Graft-versus-host Disease Scores Research Articles

A Calcineurin Inhibitor Does Not Affect the Post-Transplantation Cyclophosphamide -Induced Regulatory T Cell Expansion after Bone Marrow Transplantation in a Murine Chronic Gvhd Model

Chronic graft-versus-host disease (GVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Prolonged administration of a calcineurin inhibitor, such as cyclosporin A (CsA), does not decrease the risk of chronic GVHD. Indeed, we and others have reported that long-term use of CsA impairs reconstitution of bone marrow-derived regulatory T cells (Tregs) and increases susceptibility to chronic GVHD. Recently, the administration of post-transplantation cyclophosphamide (PTCY) has been used widely in GVHD prophylaxis and lower incidences of acute and chronic GVHD have been reported. Here, we evaluated the effects of PTCY and CsA on Tregs and cytokine production in effector T cells using the well-defined chronic GVHD mouse model of B10.D2 (H2d) cells into BALB/c (H2d) mice. Sublethally-irradiated recipient BALB/c mice were transplanted with 8.0 × 106 T cell-depleted bone marrow cells and 2.0 × 106 T cell-replete spleen cells from donor B10.D2 mice. PTCY was administered by intraperitoneal injection at days 3 and 4 post bone marrow transplantation (BMT). CsA was injected peritoneally once daily, starting on day 0 of BMT until the day of analysis. Administration of PTCY, with or without CsA, improved the clinical chronic GVHD score significantly at day 50 versus the controls and the CsA-alone group (controls: 2.8 ± 0.34, PTCY: 0.5 ± 0.0, p < 0.01; CsA alone: 1.8 ± 0.73, PTCY-CsA: 0.5 ± 0.0, p < 0.01). There was no significant difference in the GVHD score between the PTCY and PTCY-CsA groups. Histopathological examinations of the skin at day 50 after BMT also showed significantly reduced chronic GVHD damage in the PTCY group versus the controls and the CsA-alone group (controls vs. PTCY, p < 0.001; CsA alone vs. PTCY, p < 0.01). Flow cytometry analysis of the cells isolated from the peripheral lymph nodes 28 days after BMT revealed that the proportions of Tregs to CD4+ T cells in the PTCY (25.8 ± 2.1%) and PTCY-CsA (29.5 ± 2.2%) groups were remarkably higher than the controls (8.3 ± 2.7%) and CsA-alone (6.7 ± 1.6%) groups (p < 0.05). There was no significant difference in the Treg ratio between the PTCY and PTCY-CsA groups. Intracellular staining showed that IFN-γ single-positive and IFN-γ/IL-17 double-positive CD4 T cells were well suppressed in the PTCY and PTCY-CsA groups versus the controls and CsA-alone group (controls: 38.2 ± 3.5%, PTCY: 11.9 ± 3.1%, p < 0.01; CsA alone: 46.9 ± 2.2%, PTCY-CsA: 17.9%, p < 0.01). Our data suggest that PTCY treatment attenuated chronic GVHD via enhanced reconstitution of donor Tregs and suppression of IFN-γ/IL-17-producing effector T cells. Moreover, CsA does not appear to hinder this suppressive effect of PTCY. This result seems helpful for understanding how PTCY prevents chronic GVHD in clinical settings. DisclosuresMaeda:Mundipharma KK: Research Funding.

Murine Donor 1928z CAR T Cells Exert Potent Graft-Versus-Lymphoma Activity without Graft-Versus-Host-Disease

Chimeric antigen receptors (CARs) promote tumor cell target recognition of adoptively transferred T cellsand clinical studies have shown that autologous T cells transduced with CARs targeting CD19, expressed on normal B cells and several B cell malignancies, are effective against CD19+ malignancies. The potential of donor-derived CD19-CAR T cells to treat relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) has been relatively unexplored due to concerns for graft-versus-host disease (GVHD) and other toxicities. However, recent clinical trials of donor-derived CD19-CAR T cells in allo-HSCT have noted a surprisingly low incidence of GVHD. We studied the GVHD and anti-tumor potential of donor-derived mouse CD19-specific CAR 1928z (m1928z) T cells in preclinical allo-HSCT and lymphoma models.We constructed a panel of retroviral vectors targeting mouse CD19 (Figure 1A) to test various aspects of CAR stimulation and co-stimulation and generated T cells expressing these constructs. We then used these constructs in mouse allo-HSCT models comprising BALB/c recipients and B6 donors (Figure 1B).A20 lymphoma-inoculated BALB/c recipients of B6 BM treated with donor B6 m1928z T cells had significantly higher tumor-free survival and improved survival compared to m19delta treated recipients (Figure 1C, P < 0.0001). Adoptive transfer of B6 m1928z T cells caused significantly less GVHD compared to B6 m19delta T cells (P < 0.001) in BALB/c recipients of B6 BM confirming that m1928z T cells cause less GVHD. We explored the requirement of CAR signaling for attenuated GVHD using hum1928z (P < 0.001) (anti-human CD19-CAR, non reactive in mice) T cells and noted significantly worse survival and GVHD scores in mice as compared to with m1928z indicating that CAR signaling was necessary for this protection (Figure 1D). We also examined the importance of co-stimulation and noted increased GVHD mediated by m19z T cells (P < 0.05) (CAR with no co-stimulation) (Figure 1E) and m19BBz (P < 0.01) (CAR with 4-1BB co-stimulation) (Figure 1F) indicating that CD28 co-stimulation was also vital for attenuated GVHD. To study the effect of concurrent TCR and CAR signaling in vitro, we used T cells engineered to co-express 1928z CAR and OT-1 TCR in a cold-target inhibition killing assay and noted that activation of the T cells through 1928z can impair lytic activity mediated by TCR. To elucidate the consequence of concurrent TCR and CAR activation, we studied the TCR signaling pathway and found increased phosphorylated PKCa, pERK1/2, pS6, pSTAT1, pSTAT3, and pSTAT5 in donor m1928z T cells compared to m19delta T cells, indicating a hyperactivated state. In order to further study the effect of concurrent stimulation of alloreactive TCR and m1928z CAR, we utilized B6 ABM-RAG1 knockout (ABM) T cells, which express monoclonal TCR specific to I-Abm12 expressed on B6.C-H2bm12 (BM12) mice. In accordance with our previous findings, we observed significantly less GVHD in allo-HSCT BM12 recipients of ABM m1928z T cells versus m19delta T cells (P < 0.0001). We performed global transcriptional analysis of ABM m1928z T cells and m19delta harvested from adoptively transferred BM12 recipients of B6 BM and differential upregulation of Bim, Bid, FasL and Apaf1 in m1928z T cells. Phenotypic analysis of these cells also showed increased PD-1 and Fas expression, consistent with post-stimulatory programmed cell deathIn conclusion, we show that donor m1928z T cells mediate strong GVT effects with decreased GVHD. Following adoptive transfer, alloreactive m1928z T cells (but not m19BBz) undergo progressive loss of effector function and proliferative potential, and eventual deletion, which significantly decreases graft-versus-host activity. The non-alloreactive m1928z T cells present in bulk donor T cell populations retain significant anti-lymphoma activity. Our findings support the administration of donor 1928z T cells to prevent relapse of CD19+ malignancies after allo-HSCT and provide mechanistic insight into recent clinical trial results demonstrating that allogeneic CD19-specific 1928z CAR T cells promote anti-lymphoma activity while causing minimal GVHD. [Display omitted] DisclosuresDavila:Adaptive Biotechnologies: Membership on an entity’s Board of Directors or advisory committees; Celyad: Consultancy; Precision Biosciences: Membership on an entity’s Board of Directors or advisory committees; Kite Pharma: Membership on an entity’s Board of Directors or advisory committees; Servier: Membership on an entity’s Board of Directors or advisory committees. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

