Irradiated B6D2F1 Mice Research Articles

Essential Role of Regulatory T Cells on Early B Cell Reconstitution after Haploidentical BMT with Posttransplant Cyclophosphamide

Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies reported that PTCy has been associated with low incidence of viral infections or EBV-related posttransplantation lymphoproliferative disease (EB-LPD), suggesting PTCy-based immune modulation leads the favorable immune reconstitution after transplant. Recently, we studied the immune reconstitution dynamics of each lymphocyte subset after transplant using PTCy and reported that PTCy-based immune-modulation strongly promoted Treg-dominant T-cell reconstitution and stem cell-derived mature B-cell generation by using murine model of haploidentical HSCT with PTCy. TCR- and BCR- repertoire analysis demonstrated that PTCy resulted in the broader diversity of both T cell and B cell repertoire than non-PTCy controls. However, the mechanism of coordination of increased Tregs and B cells after PTCy-based HSCT has not well studied. To address this issue, we here investgated the impact and role of PTCy on Tregs and B cell recovery and the mutual influence between the two important subsets by using murine BMT model. Irradiated B6D2F1 mice were transplanted with 5x106 spleen cells from the CD45.1 B6 mice together with 5x106 TCD-BM from CD45.2 B6 donors. Cyclophosphamide 100mg/kg or control vehicle was administered at day 3 after transplant. Peripheral blood mononuclear cells (PBMCs) and splenic cells were sequentially obtained at day21. The chimeric balances among host-residual H-2kd+ cells, donor graft-derived cells and donor BM-derived cells in Tregs and B cells were monitored separately. To evaluate the homeostatic stability, proliferation marker Ki-67 and anti-apoptotic BCL-2 were also quantitatively examined respectively. To examine the detailed phenotype of Tcells, CD4, CD8, CD25, Foxp3, ICOS, CXCR5 were tested. Also, to examine the detailed phenotype of B cells, the expression of B220, CD21, CD23, CD24 were tested. At day21 after HSCT, the number of Treg cell in spleen was significantly higher on PTCy-treated recipients than that of non-PTCy control (P < 0.05). We also checked the emergence of donor-derived follicular helper T cells (Tfh) in lymph node because recent studies have shown that Tfh could contribute B cell differentiation after HSCT. In our analysis at day21 in this PTCy model, Tfh cells were still few in both PTCy group and non-PTCy group, suggesting the early T cell recovery after PTCy did not promote B cell differentiation into pathological plasmablast. The number of B cell was markedly higher in PTCy-treated recipients than that of non-PTCy control (P < 0.01). The increased B cell population was mainly transitional-1 phenotype suggesting these B cells were recent emigrants from bone marrow. In consistent with low recovery of Tfh cells, plasmablast-like phenotype of B cells were not observed. Next, to evaluate the impact of graft-derived Treg recovery on B cell reconstitution, we depleted Treg from spleen cells of B6 donors by using CD25+ magnetic beads. After the depletion, residual Treg cell in the graft was under 1% of total CD4 T cells. At day21, splenic Treg cells is under 3% of total CD4 T cells in Treg-depleted group, that is much lower than Treg in Treg-repleted group. Interestingly, the number of B cells at day21 in PTCy-treated recipients was equivalent to that of non-PTCy recipients when we depleted Treg from donor graft, suggesting that well-balaced B cell recovery promoted by PTCy was completely abolished by Treg-depletion. Taken together, our data suggested that PTCy could induce the expansion of Treg recovery and naïve B cell recovery without urging early emergence of Tfh cells in lymph node, resulting in well-diversed T and B cell reconstitution. Treg appears to contribute to promote naïve B cell emergence from bone marrow, indicating the T and B cell recovery might be mutually influenced. Moreover, slow recovery of Tfh cells in lymph node could be advantage for preventing B cell from forced differentiation into plasmablast causing GVHD. These data provide the important information for understanding the immunological reconstitution after PTCy-based haploidentical HSCT. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

Liposomal Alpha-Galactosylceramide Ameliorates Graft-Versus-Host Disease with Remaining Intensified GVL Gained By Dose-Reduction of Posttransplant Cyclophosphamaide after Haploidentical HSCT

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment for many hematological malignancies. Clinical studies have shown that HSCT from haplo-identical donors with posttransplant cyclophosphamide (PTCy) can widely provide alternative donor sources and is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD). However, on the other hands, it is concerned that the immune suppressive effect by PTCy also might diminish graft-versus-leukemia (GVL) effect and increase the relapse rate in patients with high tumor burden at transplant. Indeed, recent clinical studies suggested that a certain extent of dose-reduction of PTCy could sufficiently prevent GVHD, but whether the dose-reduction can lead to enhance GVL effect has not been clarified. In this study, we investigated the effects of dose-reduction of PTCy on GVHD and GVL by using murine haploidentical BMT model. Also, we explored the immune-modulating impacts of liposomal alpha-galactosylceramide (liposomal a-galcer; RGI2001) on reduced PTCy. Liposomal a-galcer is a synthetic glycolipid, a ligand stimulating invariant natural killer T (NKT) cell in CD1d restricted manner, which is shown to prevent GVHD via expansion of regulatory T cell (Treg) in murine experiments. First, we tested the effects of dose-reduction of PTCy on GVL effect, we transplanted from C57BL/6J donor to B6D2F1 recipients bearing with luciferase-expressing P815 leukemia cells. The impact of PTCy dose-reduction on GVL was evaluated by using in vivo bioluminescence system. Lethally irradiated B6D2F1 mice were transplanted with 5x106 T cell depleted (TCD) BM, 10x106 whole splenic cells from C57BL/6J donor mice and 5x105 luciferase-expressing P815 leukemia cells. PTCy or control vehicle was administered at day3 after BMT. As for PTCy, 50mg/kg of cyclophosphamide (Cy) has already shown as standard dose to sufficiently suppress GVHD, thus we tested effect of 25mg/kg of Cy comparing with 50mg/kg of Cy. In vivo imaging system (IVIS) was followed weekly since day10 after BMT to evaluate tumor burden. A trend toward lower levels of radiance was measured by IVIS in reduced-dose group than full-dose group through the experimental period, suggesting reduced-dose of Cy contribute to leave alloagressive T cell activity and result in the enhancement of GVL effect. However, the benefit of GVL enhancement in reduced PTCy group was counterbalanced by the deterioration of GVHD when we used higher dose of total body irradiation (TBI) for the aggressive GVHD-prone experimental setting. To overcome the negative impact of PTCy-reduction on GVHD, we next tested the immune-modulating effect of liposomal a-galcer on this system. Liposomal a-galcer 1mg/kg or control vehicle was administered at day3, 5, 7, after BMT. Our data indicated that reduced PTCy plus liposomal a-galcer significantly improved acute GVHD in overall survival (vs control group, p < 0.01). In comparison with those treated with PTCy plus liposomal a-galcer, the others kept on losing weight and worsening GVHD score to the end beyond the third week without recovery. Importantly, liposomal a-galcer did not influence the effect of GVL when we evaluated tumor-bearing recipents with IVIS, suggesting that add-on of liposomal a-galcer improved GVHD with remaining intensified GVL effect which was gained by dose-reduction of PTCy. Flowcytometric analysis of splenic cells at day 14 showed that %Treg of CD4+ Tcell was significantly higher in reduced PTCy plus liposomal a-galcer group than control group (5.30 vs 2.75, p < 0.05) and reduced PTCy alone group (5.30 vs 2.57, p < 0.05). Additionally, in PTCy plus liposomal a-galcer group, both Treg/CD4+ conventional Tcell (Tcon) ratio and Treg/CD8+ Tcell ratio were significantly higher than control group (p < 0.05) and reduced PTCy alone group (p < 0.05), respectively. Analysis of MLN cells also showed the selective increase of Treg in PTCy plus liposomal a-galcer group more than in other groups. Analysis of splenic cells and MLN cells at day28 showed similar trends with at day14. Taken together, these data demonstarated that liposomal a-galcer could efficiently compensate the immune-suppressive effect after reduced PTCy by preferentially promoting Treg recovery with remaining GVL. Our data might provide the important information for developing new strategy of haploidentical HSCT with PTCy for patient with high-tumor burden. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

