image_1_Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.TIF

<p>Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.</p

table_2_Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.PDF

<p>Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.</p

table_1_Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.PDF

<p>Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.</p

image_2_Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.TIF

<p>Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.</p

Pneumovirus-Induced Lung Disease in Mice Is Independent of Neutrophil-Driven Inflammation

<div><p>The human pneumovirus respiratory syncytial virus (RSV) is the most common pathogen causing lower respiratory tract disease in young children worldwide. A hallmark of severe human RSV infection is the strong neutrophil recruitment to the airways and lungs. Massive neutrophil activation has been proven detrimental in numerous diseases, yet in RSV the contribution of neutrophils to disease severity, and thereby, the relevance of targeting them, is largely unknown. To determine the relevance of potential neutrophil targeting therapies, we implemented antibody-mediated neutrophil depletion in a mouse pneumonia virus of mice (PVM) model. PVM is a host specific murine pneumovirus closely related to human RSV, which reproduces many of the features of RSV infection, such as high viral replication and neutrophil recruitment. Clinical disease and markers of lung inflammation and injury were studied in PVM-infected mice treated with either depleting or isotype control antibodies. To confirm our results we performed all experiments in two mice strains: C57Bl6 and BALBc mice. Neutrophil depletion in blood and lungs was efficient throughout the disease. Remarkably, in both mouse strains we found no difference in clinical disease severity between neutrophil-depleted and control arms. In line with this observation, we found no differences between groups in histopathological lung injury and lung viral loads. In conclusion, our study shows that in mice neutrophil recruitment to the lungs does not affect disease outcome or viral clearance during severe PVM infection. As such, this model does not support the notion that neutrophils play a key role in mouse pneumovirus disease.</p></div

BAL cellularity, cytokines and neutrophil activation.

<p>BAL cellularity, cytokines and neutrophil activation.</p

Lung histopathology.

<p><b>(A,C)</b> Representative image of HE-staining of C57Bl6 mice (day 8), showing interstitial cellular infiltrates and proteinacious debris <b>(B,D)</b> HE-staining of BALBc mice (day 7), showing hemorrhaging and proteinacious debris (magnification 400×).</p

NET production.

<p><b>(A)</b> Citrullinated histone H3 staining of a non-infected lung tissue section, no NETs are visible (magnification 200×). <b>(B,C)</b> Citrullinated histone H3 staining of PVM infected C57Bl6 (day 8) and BALBc (day 7) mice (non-lavaged lung sections, magnification 200×) shows scarce NET formation (insets, magnification 1200×) without airway occlusion (asterisk).</p

Semi-survival experiment.

<p>Kaplan-Meier curves showing the percentages of BALBc mice (solid lines) and C57Bl6 mice (dashed lines) treated with either 1A8 mAb (black, N = 5-6/group) or isotype control antibody (grey, N = 5-6/group) reaching the end point of a clinical score of ≥ 4 and/or > 20% weight loss after PVM inoculation (not significant).</p

Clinical PVM disease severity.

<p><b>(A-B)</b> Weight loss and clinical score of illness as measured by the modified Cook’s score <b>(C-D)</b> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168779#pone.0168779.ref025" target="_blank">25</a>] in C57Bl6 and BALBc mice treated with either 1A8 mAb (grey circles, N = 6) or isotype control antibody (white circles, N = 6) during the course of severe PVM disease. No significant differences between groups. Data are shown as median with bars depicting IQR.</p