Analysis of the Impact of KIR B Haplotype Donors on Outcomes after Haploidentical (HI) and Matched Related (MR) Hematopoietic Stem Cell Transplantation (HSCT)

Introduction:Natural killer (NK) cell alloreactivity has been associated with graft versus leukemia effects in HSCT. These effects are in part mediated through killer cell immunoglobulin-like receptors (KIR) on the surface of NK cells. The absence of inhibition of NK cells via inhibitory KIR in KIR ligand mismatched T-cell depleted HI HSCT, and the use of unrelated donors possessing activating KIR haplotypes (KIR-B) have been associated with improved overall survival (OS) due to relapse protection. Inhibitory KIR ligand mismatch in the setting of T-cell replete HI HSCT has not consistently been shown to be beneficial, and data regarding the impact of using KIR-B donors in T-replete HI and MR HSCT is limited. In this preliminary retrospective review, we compared outcomes between patients with KIR-B donors versus KIR A haplotype (KIR-A) donors after HI and MR HSCT to help inform donor selection.Methods:All patients underwent HSCT on our 2-step approach in which after conditioning, a fixed T cell dose (2 x 108/kg) is administered (step 1), followed 2-3 days later by cyclophosphamide (CY) for bidirectional tolerization. Two days after CY, patients receive a CD 34-selected donor product (step 2). To be included in the analysis, patients had to be alive for at least 1 month post HSCT and be without evidence of graft rejection. Recipients were divided into 2 groups based on whether they were transplanted from a KIR-B (possessing one or more non-framework activating KIR genes) versus a KIR-A donor (genotypically negative for non-framework activating KIR genes). The relationship between the use of a KIR-B donor and the end points of OS, relapse, grades II-IV acute graft versus host disease (GVHD), chronic GVHD and non-relapse mortality (NRM) was examined. Probability of OS was estimated by Kaplan-Meier (KM) method with log-rank analysis and multivariate analyses using Cox proportional hazards. Donor and recipient age and gender, HCT-CI score, Revised Disease Risk Index, presence of any inhibitory KIR ligand mismatch between donor and recipient and CMV reactivation after HSCT were considered as confounding variables. Cumulative incidence functions with competing risk analyses were used to estimate the probabilities of relapse, NRM, acute and chronic GVHD.Results:The study group consisted of 128 consecutive patients with myeloid malignancies (AML=91 MDS=27, CML=1, MPD=9) undergoing HI [n=84] or MR [n=44] HSCT. The median recipient age and follow-up of the study group was 58 (range 19-78) years and 40.6 (range: 1.07-117.27) months respectively. Only 13 (10.2%) patients had grade III or higher acute GVHD and only 7 (5.5%) patients developed severe, chronic GVHD (score of 3) respectively. Seventy-four (58%) and 54 (42%) of recipients had KIR-B versus KIR-A donors respectively. Fifty-five (74.3%) KIR-B donors possessed HLA C1 or C1/2 alleles in the presence of KIR2L2/3/2DS2. On multivariate Cox regression analysis, OS (HR, 0.579; 95% CI, 0.337-0.995; P= 0.048) was significantly higher in patients who received HSCT from KIR-B donors. On competing risk analysis, relapse (HR, 0.756; P=0.403) and NRM (HR, 0.466; P=0.128) were lower in patients who had KIR haplotype B donors though not statistically significant. In contrast, the risk of grades II to IV acute GVHD (with death as a competing event) was higher in patients with KIR-B donors (HR, 1.972; 95% CI, 1.009-3.852; P=0.047). Similarly transplants from KIR-B donors were associated with a higher risk of chronic GVHD (HR, 2.89; P=0.053).Discussion:Our results show that within the context of the 2-step approach, the use of a KIR-B donor is associated with improved OS and an increased risk of acute and chronic GVHD in patients with myeloid neoplasms receiving HI and MR HSCT. While not reaching significance, the results for relapse and NRM suggest that the OS benefit in patients with KIR-B donors was derived from a combination of lower relapse rates and NRM that was not adversely impacted by GVHD which was mostly lower grade in this population. Our findings support the concept of KIR-B donors as more alloreactive than KIR-A donors, specifically those possessing a C1-C1/2/2DL2/3/2DS2 repertoire, the primary type possessed by KIR-B donors in this analysis. The data argues for the continued use of KIR genotyping at our institution and analyses of HSCT outcomes based on donor KIR repertoire and other donor characteristics to optimize donor selection in HI and MR HSCT. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DR3 Signaling Modulates the Function of Foxp3+ regulatory T Cells and the Severity of Acute Graft and Host Disease