The effects of secondary iron overload and iron chelation on a radiation-induced acute myeloid leukemia mouse model

BackgroundPatients with myelodysplastic syndrome (MDS) require chronic red blood cell (RBC) transfusion due to anemia. Multiple RBC transfusions cause secondary iron overload and subsequent excessive generation of reactive oxygen species (ROS), which leads to mutations, cell death, organ failure, and inferior disease outcomes. We hypothesize that iron loading promotes AML development by increasing oxidative stress and disrupting important signaling pathways in the bone marrow cells (BMCs). Conversely, iron chelation therapy (ICT) may reduce AML risk by lowering iron burden in the iron-loaded animals.MethodsWe utilized a radiation-induced acute myeloid leukemia (RI-AML) animal model. Iron overload was introduced via intraperitoneal injection of iron dextran, and iron chelation via oral gavage of deferasirox. A total of 86 irradiated B6D2F1 mice with various levels of iron burden were monitored for leukemia development over a period of 70 weeks. The Kaplan-Meier estimator was utilized to assess AML free survival. In addition, a second cohort of 30 mice was assigned for early analysis at 5 and 7 months post-irradiation. The BMCs of the early cohort were assessed for alterations of signaling pathways, DNA damage response and gene expression. Statistical significance was established using Student’s t-test or ANOVA.ResultsIron loading in irradiated B6D2F1 mice accelerated RI-AML development. However, there was a progressive decrease in AML risk for irradiated mice with increase in iron burden from 7.5 to 15 to 30 mg. In addition, ICT decreased AML incidence in the 7.5 mg iron-loaded irradiated mice, while AML onset was earlier for the 30 mg iron-loaded irradiated mice that received ICT. Furthermore, analysis of BMCs from irradiated mice at earlier intervals revealed accelerated dysregulation of signaling pathways upon iron loading, while ICT partially mitigated the effects.ConclusionsWe concluded that iron is a promoter of leukemogenesis in vivo up to a peak iron dose, but further iron loading decreases AML risk by increasing cell death. ICT can partially mitigate the adverse effects of iron overload, and to maximize its benefit this intervention should be undertaken prior to the development of extreme iron overload.

PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD.Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores.Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients.Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide Ameliorates Lethal Thymic GVHD By Restoring Regulatory and Effector T Cell Homeostasis in Recipients with Prior PD-1 Blockade Therapy

Allogeneic HSCT has a curative potential for patients with hematological malignancies. However, graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after HSCT. Regulatory T cells (Tregs) are critical mediator for immune tolerance after HSCT and we recently reported that PD-1 plays an essential role for Treg survival (Asano et al, Blood 2017). Clinical studies suggested that PD-1 blockade prior to HSCT could be a risk of increasing severe GVHD. However, the mechanisms about GVHD induced by PD-1 blockade have largely unclear and there remains a paucity of data on appropriate GVHD prophylaxis for patients who undergo HSCT after PD-1 blockade. To address these issues, we investigated the impact of PD-1 expression on donor T cells on immune reconstitution with murine BMT models. First, lethally irradiated B6D2F1 mice were transplanted with 10 million of C57BL/6-background PD-1+/+ or PD-1-/- spleen cells with 5 million of bone marrow cells from normal C57BL/6, and GVHD scores and overall survival was monitored. Recipients receiving PD-1-/- graft developed severe GVHD resulting in a significant shorter survival than recipients receiving PD-1-/- graft (P<0.0001). We analyzed lymphocytes in spleen and thymus on day3, 7, and 14. We found that CD8 T cells in PD-1-/- group showed markedly higher Ki67 expression and CFSE-dilution until day3. Interestingly, PD-1-/- Tregs increased aggressively at day3 but it could not maintain until day14, while PD-1-/- CD8 T cells and conventional CD4 T cells (CD4 Tcons) continued to increase until day+14, resulting in the significant higher CD8/Treg ratio in PD-1-/- group (P<0.05, vs PD-1+/+ group). PD-1-/- Tregs showed significantly higher expression of Annexin V on day+7 and thymus CD4- and CD8- double-positive (DP) cells were in the extremely low levels in PD-1-/- group on day+14 (P<0.05, vs PD-1+/+ group). Thymic analysis showed that donor PD-1-/- graft-derived CD8 T cells infiltrated thymus in PD-1-/- group, suggesting reconstruction of thymic function was critically disturbed by severe GVHD. These data suggest that loss of PD-1 signaling resulted in unbalanced reconstitution of donor-derived T cell subsets as a consequence of continuous CTL expansion and increased Treg apoptosis. Next, to evaluate the impact of post-transplant cyclophosphamide (PTCy) on the abnormal reconstitution after PD-1 blockade, we administered 50mg/kg of Cy or control vehicle on day3. PTCy efficiently ameliorated GVHD in PD-1-/- group and extended overall survival by safely regulating the proliferation and apoptosis of T cell subsets. Of note, after PTCy, Tregs regained the ability of continuous proliferation in the first 2 weeks, resulting in well-balanced reconstitution of donor-derived T cell subsets. Thymic DP cells on day 14 was markedly increased in PD-1-/- group with PTCy intervention as compared to without PTCy, suggesting PTCy could rescue thymus from PD-1 blockade-related severe GVHD. Finally, to evaluate GVL activity, we performed BMT with co-infusion of P815L tumor cells on day0 and we confirmed that PTCy treatment for PD-1-/- recipients reduced the severity of GVHD with maintaining sufficient GVL effect. In summary, our data suggested three insights about the impact of PD-1 signaling on immune reconstitution. First, PD-1 inhibition influenced graft-derived T cells very differently within T cell subsets. PD-1-/- Tregs increased transiently but it was counterbalanced by accelerated apoptosis, while PD-1-/- CD4+Tcons and CD8 T cells continued the drastic expansion. Second, we found that PD-1-/- donor T cells developed severe GVHD in thymus. Few reports have concentrated on the impact of donor graft PD-1 expression to thymus after BMT and acute GVHD in thymus could lead late central immune disturbance. Third, PTCy successfully ameliorated GVHD induced by PD-1-/- donor T cells preserving GVL effect. Cell proliferation study implied that PD-1-/- graft-derived CD8 T cells might be more susceptible for PTCy because of the high-rate proliferation. In conclusion, PD-1-/- graft cause lethal thymic GVHD and PTCy successfully ameliorated it. The influence of PD-1 inhibition was different within T cell subtypes. PTCy might be appropriate GVHD prophylaxis strategy for patients who had prior usage of PD-1 blockade. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Analyses of Early Lymphocyte Reconstitution after Haploidentical HSCT with Posttransplant Cyclophosphamide: Coordinated Treg-Dominant T-Cell Reconstitution and Stem Cell-Derived Mature B-Cell with Broad BCR-Repertoir Diversity

Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies reported that PTCy has been associated with low incidence of viral infections and EB-LPD, suggesting PTCy-based immune modulation leads the favorable immune reconstitution after transplant. However, the immune reconstitution dynamics of each subset after HSCT using PTCy remains poorly understood. To address this issue, we explored the impact and role of PTCy on the early lymphocyte reconstitution by using murine BMT model. Irradiated B6D2F1 mice were transplanted with 5x106 spleen cells from the CD45.1 B6 mice together with 5x106 TCD-BM from CD45.2 B6 donors. Cyclophosphamide 100mg/kg or control vehicle was administered at day 3 after transplant. Peripheral blood mononuclear cells (PBMCs) and splenic cells were sequentially obtained at day7, 14 and 21.The chimeric balances among host-residual H-2kd+ cells, donor graft-derived cells and donor BM-derived cells in CD8+ T cells, CD4+ Tcons, Tregs, B cells and NK cells were monitored separately. To evaluate the homeostatic stability of each lymphocyte subset at various time points, proliferation marker Ki-67 and anti-apoptotic BCL-2 were also quantitatively examined in each subset. To evaluate the clonal diversity of T and B cells, we performed the TCR- and BCR- repertoire analysis at day 21. Between day 0, transplanted recipients were developed severe acute GVHD, however, recipients received PTCy at day 3 promptly showed the recovery of the weight and improvement of the clinical GVHD score after day 5, whereas control continue to lose weight, suggesting the effect of PTCy to ameliorate acute GVHD. At day 7, all T cell subsets were critically depleted from both peripheral blood and spleen. The number of T cells was markedly lower in PTCy group than in control group (CD8 T+ cells; 5.1 vs 155.1/mm2, P<0.01: CD4 Tcons; 5.2 vs 61.2/mm2, P<0.01: Treg; 0.02 vs 0.52/mm2, P<0.01, respectively). Especially, Ki-67+ proliferating cells, including Tcons and Tregs, were completely depleted, indicating these activated cells are very sensitive to cyclophosphamide intervention. However, interestingly, surviving T cells in recipients just after cyclophosphamide intervention showed significantly high-levels of BCL-2 expression than control recipients (MFI: CD8 T cells; 2.8 vs 10.0: CD4 Tcns; 2.9 vs 9.9: Treg; 1.8 vs 5.3, respectively). Based on the elevated anti-apoptotic elements, T cell in PTCy-treated recipients undergo aggressive homeostatic proliferation and the number of CD4 T cell subset, especially Tregs, took over that of control recipient by day 14. CD8+ T cell proliferation after PTCy was less aggressive than CD4 T cells, resulting Treg ratio to CD8 T cells in PTCy recipents was greatly higher than in control (Treg/CD8: 0.061 vs 0.031, P<0.05). During 3 weeks, T cell recovery was basically maintained by donor graft-derived cell, though PTCy recipents involved averagely 10% of host-residual T cells. In comparison to T cells, main reconstitution of B cells was maintained by donor stem cell-derived cell. In PTCy recipients, CD23+CD24+ Transitional-2 naïve B cell and CD21-CD24+ mature follicular B cell overwhelmingly increased by Day 21(Follicular B cells in PTCy group and control; 1.22e6 vs 1.31e5, P<0.0001). BCR-repertoire diversity analysis demostrated that PTCy resulted in the broad diversity of B cell repertoire (Inverse Simpson Index; 40.5 vs 13.9). Our data clearly indicated that PTCy contributes the favorable immune reconstitution by modulating coordinate T and B cell recovery. These findings might provide important information to promote immune tolerance after PTCy-based transplant. DisclosuresMaeda:Mundipharma KK: Research Funding.

R-spondin1 Promotes Paneth Cell Growth, Maintains Intestinal Microbial Ecology, and Ameliorates GvHD

