagonistic anti-TIM-1 mAb (Tcon+BM+3D10 group, n¼10) showed a survival advantage (p¼0.02) compared to mice that received allogeneic T cells alone (Tcon+BM group, n¼10).
Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
Our analysis highlights HDCTX as a compelling option for GVHD prophylaxis, but with nuances. Rates of ANC and Platelet recovery slightly lag behind Tac/MTX but HDCTX is associated with lower rates of severe GVHD, improved TRM and decreased admissions and inpatient hospital days. This is more impressive when taking into account that the HDCTX included more high risk patients (the haplo transplants) compared to the Tax/MTX group. Thus, HDCTX may have a significant positive impact on transplant outcomes and the long-term costs of allogeneic transplants. Longer follow-up and analysis of a larger pool of patients will be required to further expand on the observations made in our study.
598 Neutrophil Granulocytes Mediate Cellular Communication Between the Damaged Intestinal Tract and Mesenteric Lymph Nodes at the Onset of Intestinal Graft-Versus-Host Disease Jan Hülsdünker1, 2, 3, Gabriele Prinz1, Katja Ottmüller4, 5, Heide Dierbach1, Marie Follo 1, Susanne Kirschnek 6, Andreas Beilhack 4, 7, 8, Georg Häcker 6, Robert Zeiser 1. 1 Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Albert Ludwigs University (ALU) Freiburg, Freiburg, Germany; 2 Faculty of Biology, University Freiburg, Freiburg, Germany; 3 Spemann Graduate School of Biology and Medicine (SGBM), University Freiburg, Freiburg, Germany; 4 Interdisciplinary Center for Clinical Research (IZKF), Würzburg, Germany; 5 University of Würzburg Graduate School of Life Sciences, Würzburg, Germany; 6 Institute of Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany; 7 Else-Kröner-Forschungskolleg for Interdisciplinary Translational Immunology, Würzburg, Germany; 8 Department of Medicine II, Würzburg University Clinics, Würzburg, Germany Multiple studies have shown the critical role of conditioninginduced damage of the intestinal tract and the intestinal immune system in the onset of graft-versus-host disease (GVHD). However, it is unclear how and where the tissue damage signals are transmitted from the peri-intestinal tissue into the mesenteric lymph nodes (mLNs), where the adaptive T cell response leading to GVHD is very likely initiated. We have recently described a critical role for neutrophil granulocytes (neutrophils) in the initiation of GvHD. We now report that neutrophils are not normally found in mLN, but that neutrophils of recipient origin accumulate in mLN upon conditioning of mice by total body irradiation. This was in parallel to the appearance of bacterial 16S RNA in these LN, supporting the concept of an influx of bacteria or their components via the lymph system, possibly within myeloid cells. Using 3D light sheet fluorescence microcopy (LSFM) of the terminal ileum and the mLN we found neutrophil-containing clusters in the terminal ileum in untreated mice, which enlarged upon irradiation, together with the appearance of infiltrates in the marginal zone of the mLN. Reduction of the bacterial load in the intestinal tract reduced neutrophil recruitment by 90% in the terminal ileum and 70% in the mLN. We then analyzed candidate molecules that could be responsible for the selective recruitment of neutrophils into the ileum/mLN. Using gene-targeted mice and pharmacological inhibitors we could exclude a role for the following molecules on the recipient side: TNF, CCL2 and P2Y2 as well as TLR4 signal transduction, sphingosin-1phosphate and JAK1/2 signaling. Importantly, we found increased levels of MHC class II and the integrin CD11b on neutrophils residing in the ileum and mLN compared to
blood-derived neutrophils. This indicates a different biological function for these LN-residing neutrophils compared to their blood-derived counterparts. One possible function is the direct, MHC II-dependent antigen presentation by neutrophils towards donor T cells. In summary, our data support the concept that neutrophil granulocytes mediate cellular communication between the damaged intestinal tract and mesenteric lymph nodes, where donor T cells are being primed during the early phase of intestinal GVHD.
599 TIM-1 Blockade of the Donor Graft Provides Protection Against Lethal Gvhd Bettina P. Iliopoulou 1, Katie Hsu 1, Antonio Pierini 1, Gordon J. Freeman 2, Rosemarie H. Dekruyff 3, Everett Meyer 1. 1 Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, Stanford, CA; 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; 3 Division of Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA TIM-1 blockade of the donor graft provides protection against lethal GVHD Bettina P. Iliopoulou1, Katie Hsu1, Antonio Pierini1, Gordon J. Freeman2, Rosemarie H. Dekruyff1, Everett H. Meyer1 1 Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA, USA 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. Graft-versus-host disease (GVHD) continues to be a common cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) and new therapeutic approaches are needed to prevent or treat this disease. The T cell immunoglobulin and mucin (TIM) proteins represent a family of molecules that act in concert with Tecell receptor and costimulatory signals to regulate expansion, differentiation and effector function of T and NKT cells. More recently it has been shown that TIM-1 protein is also expressed on regulatory B cells as well plasmacytoid dendritic cells. TIM-1, a member of the TIM gene family, plays a critical role in regulating immune responses including allergy, asthma, autoimmunity and solid organ transplant tolerance. TIM-1 binds to phosphytidylserine, exposed on the surface of apoptotic cells. Given the immunoregulatory role of TIM-1 and the proapoptotic state inherent to HSCT, we explored how TIM-1 influences GVHD in a murine model of allogeneic HSCT. Here
Figure. Antagonistic anti-TIM-1 mAb protects from lethal GVHD. Survival was assessed 70 days post BMT of BALB/c recipient mice treated with lethal body irradiation before receiving allogeneic bone marrow and T cells (Tcon) from C57BL/6 mice. Mice that in addition received five doses (days -1, 3, 7, 11, 15; 400 mg) of the antagonistic anti-TIM-1 mAb (Tcon+BM+3D10 group, n¼10) showed a survival advantage (p¼0.02) compared to mice that received allogeneic T cells alone (Tcon+BM group, n¼10).