Background:CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft versus host disease (GVHD) in murine models of allogeneic hematopoietic cell transplantation (HCT). We recently demonstrated that a single treatment of the agonistic antibody to DR3 (Death receptor 3, aDR3) to donor mice resulted in the expansion/activation of donor derived Treg and prevented acute GVHD (Blood 126:546, 2015), although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we investigated the efficacy of αDR3 treatment to recipient mice in model of murine GVHD.MethodsTo analyze the DR3 expression in immune cells with or without TCR stimulation, we comprehensively analyzed the cells with multicolor cytometry using viSNE (visualization of stochastic neighbor embedding algorithm). In transplantation experiments, 5x10e6 T cell depleted bone marrow (from WT C57BL/6 mice, H2kb) and 1x10e6 T cells (C57BL/6-Luciferase mice, H2kb) were injected intravenously into lethally irradiated (8Gy in total) BALB/c recipient mice (H2kd). aDR3 was intraperitonealy injected at different time point after transplantation. The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking, and survival time. To investigate the role of donor or recipient derived Treg in this model, in vivo Treg depletion using B6-Foxp3DTR mice was also performed.ResultsviSNE analysis demonstrated that DR3 was preferentially expressed on resting-Treg (79%), although a subpopulation of CD4+Foxp3-T cells (59%), CD8+T cells (24%), and NK1.1+TCRb+NKT celsl (42%) also expressed DR3. However, DR3 expressions in CD4+Foxp3-T cells and CD8+T cells were elevated after TCR stimulation in vitro (p<0.01). These data suggested that activation of DR3 signaling would also affect the function of conventional T cell upon activation. In the mixed lymphocyte reaction using allogeneic T cells (from WT C57BL/6 mice) and irradiated splenocytes (from BALB/c mice), the activation of DR3 promoted allogeneic T cell proliferation (p<0.01). In transplantation experiments, aDR3 treatment (day 3 after transplant) to animals with ongoing GVHD failed to expand Treg and further promoted donor T cell activation/proliferation with worse outcomes (p<0.05 in BLI study, p<0.01 in survival).However, the prophylactic treatment of animals with aDR3 (day 0 αDR3 and day 2 allogeneic T cells) resulted in the expansion of recipient derived Treg (H2kd+CD4+Foxp3+ cells, p<0.01) and reduced the severity of GVHD with markedly prolonged survival (p<0.001). These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. This survival benefit could be observed even if Treg were depleted from the donor allogeneic T cells (from diphtheria toxin treated B6-Foxp3DTR mice), suggesting that host derived Treg, rather than donor cells, play a critical role in abrogating GVHD in this model.ConclusionIn conclusion, we demonstrated that activation through DR3 signaling not only expands and activates Treg, but also further activates conventional alloreactive T cells and has very different clinical impact depending upon the timing of administration. These data provide important information for future clinical translation using modification of DR3 signaling. DisclosuresNegrin:Stanford University: Patents & Royalties.

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.

The Use of Monoclonal Antibody Directed Chimeric Antigen Receptors to Facilitate Conventional T Cell and Treg Control of GvHD and Tissue Tolerance in Murine Models

INTRODUCTIONRegulatory T cells (Treg) modulate allograft immune responses and Treg-based cellular therapies can be used for prevention of graft-versus-host disease (GvHD) following hematopoietic cell transplantation and for prevention of allograft rejection following tissue or organ transplantation. Treg adoptive transfer has limitations including that Treg do not necessarily home to sites where they are needed and can become inactivated in inflammatory milieus.METHODSWe used new technologies of T cell engineering to force the expression of a chimeric antigen receptor on T cells and Treg that recognizes labeled therapeutic monoclonal antibodies (mabCAR), allowing for precise control of their localization in vivo. The mabCAR recognizes fluorescein isothiocyanate (FITC) through a FITC-specific single-chain variable fragment fused to a CD28 and TCRζ costimulatory domain. Any monoclonal antibody (mab) coupled to FITC within its Fc domain can be recognized. We tested this approach with T cells and Treg to ameliorate GvHD and induce tolerance to pancreatic islet grafts.RESULTSWe first tested our mabCAR construct in conventional T cells (Tcon) which when transfected and stimulated with a FITC-mab become activated and expressed higher levels of CD44 (p=0.0003), CD25 (p=0.009) and produced more IFNγ (p=0.04). To test if mabCAR transient transfection alters Tcon homing after adoptive transfer, we injected luc+ mabCAR Tcon directed against MAdCAM1 (a gut and lymph node endothelial integrin) or SDF1 (a chemokine mainly expressed in the bone marrow) into allogeneic hosts. MAdCAM1-directed Tcon mainly homed to the gut and lymph nodes, while SDF1-directed Tcon homed to bones and spleen. SDF1-directed Tcon induced a milder GvHD (p<0.001), demonstrating that cell homing impacts GvHD severity.We then tested mabCAR Treg ability to maintain their suppressive activity in vitro and in vivo. We found that mabCAR transiently transfected into Treg have increased ability to proliferate in response to anti-CD3/CD28 stimulatory beads (p<0.01) and highly suppress Tcon proliferation when co-cultured with allogeneic irradiated splenocytes. We injected MAdCAM1 directed Treg in an allogeneic GvHD model and these mabCAR Treg prolonged survival (p=0.03), improved GvHD score (p<0.001) and mouse weight profile (p<0.001), thus demonstrating that mabCAR Treg retain regulatory functions.Finally, we tested if mabCAR Treg could induce tolerance to allogeneic pancreatic islet grafts in sublethally irradiated hosts. Luc+gfp+ mabCAR Treg homed and expanded over time (p<0.05) to the site of the allogeneic islet grafts (right kidney capsule) when FITC-anti-allogeneic MHC-I mab directed the Treg as compared to isotype mab controls (see figure). Allo-MHC-I directed mabCAR-Treg prolonged allogeneic islet graft survival in comparison to isotype-mabCAR Treg (p=0.002) allowing for production of higher insulin levels. To assess if allo-MHC-I Treg promoted antigen-specific tolerance, we performed secondary skin graft transplantation. We found that mice which received MHC-I Treg showed a significant delay in the rejection of skin grafts from mice with the same MHC mismatch as the previous islet-allografts in comparison to third-party skin grafts (p=0.02) offering strong evidence that CAR-Treg could be used to enhance antigen-specific graft protection.CONCLUSIONMabCAR expression can be used to control immune cell homing after transfer in different models according to localizing mab availability. We believe that the mabCAR approach may represent a new tool for optimizing cellular therapies to modulate GvHD and for inducing tolerance in islet and organ transplantation. [Display omitted] DisclosuresPierini:Stanford University: Patents & Royalties. Iliopoulou:Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties. Kim:Stanford University: Patents & Royalties. Meyer:Stanford University: Patents & Royalties.