Introduction: Graft-versus-host disease (GVHD) and infections are the major obstacles to perform allogeneic hematopoietic stem cell transplantation (HSCT). We previously demonstrated that intestinal stem cells (ISCs) and their niche Paneth cells were targeted in GVHD. The intestinal microbial ecology is regulated by Paneth cells through secretion of antimicrobial molecules. Loss of Paneth cells by GVHD frequently leads to intestinal dysbiosis (Eriguchi et al, Blood, 2012). R-spondin1 (R-Spo1) is a growth factor of ISCs and protects ISCs from the damages by conditioning and GVHD (Takashima et al, J Exp Med. 2011). However, the effects of R-Spo1 on Paneth cell biology and intestinal microbial ecology have never been demonstrated.Method: Naïve B6D2F1 (H-2b/d) mice were intravenously injected with R-Spo1 (200 μg/day) or diluent for 6 days. In HSCT models, lethally irradiated B6D2F1 mice were transplanted with 5 × 106 splenocytes plus 5 × 106 bone marrow cells from allogeneic B6 (H-2b) or syngeneic B6D2F1 donors on day 0. Mice were injected with R-Spo1 or diluent from day -3 to -1 and day +1 to +3 after HSCT. On days +4 and +7, small intestine was harvested to evaluate histology and fecal samples were collected to measure fecal levels of alpha-defensin and to determine composition of microbiota by 16S rRNA gene sequencing.Result: In naïve mice, R-Spo1 administration significantly increased Paneth cells in the small intestine (Table). Fate mapping analysis using LGR5-creERT2 × Rosa26Tomato mice to visualize LGR5+ ISCs and their progenies revealed that R-Spo1 did not expand pre-existing Paneth cells but stimulated de novo differentiation of Paneth cells from ISCs. R-Spo1-induced Paneth cells produced antimicrobial peptide alpha-defensin and its activator matrix metalloproteinase-7, leading to a significant increase in fecal levels of alpha-defensin (Table). Next, we evaluated the effects of R-Spo1 on Paneth cells and intestinal microbial ecology in a HSCT model. The numbers of Paneth cells in the small intestine and fecal levels of alpha-defensin were significantly reduced in allogeneic controls compared to those in syngeneic controls on day +7 (Table), resulting in dysbiosis with a significant outgrowth of the family Enterobacteriaceae, including the genus Escherichia, and the family Bacteroidaceae in allogeneic animals (Figure). R-Spo1 protected Paneth cells from GVHD, resulting in significantly more Paneth cells persisting on days +4 and +7 compared to allogeneic controls (Table). Importantly, R-Spo1 restored the fecal levels of alpha-defensin in allogeneic recipients (Table), resulted in preservation of intestinal commensals (Figure). R-Spo1 reduced GVHD mortality as previously reported, but this survival benefit mediated by R-Spo1 was abrogated when mice were treated with broad-spectrum antibiotics including ampicillin, streptomycin, vancomycin and metronidazole, suggesting that R-Spo1-mediated GVHD suppression depended on intestinal commensals.Conclusion: We demonstrated for the first time that R-Spo1 is a growth factor of Paneth cells. R-Spo1 administration in allogeneic recipients restored fecal levels of alpha-defensin, prevented intestinal dysbiosis, and improved the outcome of HSCT in the presence of intestinal commensals. Brief administration of R-Spo1 represents a novel pharmacological approach to modify intestinal microbial ecology. Such strategies to target the intestinal microbiota may have benefits in the treatments of inflammatory bowel diseases other than GVHD.TableNo. of Paneth cells / cryptFecal levels of alpha-defensin4(ng / g feces)Naïve B6D2F1Control R-Spo13.6 ± 0.2 10.2 ± 0.7*46.3 ± 5.3 105.3 ± 28.8*Transplant (day4)Allo Allo+R-Spo12.2 ± 0.1 3.9 ± 0.2*25.9 ± 4.0 108.2 ± 12.6*Transplant (day7)Syn Allo Allo+R-Spo13.1 ± 0.1 0.7 ± 0.1 1.3 ± 0.2*18.9 ± 3.8 7.1 ± 2.9 25.6 ± 4.2*Data are shown as mean ± SE. NA; not assessed, *P<0.05 (Control vs R-Spo1, Allo vs Allo+R-Spo1) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human mesenchymal stromal cells attenuate graft-versus-host disease and maintain graft-versus-leukemia activity following experimental allogeneic bone marrow transplantation.

We sought to define the effects and underlying mechanisms of human, marrow-derived mesenchymal stromal cells (hMSCs) on graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T-cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC-treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T-cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T-cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T-cell proliferation, reduced TNFα, IFNγ, and IL-10 but increased PGE2 levels. Indomethacin and E-prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC-mediated in vitro T-cell suppression, confirming the role for PGE2 . Furthermore, cyclo-oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T-cell proliferation likely through PGE2 induction.

Enhanced Expression of PD-1 Modulates CD4+Foxp3+ Regulatory T Cell Homeostasis during Low-Dose IL-2 Therapy in Patients with Chronic Graft-Versus-Host Disease

CD4+Foxp3+ regulatory T cells (Treg) play a central role in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously reported that low-dose IL-2 administration preferentially increased Treg in patients with active cGVHD and resulted in clinical improvement with only minor toxicities (NEJM 2011). In the clinical trial, IL-2 induced selective and rapid proliferation of Treg in the first week of therapy but proliferation subsequently returned to baseline levels despite continued daily administration of IL-2 (Matsuoka et al. Sci Trans Med 2013). Mechanisms that limit Treg proliferation may play an important role in Treg homeostasis as continuous high-level proliferation may increase Treg susceptibility to apoptosis resulting in overall reduction of the Treg population (Matsuoka et al. JCI 2010). To examine mechanisms for negative regulation of Treg proliferation we examined expression of Programed Death-1 (PD-1) on Treg during IL-2 therapy. Serial blood samples from 14 patients who received daily subcutaneous IL-2 (3x105-3x106IU/m2/day) for 8 weeks were studied before and at 1, 2, 4, 6, 8, 10 and 12 weeks after starting IL-2. Treg were compared to conventional CD4+Foxp3- T cells (Tcon) within individual patient samples and examined for expression of PD-1. Treg and Tcon were further divided into subpopulations by the expression of CD45RA. Before IL-2 therapy, both CD45RA+ naïve Treg and Tcon showed little expression of PD-1 (%PD-1+ cells; median 2.1%, 1.8%, respectively). However, both CD45RA- activated/memory Treg and Tcon showed significantly higher expression of PD-1 than their naïve counterparts (%PD-1+ cells: median 22%, p<0.0001; median 19%, p<0.0001, respectively) and there was no significant difference in PD-1 expression between CD45RA- Treg and Tcon. After starting IL-2, expression of PD-1 rapidly increased, mainly on the CD45RA- activated/memory Treg subpopulation and this was sustained during 8 weeks of IL-2 therapy (%PD-1+ Treg; median 38.7% at 4 weeks, p<0.002 vs baseline). In contrast, Tcon PD-1 expression did not change during IL-2 therapy. To confirm the inhibitory function of PD-1 on Treg in patients receiving IL-2, we purified Treg and Tcon by cell-sorting, labeled cells with CFSE and cultured each subset separately with or without blocking anti-PD-L1 antibody, in the presence of anti-CD3 and anti-CD28 stimulation for 4 days. Treg obtained during IL-2 therapy showed resistance to in vitro proliferation, but vigorous proliferation was regained in the presence of anti-PD-L1 mAb. In contrast the effect of PD-L1 blockade on Tcon was relatively small. These data suggest that the PD-1 selectively limits Treg proliferation during IL-2 therapy. Based on these findings, we explored the possibility of a novel immune strategy to enhance Treg expansion during low-dose IL-2 by combination with PD-1 blockade using a murine BMT model. Lethally irradiated B6D2F1 mice were transplanted with 1x106 spleen cells from the control B6 mice together with 5x106 TCD-BM from either PD-1 KO B6 or control B6 donors. Recipients were treated with 5x103 IU IL-2 or control vehicle from day 35 to 49. As expected, recipients transplanted with cells from control donors showed significantly higher expression of PD-1 on Treg than Tcon when treated with IL-2 (%PD-1+ cells; median 47.2%, 8.9%, respectively, p<0.0001) and the increase of Treg after IL-2 treatment was more evident in recipients transplanted from PD-1 KO donors (p<0.05). To examine this clinically, we tested whether anti-PD-1 mAb can boost Treg proliferation during IL-2 administration and we confirmed that IL-2 induced Treg proliferation was significantly enhanced with the combined use of anti-PD-1 antibody (%Ki-67 + cells; median 22.9% vs 33.2%, p<0.05). In conclusion, these findings indicate that the self-regulation of Treg homeostasis through the PD-1/PD-L1 pathway can limit the expansion of Treg mediated by exogenously administered IL-2. Blockade of PD-1/PD-L1 pathway may provide an opportunity for in vivo manipulation of Treg homeostasis. Our data provide important information for developing therapeutic strategies to modulate Treg homeostasis in vivo to promote immune tolerance. DisclosuresNo relevant conflicts of interest to declare.