Vitamin A-Coupled Liposomes Carrying siRNA Against HSP47 Ameliorate Skin Fibrosis in Chronic Graft-Versus-Host Disease

Introduction: Chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is characterized by multiple-organ fibrosis and often refractory to conventional immunosuppressive therapies, profoundly affecting quality of life of SCT survivors. Heat shock protein 47 (HSP47), a collagen specific molecular chaperone, plays a critical role in collagen synthesis in myofibroblasts. Vitamin A-coupled liposomes carrying HSP47 siRNA (VA-Lip HSP47) have been shown to deliver HSP47 siRNA to pathogenic myofibroblasts and ameliorate mouse liver cirrhosis. We tested if VA-Lip HSP47 could inhibit collagen synthesis in myofibroblasts and ameliorate skin fibrosis in chronic GVHD, using a well-established murine model of cutaneous chronic GVHD.Methods: VA-Lip HSP47 was provided by Nitto Denko Corporation (Osaka, Japan). BALB/c mice were exposed to a single dose of 6 Gy total body irradiation, followed by intravenous injection with 8 x 106 bone marrow cells plus 2.5 x 107 splenocytes from minor histocompatibility antigen mismatched B10.D2 or syngeneic BALB/c donors on day 0. Recipient mice were intravenously injected with 4.5 mg/kg VA-Lip HSP47 or vehicle thrice a week from day +1 to +41 after bone marrow transplantation (BMT). To deplete macrophages, a group of recipients were intraperitoneally injected with 0.5 mg/body of anti-CSF1 receptor monoclonal antibodies (aCSF1R) thrice a week from day +7 after BMT. Skin samples were harvested on day +42. For in vitro analysis, primary fibroblasts isolated from mouse skin were stimulated by 5 ng/ml TGF-b in the presence or absence of 50 nM of VA-Lip HSP47 or VA-Lip containing scrambled siRNA. HSP47 expressions were measured by quantitative PCR.Results:Masson’s trichrome staining of the skin sections demonstrated skin thickening with massive fibrosis of the dermis 42 days after BMT. Quantitative collagen assay confirmed collagen volume was significantly increased in the skin of allogeneic recipients compared to syngeneic controls. Immunofluorescent study showed myofibroblasts expressing HSP47 were accumulated in the dermis of allogeneic animals, while these cells were hardly detected in the skin of syngeneic controls. Macrophage-depletion with repeated administration of aCSF1R dramatically reduced HSP47+ myofibroblasts, suggesting a critical role of macrophages in differentiation of HSP47+ myofibroblasts. In vitro culture showed VA-Lip HSP47 knocked down HSP47 in mouse skin myofibroblasts, while VA-Lip carrying scrambled siRNA did not alter HSP47 expression (Figure 1). To confirm in vivo distribution of VA-Lip HSP47 specifically to the fibrotic skin lesion, mice with bleomycin-induced localized skin fibrosis were injected with VA-Lip HSP47 labeled with fluorescent dye. We confirmed VA-Lip HSP47 was distributed specifically to fibrotic skin, whereas no VA-Lip HSP47 particle was found in non-fibrotic skin of the same mouse. Next, recipients of allogeneic BMT were treated with VA-Lip HSP47 from day +1 to +41. VA-Lip HSP47 knocked down HSP47 expression in the skin of allogeneic mice, resulting in significantly reduced collagen-deposition and skin thickness (Figure 2). In contrast, VA-Lip HSP47 had no effects on collagen volumes in the skin of naïve mice. Finally, allogeneic recipients were injected with VA-Lip HSP47 thrice a week from day +21 to test if VA-Lip HSP47 could promote regression of established skin fibrosis in chronic GVHD. Skin thickness and collagen volume on day +42 were again reduced in allogeneic mice treated with VA-Lip HSP47 compared to allogeneic controls, indicating that VA-Lip HSP47 is a promising therapeutic agent for established skin fibrosis. Because VA-Lip HSP47 did not affect donor T-cell expansion in the spleen or systemic GVHD scores after allogeneic BMT, VA-Lip HSP47 ameliorated skin fibrosis independently of systemic immunosuppression.Conclusion: VA-Lip HSP47 abrogates HSP47 expression in pathogenic myofibroblasts in the skin and ameliorates skin fibrosis in chronic GVHD without affecting collagen synthesis in the normal skin. VA-Lip HSP47 is a safe and promising anti-fibrotic agent for skin fibrosis in chronic GVHD without inducing systemic immunosuppression. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacologic Co-Blockade of IFNγR and IL6R Pathways to Prevent and Treat GvHD

The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from an anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT.We have previously published that in vivo administration of JAK1/JAK2 inhibitors to murine allo-HSCT recipients of interferon gamma receptor deficient (IFNγR-/-) T cells results in 100% survival in a fully MHC-mismatched B6 to Balb/c allo-HSCT model (Choi et al., 2014, PLoS ONE). Since the infusion of IFNγR-/- T cells alone is associated with only ~70% survival, we hypothesize that JAK1/JAK2 inhibitors have either additional off-target effects or are inhibiting other non-IFNγR signaling pathways which are themselves dependent on JAK1/JAK2. The major other cytokine receptor signaling pathway mediated via both JAK1 and JAK2 is the interleukin 6 receptor (IL6R) signaling pathway. Thus, it is possible that JAK1/JAK2 inhibitors also block signaling through IL6R in addition to IFNγR. In addition, Alam et al. have recently reported that single nucleotide polymorphisms of donor IFNγ and IL6 are closely linked to gastrointestinal GvHD in patients (2015, BMT). Therefore, we examined if blockade of both IFNγR and IL6R signaling results in complete elimination of GvHD after a fully MHC-mismatched allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with B6 pan T cells (5x105) are intravenously injected into lethally irradiated Balb/c mice (H-2d). As shown in Fig. 1, we have found that blocking both IFNγR (IFNγR-/- T cells) and IL6R (α-IL6R Ab) signaling dramatically reduces GvHD and results in >95% survival. In addition, we found that blocking both IFNγR and IL6R signaling significantly increased regulatory T cells (Tregs) in peripheral blood (23.2% Foxp3+ in CD4+ T cells (n=17) vs 2.5% in WT T cell control (n=16) at day 27 after allo-HSCT), suggesting that increase in Tregs might be a potential mechanism underlying the reduced GvHD after dual blockade of IFNγR and IL6R signaling.Baricitinib is a potent and balanced JAK1/JAK2 inhibitor currently being clinically developed by Eli Lilly for the treatment of inflammatory diseases. We hypothesize that baricitinib will optimally block both IFNγR and IL6R signaling pathways and prevent GvHD. We found that that baricitinib is a potent suppressor of GvHD in B6 to Balb/c allo-HSCT models (100% survival), superior to ruxolitinib and similar to blockade of both IFNγR and IL6R signaling (Fig. 2A). Baricitinib increases Tregs in vivo (Fig. 2B) and reduces the ratio of IL5 (Th2 cytokine) to IL2 (cytokine for Treg induction) in plasma (p=0.0046), a potential diagnostic marker for GvHD (Fujii et al., 2006, Int J Mol Med), significantly better than ruxolitinib. Lastly, we found that baricitinib inhibits the expression of T-bet (Fig. 2C), which is the master transcription factor of Th1 cells, that are primary effector T cells in inducing GvHD. These data suggest that the suppression of GvHD by baricitinib results from increased Tregs and decreased Th1 and Th2 cells.We next examined if the prevention of GvHD by baricitinib is dependent on natural regulatory T cells (Tregs) in donor grafts. Tregs were depleted from donor pan T cells before allo-HSCT (B6 to Balb/c). We found that in vivo administration of baricitinib resulted in 70% survival (0% control, p<0.0001; 100% Treg-replete T cells + baricitinib. In addition, based on clinical GvHD score when recipients of Treg-replete T cells were compared with those of Treg-deplete T cells, the beneficial effect of Tregs in the donor grafts for the prevention of GvHD was observed only for the first two weeks after allo-HSCT (p≤0.01).Lastly, we examined whether baricitinib can cure ongoing GvHD by administering baricitinib starting at day 10 after allo-HSCT when GvHD is established (B6 to Balb/c). We found that baricitinib treatment results in a significant reduction of GvHD and 100% survival (10% control, p<0.0001).All of these data suggest that pharmacologic co-blockade of IFNγR and IL6R pathways is a promising therapeutic strategy to prevent and effectively treat established GvHD. The inhibitory effect of baricitinib, ruxolitinib, and blockade of IFNγR and IL6R on JAK-STAT signaling using JAK/STAT phosphorylation antibody arrays is currently being investigated and will be presented. [Display omitted] [Display omitted] DisclosuresDiPersio:Incyte Corporation: Research Funding.

The Chemerin/ChemR23 Axis Plays a Pivotal Role in the Pathogenesis of Intestinal Damage in a Murine Model of Graft Versus Host Disease

Allogeneic haematopoietic-stem-cell transplantation (HSCT) is the treatment of choice for many malignant and non-malignant disorders. However, Graft-versus -Host Disease (GvHD), the major complication of allogeneic HSCT, limits its wider application. Among different sites that can be involved, gastrointestinal GvHD represents the major cause of patients morbidity and mortality.The infiltration of different cell subsets into target organs is an important step in GvHD pathogenesis, and the modulation of cell trafficking could represent a promising strategy for GvHD prophylaxis and treatment. Chemerin has been recently identified as a chemotactic protein, which is produced by several tissues during inflammation and binds the G protein-coupled receptor ChemR23, expressed by immature myeloid and plasmacytoid Dendritic Cells, macrophages and natural killer cells.The aim of this study was to evaluate the potential role of Chemerin/ChemR23 axis in the pathogenesis of GvHD, in order to identify disease-specific pathways exploitable for developing new potential therapeutic targets. For this purpose, lethally irradiated Balb/C recipient mice were transplanted with bone marrow cells and splenocytes obtained from ChemR23deficient (ChemR23-/-) C57BL6 mice. After transplantation, mice were monitored daily for survival and GvHD severity. Recipient mice were sacrificed at different time points to evaluate Chemerin production and leukocytes infiltration in skin, lung, liver, and gut by using different techniques, such as ELISA, histopathology, FACS and PCR.Starting from day +6 after transplantation, Chemerin plasma levels appeared significantly higher in both wild type (WT) and ChemR23-/- mice who developed GvHD (WT mean level=86.8 ng/ml; range=80.1-91.9; ChemR23-/- mean level=83.8 ng/ml; range=70.5-103.6, n=6, day+7) compared to syngeneic controls (mean level=62.8 ng/ml; range=49.2-82.5, n=6, day+7), p=0.02. Interestingly, ChemR23-/- mice developed a more severe GvHD compared to mice transplanted with WT cells. In particular, ChemR23-/- transplantedmice showed a higher mortality rate (at day +28 after HSCT: 85% mortality in ChemR23-/- vs 25% in WT, p=0.0004, n=45) (Fig. 1). Differences in GvHD score between ChemR23-/- and WT transplanted mice resulted by a significantly increase in weight loss, associated to severe diarrhea. In accordance, histopathologic analysis performed on GvHD target organs showed a significantly higher GvHD score in large intestine of ChemR23-/- transplanted mice, whereas no differences were found in other GvHD target organs. In addition, a deeper histological analysis on large intestine showed that tissue damage is characterized by crypt hyperplasia and atrophy, epithelium apoptosis and colitis. Moreover, FACS analysis of large intestine infiltrating leukocytes showed an higher neutrophils frequency in ChemR23-/- transplantedmice compared to WT (neutrophils=10.39%, range 6.5%-15.1% versus neutrophils=5.84%, range 2.6%-12.8%, respectively, p=0.001, n=10). This observation was also obtained by analyzing the mesenteric lymphnode. The higher neutrophils infiltration was also confirmed by immunohistochemistry evaluating the number of myeloperoxidase (MPO) positive cells and by quantitative PCR detecting MPO expression (ChemR23-/- mean 2-ΔΔCt =22.35, range 2.43-46.05; WT mean 2-ΔΔCt =6.42, range 0.20-19.23; p=0.04, n=8).All these findings suggest that the Chemerin/ChemR23 axis play a crucial role in intestinal GVHD. Further studies are needed to better understand the mechanisms underling the severe damage observed in the gastrointestinal tract of mice transplanted with ChemR23-/- cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