Host Immune Status Determines the Effects of Therapeutic Interleukin-2 Administration: Enhancement of GVL or Induction of Tolerance?

Interleukin-2 (IL-2) has a central role in immune tolerance thorough maintaining the homeostasis of CD4+CD25+Foxp3+ regulatory T cell (Treg). We recently reported that administration of low-dose IL-2 could preferentially enhance Treg in vivo and suppress clinical manifestations of chronic GVHD after allogeneic hematopoietic stem cell transplantation (NEJM2011). On the other hand, IL-2 is also necessary for the development of cytotoxic T cell function and has been used for the systemic immune therapy to amplify anti-tumor immunity. Thus, IL-2 administration after transplantation can induce the bipolar effects, namely, enhancement of Graft-versus-Leukemia effect (GVL effect) or prevention of Graft-versus-Host Disease (GVHD). For the appropriate therapeutic use of IL-2 for different purpose, we need to elucidate the factors to predict the effect of IL-2, however, the determinants except for the IL-2 dosage has not been characterized. To address this issue, we explored the impact of the immunological reconstitution after transplant on the effect of IL-2 therapy. First, we examined the in vivo reactivity to exogenous IL-2 in different lymphocyte subsets; CD8+ T cells, CD4+ conventional T cells (Tcon) and CD4+ regulatory T cells (Treg). We purified CD62L+ naïve lymphocytes and CD62L- lymphocytes from B6 spleen by cell-sorting and labeled with CFSE, adaptively transferred cells into irradiated B6 host and subcutaneously administrated 5000 IU of IL-2 or control vehicle from day1 to 5. At day6, spleen cells were harvested and the in vivo proliferation of each lymphocyte were evaluated by CFSE dilution assay. Proliferation of Tcons was just limited but both CD8 T cells and Tregs showed vigorous proliferations in response to IL-2. As expected, IL-2 induced the proliferation of CD62L- activated/memory CD8 T cells more than CD62L+ naïve CD8 T cells. In contrast, oppositely to CD8+ subsets, IL-2 induced the proliferation of CD62L+ naïve Tregs more than CD62L- Tregs, suggesting naïve Treg might be “primed” naturally and be ready to respond to IL-2 without antigenic TCR stimulation. Next, we examined the balance between naïve and memory phenotype in each lymphocyte subsets after allogeneic HSCT using murine BMT model. Lethally irradiated B6D2F1 mice were transplanted with 5x106 spleen cells from the CD45.2 B6 mice together with 5x106 TCD-BM from CD45.1 B6 donors. The balance between graft-derived cells (CD45.2) and BM-derived cells (CD45.1) in CD8+ T cell, CD4+ Tcon and Treg were monitored separately at day7, 14, 21,28 and 35. In the early phase of transplant, graft-derived cells showing CD62- were predominant, peaked at day21 and thereafter decreased in each subset. After day28, BM-derived cells generally emerged in each subset. Using this model, we compared the effects of IL-2 administration in the early phase (Day5-12) and in the late phase (Day35-42). Interestingly, IL-2 administration of daily 5000 IU of IL-2 in the early phase resulted in the dominant expansion of circulating CD62-CD44+ effector/memory CD8 T cell without Treg increase (CD8+T; 162 cells vs 75 cells/uL, P<0.05: Treg; 0.42 cells vs 0.48 cells/uL, NS). In contrast, the same dose of IL-2 administration in the late phase resulted in the vigorous increase of CD62+ Treg without CD8 T cell increase (CD8+T; 205 cells vs 185 cells/uL, NS: Treg; 57.1 cells vs 21.0 cells/uL, P<0.05). GVL experiments using 2.5x104 host-type P815 leukemia cells, that were uniformly lethal to the recipients of syngeneic BMT by day 15 after BMT, showed that IL-2 injection from day5 delayed the leukemia relapse (P<0.05), indicating IL-2 enhanced CD8+ CTL might mediated anti-tumor immunity. In conclusion, these data suggests that not only the dose of IL-2 but also host immune status at IL-2 administration is a critical factor to determine the in vivo effect of IL-2 therapy. From the point of view, the adjusted dose and timing of IL-2 infusion based on the detailed immune monitoring might enable to optimize and personalize this cytokine therapy. Our findings provide important information for developing therapeutic strategies to modulate immune cells in vivo and promote well-balanced immune recovery after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Preventive Azithromycin Treatment Reduces Noninfectious Lung Injury and Acute Graft-versus-Host Disease in a Murine Model of Allogeneic Hematopoietic Cell Transplantation

Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)– dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c+ dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.

MyD88-dependent expansion of an immature GR-1+CD11b+ population from donor bone marrow reduces early mortality after allogeneic hematopoietic stem cell transplantation (P2166)

Abstract Myeloid-derived suppressor cells coexpressing both CD11b and Gr-1 antigens are a heterogeneous population of immature myeloid cells. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs. We investigated how the MyD88-dependent expansion of MDSCs from donor BM contributes to protection of GVHD. Lethally irradiated B6D2F1 mice were transplanted with C57BL/6 WT or MyD88 KO mice TCD-BM together with WT spleen T cells. Morbidity and mortality of GVHD were significantly worse in recipients of KO TCD-BM. The expression of MDSCs in blood, MLN and liver was reduced in the recipients of KO TCD-BM, whereas the percentage of donor T cells infiltrating gut and liver was markedly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of KO TCD-BM. Moreover, in vivo administration of isolated MDSCs to recipients of KO TCD-BM attenuated the severity of GVHD. Administration of LPS to donor mice expanded MDSCs in BM with enhanced expression of MyD88 mRNAs. Recipients of TCD-BM from WT mice injected LPS also showed attenuated GVHD severity. In summary, MyD88-dependent expansion of MDSCs population from donor non T-cell component appears to be critical for attenuating GVHD severity. Incomplete expansion of MDSCs in target organs correlates with decreased apoptosis and increased infiltration of donor T cells.