Intermediate Filament (IF) Protein Vimentin Regulates T Cell Mediated Immune Response in Gvhd

Graft-versus-host-disease (GVHD) is an alloimmune response complicating allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T lymphocytes and recipient antigen presenting cells (APCs) are the primarily mediators of GVHD, the molecular and cellular basis are not well understood. Recent published studies investigated T cell migration and homing in GVHD, especially effects of adhesion molecules including chemokines and integrins. Intermediate filaments (IFs) are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support. As the most abundant IF protein and the only known IF protein in leukocytes, vimentin plays an important role in stabilizing intracellular architecture, maintaining cellular integrity and providing resistance against stress. Vimentin-deficient (Vim-/-) mice are viable with impaired wound healing, and defects in PMN and lymphocyte adhesion to endothelial cells. In addition, Vimentin has been reported to be involved in colitis, Crohn's disease and allograft rejection. Herein, we report that vimentin regulates the organization of proteins in cell adhesion, migration and signaling, all of which are important for T cell activation in GVHD.Using homotypic aggregation assay, we found there was significantly reduced aggregation of primary T cells isolated from Vim-/- mice compared to WT control. However, the expression of both LFA-1 and ICAM-1 are similar between WT and Vim-/- T cells, thus the reduced T cell adhesion is not attributed to LFA-1 expression. We further investigated whether vimentin regulates mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and demonstrated there was a significant reduction of both CD4+ and CD8+ T cell proliferation in the absence of vimentin (Vim-/-) compared to WT control. Moreover, the frequency of IFN-γ (Th1) and IL-17 (Th17) producing CD4+ cells was significantly reduced in T cells isolated from Vim-/- mice compared to WT control. To investigate the role of vimentin in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells, and found vimentin was enriched proximal to the membrane and associated with the prominent CD3/LFA-1 cluster upon TCR stimulation. In the control without any TCR stimulation, the predominant pattern was different with vimentin evenly distributed beneath the cell membrane. In the absence of vimentin (Vim-/-), immune synapse failed to form in mouse CD8+ T cells upon TCR stimulation. The data demonstrate that vimentin can regulate immunological synapse function and signal transduction in T cell activation and proliferation.To determine whether vimentin expression in T cells plays a role in GVHD, the MHC class I and II disparate model, C57BL/6 (H-2b) to BALB/c (H-2d) transplantation, was used to establish GVHD. T cells from WT or Vim-/- (C57BL/6 background) mice were used as donors and Balb/c mice as recipients. Irradiated BALB/c mice received 5x106 T cell-depleted bone marrow cells (WT) and 10×106 T cells (WT or Vim-/-). In comparison to WT control, mice received Vim-/- T cells showed a lower mortality rate. Within 8 weeks post-transplantation, about 65% of mice received Vim-/- T cells survived, compared with only less than 10% of control mice received WT T cells (P=0.036; n=15 in each group). Control recipients had severe GVHD in the skin, intestine and liver. Mice received Vim-/- T cells exhibited only mild changes in these organs, reflected in their significantly lower GVHD scores. There were significantly reduced donor-derived CD4+ and CD8+ T cells in secondary lymphoid organs. Thus, the reduced homing and proliferation of Vim-/- T cells in vivo led to the reduced mortality and morbidity associated with GVHD.In summary, we have shown that there are significantly reduced T cell adhesion, proliferation and Th1/17 polarization in Vim-/- T cells, and vimentin participates in TCR clustering and immunological synapse formation in CD8+ T cells. Furthermore, vimentin plays an important role in GVHD through regulating donor T cell adhesion, proliferation and activation. Our data will not only significantly advance our knowledge of GVHD, but also define a new function of vimentin and IF proteins in health and diseases, and thus provide a rationale for using vimentin inhibitors as potential novel therapeutic interventions for GVHD. DisclosuresNo relevant conflicts of interest to declare.

Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo

Background aimsHuman decidual stromal cells (hDSCs) may cure acute graft-versus-host disease (GVHD) in humans. We evaluated immunosuppression by xenogenic hDSCs in mice, both in vitro and in vivo. MethodshDSCs inhibited mouse lymphocyte proliferation in allo- and xeno-stimulation assays in mixed lymphocyte culture (MLC) and after mitogenic stimulation. The immunosuppressive effect of hDSCs was dose-dependent and strain-independent. Trans-well experiments showed that hDSCs needed cell-to-cell contact to be immunosuppressive. In a GVHD model, Balb/c mice were transplanted with bone marrow and splenocytes from C57BL/6 a donor. Varying doses of hDSCs (105–106 per mouse) were infused at different time points. Recipient mice showed lower GVHD scores than untreated mice, but they did not have consistently improved survival. Histopathological investigation of liver, gastrointestinal tract and skin of animals with GVHD did not show any significant improvement from hDSC infusion. ResultshDSCs were transduced with immunosuppressive genes including those encoding interleukin-10, prostaglandin-E2 receptor, indoleamine dioxygenase, interferon-γ and programmed death ligand-1. Transduced and untransduced hDSCs showed similar effects in vitro and in vivo. At a dose of 106hDSCs per mouse, the majority of recipients died of embolism. ConclusionshDSCs inhibit allo-reactivity, xeno-reactivity and mitogen-induced stimulation in mouse lymphocytes. Although the GVHD score was reduced by hDSC infusion, survival and GVHD histopathology were not improved. One reason for failure was fatal embolism.

Correlation Between Tear Film Osmolarity and the Disease Score of the International Chronic Ocular Graft-Versus-Host-Disease Consensus Group in Hematopoietic Stem Cell Transplantation Patients.

To evaluate tear film osmolarity (TFO) as a diagnostic tool for detecting chronic ocular graft-versus-host disease (GVHD) in patients after hematopoietic stem cell transplantation and to assess its correlation with the new international chronic ocular GVHD score. A group of 204 consecutive patients who underwent hematopoietic stem cell transplantation at University Hospital Wuerzburg in Germany received an ophthalmologic examination after transplantation. TFO was measured and the chronic ocular GVHD score was calculated based on the Schirmer test, corneal fluorescein staining, conjunctival injection, Ocular Surface Disease Index questionnaire, and presence of systemic GVHD. A total of 172 patients showed no chronic ocular GVHD. Of the remaining 32 patients using the international chronic ocular GVHD score, 21 were classified as "probably" and 11 as "definite" chronic ocular GVHD. TFO was positively correlated with the new chronic ocular GVHD score (P < 0.01, r = 0.35). TFO differed significantly between patients with no ocular GVHD (300 ± 16.5 mOsm/L) and definite ocular GVHD (337 ± 36 mOsm/L)-a receiver operating characteristic analysis showed high discrimination capability (area under the curve: 0.91 ± 0.04) and suggested a threshold level of the TFO value of 312 mOsm/L yielding a sensitivity of 91% and a specificity of 82%. TFO can be used for detecting chronic ocular GVHD with high sensitivity and specificity as a noninvasive objective test in addition to traditional dry eye tests. It correlates positively with the diagnostic criteria of a recently established international consensus score for diagnosing the disease.

Ex Vivo Induced Regulatory Human/Murine Mesenchymal Stem Cells as Immune Modulators.

Over the past decade there has been a growing interest in using mesenchymal stem cells (MSCs) as an immune-regulatory agent for prevention and treatment of various immune disorders including graft-versus-host disease (GVHD), transplanted organ rejection, and autoimmune diseases. However, the high diversity in the results from clinical trials using MSCs for such disorders emphasizes the need for MSCs to be "professionalized" ex vivo to a more defined regulatory phenotype before administering to patients. To this aim, we have established an ex vivo immunomodulatory triple combination treatment (TCT) for MSCs, using IFNγ, TGFβ, and kynurenine. We show that pretreated MSCs acquire an immunomodulatory phenotype, have improved regulatory functions, and upregulate the expression of inducible nitric oxide synthase, indoleamine 2,3-dioxygenase, cyclooxygenase-2 (COX2), heme oxygenase 1, leukemia inhibitory factor (LIF), and programmed death ligand 1. We define the pathway of kynurenine induced aryl hydrocarbon receptor activation in MSCs and how it contributes to the upregulation of COX2 expression and IL-6 downregulation. The combination of reduced IL-6 secretion with enhanced LIF expression leads to the inhibition of Th17 differentiation in coculture of TCT MSCs and lymphocytes. To test the immunomodulatory function of TCT MSCs in vivo, we used the cells as GVHD prophylaxis in a GVHD mouse model. TCT MSCs administration significantly decreased GVHD score and improved mouse survival. Importantly, single administration could attenuate disease symptoms for more than 3 weeks. Based on these results, we suggest considering TCT MSCs as an improved cell therapy for systemic diseases with an underlying inflammatory and immunologic etiology. Stem Cells 2015;33:2256-2267.

Basophils inhibit proliferation of CD4+T cells in autologous and allogeneic mixed lymphocyte reactions and limit disease activity in a murine model of graft versus host disease

Basophils are known to modulate the phenotype of CD4(+) T cells and to enhance T helper type 2 responses in vitro and in vivo. In this study, we demonstrate that murine basophils inhibit proliferation of CD4(+) T cells in autologous and allogeneic mixed lymphocyte reactions. The inhibition is independent of Fas and MHC class II, but dependent on activation of basophils with subsequent release of interleukin-4 (IL-4) and IL-6. The inhibitory effect of basophils on T-cell proliferation can be blocked with antibodies against IL-4 and IL-6 and is absent in IL-4/IL-6 double-deficient mice. In addition, we show that basophils and IL-4 have beneficial effects on disease activity in a murine model of acute graft-versus-host disease (GvHD). When basophils were depleted with the antibody MAR-1 before induction of GvHD, weight loss, GvHD score, mortality and plasma tumour necrosis factor levels were increased while injection of IL-4 improved GvHD. Basophil-depleted mice with GvHD also have increased numbers of CD4(+) T cells in the mesenteric lymph nodes. Our data show for the first time that basophils suppress autologous and allogeneic CD4(+) T-cell proliferation in an IL-4-dependent manner.

Immunosuppressive mechanisms of human bone marrow derived mesenchymal stromal cells in BALB/c host graft versus host disease murine models.

BackgroundMesenchymal stromal cells (MSCs) are proven to have immunosuppressive functions via various mechanisms. These mechanisms were demonstrated by administering bone marrow derived human MSCs (hMSCs) to graft versus host disease (GVHD) murine models.MethodsBALB/c host mice were irradiated prior to receiving C57BL/6 donor T cell depleted bone marrow (TCDBM) cells (negative control) and donor CD4+ T lymphocyte with (treatment group) or without hMSCs (positive control). The presence of hMSCs in target tissues and lymphoid organs was documented by using in vivo imaging and measuring the expression of EphB2 and ephrin-B2 by RTqPCR. Survival rate and GVHD score were also monitored. Tissue sections were obtained for histopathologic analysis. Flow cytometry was used to document donor T cell alloreactivity and expression of CCR5, CXCR3 and CCR7. ELISA was utilized to determine levels of proinflammatory cytokines, RANTES (CCL5) and phosphorylated STAT 5A/B. RTqPCR was performed to quantify expression of CCL3 and CXCL9. Western blotting was performed to qualitatively measure iNOS expression.ResultsSurvival rate and GVHD score improved with hMSC treatment. Pathologic changes of GVHD were abrogated. Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs. Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.ConclusionsThese mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-015-0007-0) contains supplementary material, which is available to authorized users.

Prophylaxis of acute graft-versus-host disease by CCR5 blockade combined with cyclosporine A in a murine model.