Oral glutamine supplementation improves intestinal permeability dysfunction in a murine acute graft-vs.-host disease model

Although a profound barrier dysfunction has been reported, little is known about the pathophysiological mechanism evoking gastrointestinal graft-vs.-host disease (GI-GvHD) and apparent therapeutic options. The aim of this study was to evaluate the influence of oral glutamine on the course of GI-GvHD in an acute semiallogenic graft-vs.-host disease (GvHD) in irradiated B6D2F1 mice. An acute semiallogenic GvHD was induced by intraperitoneal injection of lymphocytes from C57BL/6 mice to irradiated B6D2F1 mice. Half of the GvHD animals received oral glutamine supplementation for 6 days started at the time of lymphocyte transfer. Six days after induction of the semiallogenic GvHD, jejunum specimens were prepared. The expression of the proinflammatory cytokine TNF-α and the tight junction protein occludin was investigated by PCR. Histological changes along with the apoptotic response were evaluated and intestinal permeability was assessed. Animals with GvHD showed a strong increase in paracellular permeability as a sign of the disturbed barrier function. TNF-α expression was significantly increased and the expression of the tight junction protein occludin decreased. GvHD led to mucosal atrophy, crypt hyperplasia, crypt apoptosis, and a disintegration of the tight junctions. Glutamine-treated mice showed reduced expression of TNF-α, increased occludin expression, fewer histological changes in the jejunum, smaller number of apoptotic cells in the crypt, and reduced gastrointestinal permeability. In conclusion, oral glutamine seems to have beneficial effects on the severity of inflammatory changes in the course of GvHD and might be a therapeutic option.

Human Mesenchymal Stem Cells Attenuate Graft-Versus-Host Disease and Maintain Graft-Versus-Leukemia in Murine Allogeneic Bone Marrow Transplantation

Abstract 1907Defining in vivo effects and biodistribution of human bone marrow-derived mesenchymal stem cell (hMSCs) following allogeneic bone marrow transplantation (alloBMT) could impact the clinical utility of MSC therapy for the prevention and treatment of graft-versus-host disease (GvHD). Using an established model of murine alloBMT, we defined hMSC effects on GvHD and graft-versus-leukemia (GvL) activity. We first studied whether hMSC could modulate in vitro murine T-cell (TC) alloreactivity in mixed leukocyte cultures (MLCs). Specifically, hMSCs added to MLCs significantly reduced TC proliferation in a concentration-dependent manner distinct from human fibroblasts. In contrast to MLC cultures alone, MLCs containing hMSCs had significant reduction in TNFα, IFNγ, and IL-10 levels and higher levels of PGE2 and TGFβ1. Modulation in the inflammatory milieu was associated with changes in TC phenotypes, including more naïve and less activated TC surface marker expression (CD62L+CD69−) and the induction of CD4+CD25+FoxP3+ T-regulatory cells. To determine whether hMSCs could modulate in vivo mTC alloreactivity, irradiated recipient B6D2F1 (H-2bxd) mice were transplanted with allogeneic C57BL/6 (H-2b) BM and purified splenic TCs (B6→B6D2F1) and then were tail-vein injected with hMSC infusions (1 million per injection) on days one and four post-transplant. Syngeneic transplant recipients (B6D2F1→B6D2F1) were used as controls. hMSC-treated alloBMT mice had significantly prolonged survival and improved clinical GvHD scores, reduced splenic TC expansion and TNFα and IFNγ-producing TCs, and lower circulating TNFα and IFNγ levels versus untreated alloBMT mice. Bioluminescence imaging showed redistribution of labeled hMSCs from the lungs to abdominal organs within 72 hours following infusion. Importantly, GvHD target tissues (small and large bowel and liver) harvested from hMSC-treated alloBMT mice had significantly lower GvHD pathology scores than untreated alloBMT mice. We next determined the effects of hMSCs on GvL activity using the murine mastocytoma cell line, P815 (H-2d). TCs co-cultured with hMSCs maintained potent in vitro cytotoxic T-lymphocyte (CTL) activity comparable to untreated control CTLs. After challenge with P815 tumor cells, hMSCs-treated alloBMT mice had less severe GvHD, eradication of tumor burden, and improved leukemia-free survival compared to alloBMT control mice. Lastly, indomethacin (IM) added to MLC-hMSC co-cultures significantly reversed attenuation in both murine TC alloreactivity and surface activation expression. In addition, IM administered to hMSC-treated alloBMT mice reversed hMSC-associated survival advantage, suggesting that PGE2 in part mediates hMSC immunomodulatory effects. Together, our results show that hMSC infusions effectively attenuate GvHD and maintain GvL potency in alloBMT mice and reveal potential biomarkers and mechanisms of action underlying hMSC effects. Disclosures:Solchaga:Bimemetic Therapeutics: Employment. Cooke:Amgen: Provides experimental drug and central pharmacy support for 2 trials for which I am Co-PI.

APG101 - a Novel Inhibitor of CD95/CD95L Signaling Prevents Graft-Versus-Host Disease without Disabling T Cell Effector Function and Anti-Tumor Activity,

Abstract 4019Allogeneic bone marrow transplantation (BMT) is a curative treatment modality for haematological malignancies since mature allogeneic T cells in the transplant efficiently eliminate residual tumor cells (graft-versus-tumor (GVT) effect). However, these donor T cells are also responsible for the induction of graft-versus-host disease (GVHD) by attacking recipient tissue leading to significant morbidity and mortality. Interfering with T cell effector mechanisms responsible for GVHD-induction without preventing T cell functions towards third-party or opportunistic pathogens and while maintaining anti-tumor cytotoxicity might lead to new treatment strategies after allogeneic BMT. The CD95/CD95L system significantly contributes to GVHD-associated tissue damage and intervention into this signaling pathway might therefore inhibit GVHD-development without disturbing the GVT-effect. APG101 is a human soluble fusion protein consisting of the extracellular domain of CD95 and the Fc portion of IgG blocking the interaction of CD95L with its cognate receptor. We tested the effect of APG101 on T cell function, GVHD development and anti-tumor cytotoxicity in an allogeneic parent into F1 (C57BL/6 (B6) (H-2b) into B6D2F1 (H-2bxd)) BMT model with a mismatch in HLA-Class I and II molecules. In vitro, APG101 efficiently inhibited CD95L-mediated apoptosis of primary naïve mouse CD4+ and CD8+ T cells but did not interfere with their proliferative or cytotoxic capacity. To test APG101 as a therapeutic agent in BMT we induced acute GVHD in lethally irradiated B6D2F1 mice by transplantation of B6-derived BM together with allogeneic spleen cells. APG101 treatment was conducted intraperitoneally twice a week until the end of the experiment, starting either one day before BMT (= prophylactic treatment) or starting six or thirteen days after BMT (= therapeutic treatment). Prophylactic and early (day 6), but not late therapeutic treatment (day 13), effectively protected recipients from clinical GVHD and significantly improved survival. Although APG101 efficiently prevented GVHD-induction, homing and proliferation of allogeneic T cells into lymphoid organs and GVHD-target organs was unaltered compared to non-treated animals. Since CD95L is also known as a reverse signaling co-stimulator in T cell activation, APG101 might interfere with T cell function in vivo. However, no differences in phenotype, proliferation or cytokine expression of transplanted allogeneic T cells was observed in APG101-treated mice compared to non-treated mice. Most importantly, the GVT effect was preserved in APG101-treated mice. Anti-tumor cytotoxicity of transplanted allogeneic T cells was not abrogated by APG101 injection independent whether the murine mastocytoma cell line P815 or Bcr-Abl transduced primary B-ALL cells were injected. In summary, our findings convincingly show that early APG101 treatment is a promising approach to prevent GVHD without abrogating T cell effector functions and while preserving the GvT effect after allogeneic BMT. Disclosures:Strauss:Apogenix: Research Funding. Hartmann:Apogenix: Research Funding. Kluge:Apogenix: Employment. Fricke:Apogenix GmbH: Employment, Equity Ownership. Debatin:Apogenix GmbH: Patents & Royalties, Research Funding.

Steroid treatment alters adhesion molecule and chemokine expression in experimental acute graft-vs.-host disease of the intestinal tract

Acute graft-vs.-host disease (aGVHD) is a major complication after allogeneic bone marrow transplantation (allo-BMT) that is characterized by high morbidity and mortality. Systemic treatment with steroids has been the mainstay of first-line therapy of aGVHD, although controlled experimental data in this context are limited. Using a haploidentical murine BMT model, steroid effects on hepatic and intestinal inflammation during aGVHD have been investigated. Lethally irradiated B6D2F1 mice received bone marrow cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. Intraperitoneal administration of prednisolone (2 mg/kg body weight every day) early after onset of GVHD from day +10 until day +42 resulted in reduced clinical GVHD severity and improved survival of allogeneic recipients. Although the liver was barely affected by prednisolone treatment, aGVHD-related histopathologic injury of the gastrointestinal tract was strongly reduced in association with diminished expression of interferon-γ, tumor necrosis factor, CXCL 9-11, CCL2-3, mucosal addressin cell adhesion molecule-1, and intercellular adhesion molecule-1. Prednisolone-induced reduction of adhesion molecule expression in the gut manifested earlier than seen for cytokines or chemokines. Interestingly, when starting steroid treatment on day +28, the course of GVHD was unchanged and no major differences in cyto- or chemokine expression in gastrointestinal tract or liver on day +42 were seen. When started early after GVHD onset, prednisolone-related beneficial effects can affect aGVHD target organs differently, involving divergent regulation of inflammation and leukocyte migration. Specifically, a change in adhesion properties between leukocytes and endothelial cells in the gastrointestinal tract may be one of the initial steps in a cascade of steroid-related aGVHD-attenuating events.

Preventive usage of broad spectrum chemokine inhibitor NR58-3.14.3 reduces the severity of pulmonary and hepatic graft-versus-host disease

Pulmonary graft-versus-host disease (pGVHD) is a major complication after allogeneic bone marrow transplantation (BMT), which involves donor leukocyte migration into the lung along chemokine gradients, leading to pulmonary dysfunction and respiratory insufficiency. As broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3 suppresses leukocyte migration in response to various chemokines, including CCL2, CCL3, CCL5, we investigated the effects of NR58-3.14.3 on the evolution of pGVHD. Lethally irradiated B6D2F1 mice received BMT from syngeneic (B6D2F1) or allogeneic (C57BL/6) donors, and animals were treated with either NR58-3.14.3 or vehicle control from day -1 to day +14. At week 6, in allogeneic recipients that received BSCI, inflammatory cell infiltrates in the lung were decreased, and reduced histopathologic changes translated into improved pulmonary function when compared to allo-controls. Acute GVHD of the liver was also diminished, whereas no differences were seen in the gut. Alloantigen-dependent splenic T cell expansion and systemic TNF-alpha and IFN-gamma levels were comparable in NR58-3.14.3-treated animals and allo-controls. No suppressive effect of NR58-3.14.3 on CTL cytotoxicity was found, and diminished cellular infiltrates in lung and liver were most likely due to decreased migration of mononuclear cells. Therefore, novel approaches involving BSCIs may provide a promising tool in the management of pGVHD.

T Cell Toll-Like Receptors Play a Critical Role in Graft-Versus-Host Disease and Graft-Versus-Leukemia

Graft-versus-host disease (GVHD), the major complication of allogeneic hematopoietic stem cell transplantation, is mediated by donor T cells recognizing host alloantigens expressed on antigen-presenting cells (APCs). Total body irradiation (TBI) activates and damages host tissue, produces inflammatory cytokines and damage-associated molecular patterns, and allows translocation of microbial products into the systemic circulation. It has been assumed that these products bind to toll-like receptors (TLRs) on APCs and monocytes/macrophages, activate inflammatory pathways, and further amplify GVHD. On the other hand, TLRs are also expressed on T cells but their roles in the adoptive T cell response remain to be investigated in vivo. TLR signals are mediated by both MyD88-dependent and independent TRIF protein pathways. We evaluated the role of T cell TLR signaling in T cell alloreactivity in a mouse model of allogeneic bone marrow transplantation using mice with defective TLR signaling induced by deletions of the TLR adaptor proteins TRIF and MyD88 (TRIF/MyD88 DKO mice); in these mice, the TLR-dependent signaling pathway was eliminated. Lethally irradiated B6D2F1 mice were injected with 2 × 106 T cells from wild-type (wt) or TRIF/MyD88 DKO mice together with 5 × 106 T cell-depleted bone marrow (TCD-BM) from wt or DKO mice. Morbidity and mortality of GVHD was significantly less in recipients of TRIF/MyD88 DKO T cells than in recipients of wt T cells, irrespective of donor bone marrow type (Table). Reduction of GVHD was not seen in recipients of MyD88−/− T cells or TRIF−/− T cells, suggesting that inhibition of signaling through TRIF or MyD88 alone in donor cells is not sufficient to reduce GVHD. Histopathologic examination of the small intestine, liver, and skin showed significantly reduced GVHD pathology in recipients of DKO T cells (Table). Thus, effects of TLR signaling in donor cells on GVHD primarily reside in the T cell compartment of the donor graft, not in donor accessory cells. In recipients of TRIF/MyD88 DKO T cells, reduction of GVHD was associated with impairment of donor T cell expansion and production of TNF-□ and IFN-□. In marked contrast though, TRIF/MyD88 DKO T cells were activated equally to wt T cells in unirradiated B6D2F1 mice after adoptive transfer, thus activation of DKO T cells was impaired only in recipient mice heavily pretreated with TBI. These results suggest that T cell TLR signaling provides critical co-stimulation for T cells in an inflamed environment. T cell TLR signaling was also required for optimal graft-versus-leukemia activity. These results challenge the current paradigm that TLR functions are primarily attributable to their role in APCs and monocytes/macrophages in GVHD, and reveal a previously unrecognized critical role for T cell TLRs in inducing GVHD and GVL after hematopoietic stem cell transplantation.Pathological scoresTBMSurvivals(%)Clinical scoresSerum IFN □ (ng/ml)LiverIntestineSkinsynsyn1000.2±0.2*ND1.0±0.61.0±0.00.0±0.0wtwt276.2±0.94.3±1.218.3±2.316.7±3.01.7±0.8wtDKO255.5±0.8DKOwt100*1.8±0.4*0.4±0.1*7.9±1.0*10.9±1.70.1±0.1*DKODKO88*2.2±0.4**P<0.05 vs wt, ND; not detected