One characteristic feature of graft-versus-host disease (GVHD) is lymphocytes' trafficking and recruitment to target tissues, and CCR5 plays a key role in the process. Thus, blockade of lymphocytes' chemotaxis may attenuate GVHD. We tested the effects of CCR5 blockade using an established murine model. The mean survival time, body weight change, and clinical GVHD scores were assessed. Concentrations of cytokines and chemokines, the CCR5, CXCR3, and CCR7 expressions on T lymphocytes, and histological changes of visceral organs were also evaluated. Additionally, we assessed the immunophenotype of infiltration cells in liver and intestine. Mice undergoing total body irradiation and allogenic hematopoietic stem cell transplantation (allo-HSCT) developed typical GVHD. MVC increased CCR5 expression whereas CCR7 and CXCR3 expression were unaffected. MVC also increased plasma levels of the ligands of CCR5. A combination of MVC with CsA significantly alleviated the degree of visceral injuries and prolonged survival time. MVC has a synergistic effect with CsA. It can attenuate the severity of GVHD and increase survival rate of mice in our murine model. This may offer a novel therapeutic perspective for clinical GVHD after allo-HSCT.

Suppression of lentivirus-mediated transgenic dendritic cells in graft-versus-host disease after allogeneic bone marrow transplantation in mice.

We determined whether genetically engineered immature dendritic cells (imDCs) mediated by lentiviral vectors alleviate acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) in mice. We introduced the mouse chemokine receptor 7 (Ccr7) gene into the bone marrow-derived imDCs of C57BL/6 mice to construct genetically engineered imDCs. A 1:1 mixture of bone marrow and spleen cells from the donors was injected into the recipients, which were divided into four groups: radiation, transplantation, empty vector, and transgenic imDC groups. Symptoms, clinical scores, GVHD pathological changes, and survival times and rates of recipients were recorded; secretion of IFN-γ and IL-4, and allogeneic chimerism rates were detected. The survival time of the transgenic imDC group (27.5 ± 7.55 days) was significantly longer than in the other three groups (P < 0.01). The GVHD score of the imDC group mice was significantly lower than in the transplantation and empty vector groups (P < 0.05), which meant that mice in the transgenic imDC group had the lightest pathology damage in the target organs. In the transplantation group, IFN-γ increased while IL-4 decreased. In contrast, IFN-γ decreased and IL-4 increased in both empty vector and trans-imDC groups, and the difference was significant in the latter (P < 0.01). Thirty days or more following transplantation, the allogeneic chimerism rate was still 95-100%, suggesting complete donor type implantation. Ccr7 transfection into imDCs suppressed occurrence and severity of acute GVHD after allo-BMT in mice; the mechanism might be associated with IFN-γ decrease and IL-4 increase.

A prognostic score for acute graft-versus-host disease based on biomarkers: a multicentre study

Graft-versus-host disease (GVHD) is the major cause of non-relapse mortality after allogeneic haemopoietic stem-cell transplantation (SCT). The severity of symptoms at the onset of GVHD does not accurately define risk, and thus most patients are treated alike with high dose systemic corticosteroids. We aimed to define clinically meaningful risk strata for patients with newly diagnosed acute GVHD using plasma biomarkers. Between April 13, 2000, and May 7, 2013, we prospectively collected plasma from 492 SCT patients with newly diagnosed acute GVHD and randomly assigned (2:1) using a random number generator, conditional on the final two datasets having the same median day of onset, into training (n=328) and test (n=164) sets. We used the concentrations of three recently validated biomarkers (TNFR1, ST2, and Reg3α) to create an algorithm that computed the probability of non-relapse mortality 6 months after GVHD onset for individual patients in the training set alone. We rank ordered the probabilities and identified thresholds that created three distinct non-relapse mortality scores. We evaluated the algorithm in the test set, and again in an independent validation set of an additional 300 patients who underwent stem cell transplant and were enrolled on multicentre clinical trials of primary therapy for acute GVHD. In all three datasets (training, test, and validation), the cumulative incidence of 6-month non-relapse mortality significantly increased as the Ann Arbor GVHD score increased. In the multicentre validation set, scores were 8% (95% CI 3-16) for score 1, 27% (20-34) for score 2, and 46% (33-58) for score 3 (p<0·0001). Conversely, the response to primary GVHD treatment within 28 days decreased as the GVHD score increased 86% for score 1, 67% for score 2, and 46% for score 3 in the multicentre validation set, p<0·0001). Biomarker-based scores can be used to guide risk-adapted therapy at the onset of acute GVHD. High risk patients with a score of 3 are candidates for intensive primary therapy, while low risk patients with a score of 1 are candidates for rapid tapers of systemic steroid therapy. The National Cancer Institute, the National Heart, Lung, and Blood Institute, the National Institute of Allergy and Infectious Diseases, the Doris Duke Charitable Fund, the American Cancer Society, and the Judith Devries Fund.

Suppression of graft-versus-host disease and retention of graft-versus-tumour reaction by murine genetically engineered dendritic cells following bone marrow transplantation.

The effect of infusion of lentiviral vector‑mediated, genetically engineered dendritic cells (DCs) following allogeneic bone marrow transplantation (allo‑BMT) on graft‑versus‑host disease (GVHD) and graft‑versus‑leukemia (GVL) was investigated in a mouse model. Lentivirus‑mediated expression of soluble tumor necrosis factor receptor 1 (sTNFR1) converted immature DCs (imDCs) from BABL/c mice into engineered DCs in vitro. An EL4 leukemia allo‑BMT model of BABL/c to C57BL/6 mice was established. Engineered DCs with donor bone marrow cells and splenocytes were subsequently transplanted into myeloablatively irradiated recipients. The average survival duration in the sTNFR1‑ and pXZ9‑imDC groups was significantly prolonged compared with that of the allo‑BMT group (P<0.05). Mild histological changes in GVHD or leukemia were observed in the recipients in the sTNFR1‑imDC group and clinical GVHD scores in this group were significantly decreased compared with those of the transplantation and pXZ9‑imDC groups. Serum interferon‑γ levels were decreased in the pXZ9‑imDC and sTNFR1‑imDC groups compared with those in the allo‑BMT group (P<0.05), with the reduction being more significant in the sTNFR1‑imDC group (P<0.05). Serum interleukin‑4 expression levels were decreased in the allo‑BMT group, but gradually increased in the pXZ9‑imDC and sTNFR1‑imDC groups (P<0.05). Co‑injection of donor genetically‑engineered imDCs was able to efficiently protect recipient mice from lethal GVHD while preserving GVL effects during allo‑BMT.