Pulmonary Function Changes in Experimental Graft-versus-Host Disease of the Lung

Pulmonary graft-versus-host disease (p-GVHD) is a serious complication after allogeneic stem cell transplantation (allo-SCT) of high morbidity and high mortality. We characterized breathing patterns and pulmonary function changes in correlation to lung histopathology and survival by using a well-established murine model of p-GVHD. Lethally irradiated B6D2F1 mice received SCT from either syngeneic B6D2F1 or allogeneic C57BL/6 animals. Within 6 weeks, severe p-GVHD developed in allogeneic recipients characterized by progressive interstitial, alveolar, peribronchial, and periluminal inflammatory cell infiltration, whereas in syngeneic recipients lung histology remained normal. Allogeneic recipients demonstrated decreased minute ventilation (MV), reduced peak inspiratory and expiratory flow rates as early as 1 week after SCT. In addition, allo-SCT resulted in restrictive pulmonary function changes as early as 7 days after transplantation and in progressive airflow obstruction within 6 weeks. Decreased breathing abilities and pulmonary function changes of allogeneic recipients were associated with increased mortality and the severity of acute graft-versus-host disease (aGVHD). These findings show that p-GVHD can be characterized by changes in pulmonary function and functional respiratory insufficiency. Furthermore, our data strengthen the understanding, that the lung is a critical target organ of aGVHD.

350: Macrophage Migration Inhibitory Factor as a New Target in Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation

Acute graft versus host disease (GVHD) is the major complication after allogeneic (allo) stem cell transplantation (SCT) involving injury to host tissues by both inflammatory cytokines and donor-derived cellular effectors. Macrophage migration inhibitory factor (MIF) is produced by various cell types and has a broad range of proinflammatory properties. We tested, whether MIF contributes to GVHD by using a well established murine model. Lethally (1300 cGy) irradiated B6D2F1 (H-2bxd) mice received SCT either from syngeneic (syn) (B6D2F1) or allo (B6; H-2b) donors. One half of animals after either syn or allo SCT were treated with polyclonal antibodies against mouse MIF from day 0 until day 14, whereas the other halves of syn and allo recipients received control IgG. GVHD severity was assessed after SCT by survival and a clinical scoring system assessing weight changes, fur texture, skin integrity, mobility and posture. Syn recipients stayed clinically well and all animals survived regardless their treatment with control or anti-MIF. Animals receiving allo-SCT plus control IgG developed significant aGVHD and high mortality. By contrast, in allo anti-MIF treated recipients, GVHD scores (day 21: 4.4 ± 0.4 vs. 5.7 ± 0.5) and weight loss (24.6% vs. 45.4%) were reduced, and survival significantly improved (p < 0.05). In addition, target organ injury to the gut (histopathology score: 4.3 ± 0.7 vs. 8.0 ± 2.6) as well as serum IFNγ (4397 ± 995 vs. 6070 ± 525 pg/ml) and TNFα (113.9 ± 12.7 vs. 159.2 ± 22.5 pg/ml) levels of anti-MIF treated allo recipients were decreased by day 7 when compared to controls. Alloantigen-specific T cell activation in vitro was significantly suppressed in the presence of anti-MIF, as determined by a reduction in T cell proliferation, IFNγ and TNFα secretion.We next challenged syn and allo recipients with P815 lymphoblast-like mastocytoma cells (H-2d) at the time of transplantation to asses the effect of anti-MIF on graft versus leukemia (GVL) responses. Significant P815 cell infiltration was seen after syn SCT as demonstrated by FACS analysis of the spleens (67.2% infiltration) and by histopathology of the liver between day 14 and day 21 after SCT. In contrast, P815 cells were significantly cleared in allo recipients treated with either control or anti-MIF (0.81% and 12.1% infiltration).In summary, our data show an important role for MIF in GVHD pathophysiology and suggest MIF as a promising target in reducing GVHD without the loss of GVL. Acute graft versus host disease (GVHD) is the major complication after allogeneic (allo) stem cell transplantation (SCT) involving injury to host tissues by both inflammatory cytokines and donor-derived cellular effectors. Macrophage migration inhibitory factor (MIF) is produced by various cell types and has a broad range of proinflammatory properties. We tested, whether MIF contributes to GVHD by using a well established murine model. Lethally (1300 cGy) irradiated B6D2F1 (H-2bxd) mice received SCT either from syngeneic (syn) (B6D2F1) or allo (B6; H-2b) donors. One half of animals after either syn or allo SCT were treated with polyclonal antibodies against mouse MIF from day 0 until day 14, whereas the other halves of syn and allo recipients received control IgG. GVHD severity was assessed after SCT by survival and a clinical scoring system assessing weight changes, fur texture, skin integrity, mobility and posture. Syn recipients stayed clinically well and all animals survived regardless their treatment with control or anti-MIF. Animals receiving allo-SCT plus control IgG developed significant aGVHD and high mortality. By contrast, in allo anti-MIF treated recipients, GVHD scores (day 21: 4.4 ± 0.4 vs. 5.7 ± 0.5) and weight loss (24.6% vs. 45.4%) were reduced, and survival significantly improved (p < 0.05). In addition, target organ injury to the gut (histopathology score: 4.3 ± 0.7 vs. 8.0 ± 2.6) as well as serum IFNγ (4397 ± 995 vs. 6070 ± 525 pg/ml) and TNFα (113.9 ± 12.7 vs. 159.2 ± 22.5 pg/ml) levels of anti-MIF treated allo recipients were decreased by day 7 when compared to controls. Alloantigen-specific T cell activation in vitro was significantly suppressed in the presence of anti-MIF, as determined by a reduction in T cell proliferation, IFNγ and TNFα secretion. We next challenged syn and allo recipients with P815 lymphoblast-like mastocytoma cells (H-2d) at the time of transplantation to asses the effect of anti-MIF on graft versus leukemia (GVL) responses. Significant P815 cell infiltration was seen after syn SCT as demonstrated by FACS analysis of the spleens (67.2% infiltration) and by histopathology of the liver between day 14 and day 21 after SCT. In contrast, P815 cells were significantly cleared in allo recipients treated with either control or anti-MIF (0.81% and 12.1% infiltration). In summary, our data show an important role for MIF in GVHD pathophysiology and suggest MIF as a promising target in reducing GVHD without the loss of GVL.