Course Of GVHD Research Articles

Safety and Efficacy of Extracorporeal Photopheresis for Acute and Chronic Graft-Versus-Host-Disease

Introduction Graft-versus-host disease(GVHD) is a severe complication of allogeneic hematopoietic cell transplantation(alloHCT), directly linked to increased morbidity and mortality. Despite novel biologic agents under study or commercial use in this field, corticosteroids and conventional intensified immunosuppressive therapy remain the cornerstone of treatment. Extracorporeal photopheresis (ECP) has been used as an alternative treatment. We studied the safety and efficacy of ECP in a large real-world cohort of GVHD patients. Methods We enrolled consecutive patients, of our JACIE-accredited center, that received ECP post alloHCT over the last 2 decades for cGVHD and over the last decade for steroid-dependent or refractory grade II-IV aGVHD. All patients with unrelated or haploidentical donors received thymoglobulin (ATG) 5mg/kg as prophylaxis. GVHD prophylaxis included cyclosporine-methotrexate in myeloablative and cyclosporine-MMF in reduced toxicity/intensity regimens. Before ruxolitinib or ibrutinib availability, MMF, cyclosporine or ATG were commenced as second line treatment in steroid-refractory patients, depending on previous prophylaxis. ECP was mostly administered as third line treatment for cGVHD and after assessment of response of 5 days of steroid treatment according to our protocol: 1 session/week for the 1 st month for cGVHD and 2sessions/week for aGVHD, 1 session/2 weeks for 3 months, evaluation of response and 1 session/month for 6 months. Before Ruxolitinib's availability, ATG was commenced simultaneously with ECP initiation in steroid-refractory aGVHD patients. Results We studied 112 patients with moderate or severe cGVHD. Only 13 received 4 or less ECP sessions because of severe GVHD-related morbidity and were excluded from further analysis. Median ECP sessions were 17(6-49). There were no ECP-related adverse events. 67 patients presented with cutaneous sclerosis manifestations, 73 mucocutaneous disease, 36 liver, 42 visceral and 27 lung involvement. ECP was commenced as second line in 35 patients. Ruxolitinib was administered in combination with ECP in 19 patients, while ibrutinib in 2. Bacterial infections were observed in 43 patients, viral in 38 and fungal in 11 patients. Only 19 patients did not show response. Significantly lower rates of response presented in patients with visceral involvement(p=0.037) and earlier post-transplant GVHD diagnosis (p=0.001). With a follow-up of 45.2 (5.6-345.1) months, 5-year CI of cGVHD-related mortality was 21.2% and was significantly reduced in patients with ECP response (p<0.001). 5-year OS was 65.3% and was independently associated with HLA matching (p=0.011), higher number of ECP sessions(p<0.001), later initiation of ECP (p=0.002), response to ECP (p=0.036) and no relapse(p=0.001). Regarding aGVHD, we studied 28 patients, aged 45 (18-67), with myeloablative (16), reduced toxicity (8) and intensity (4) conditioning, from sibling (4), matched (11) or one locus mismatched (12) volunteer unrelated and haploidentical (1) donors. Disease risk index was very high (1), high (11), intermediate (14) and low (2). aGVHD was observed at day +17(8-50). Skin, intestine and liver involvement was evident in 9 patients, skin and intestine in 13 and skin only in 6 patients. 13 patients were steroid-dependent and 15 steroid-refractory. ECP was commenced at day +18 (8-56) for 15 (4-20) sessions. The majority of patients (19/27) presented partial (7), very good (11) or complete (1) response. With 9.9 (1.7-113) months of follow-up, immunosuppression was reduced in 12/27 and ceased in 1 patient. Clinically significant bacterial infections were found in 19 patients, fungal in 3, CMV and EBV reactivation in 19 and 12 respectively and other viral in 6 patients. Cumulative incidence of aGVHD was 56.4 at 1-year.Five-year overall survival (OS) was 34%. Reduction of immunosuppression(p=0.026) and number of ECP sessions(p<0.001) were associated with improved OS, irrespectively of other factors. In particular, optimal OS was observed in patients that received more than 19 ECP sessions (Figure). Conclusion Our data confirm that ECP is safe and effective for GVHD. Even in the era of novel biologics, ECP should be considered early in the course of GVHD, before irreversible end organ damage has been established. Combination with other treatments and individualized treatment algorithms remain important unanswered questions.

IL-35 and Rapamycin Reduce Acute Graft-Versus-Host Disease Associated with Decreased Platelet Aggregation in a Mouse Model

▪Introduction: Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) caused by the activation of donor T lymphocytes by host antigen-presenting cells and the immune-mediated inflammatory response. Epithelial cells of the skin and mucous membranes, biliary ducts, and intestinal tract crypts are the primary tissue systems damaged during the pathobiological course of GVHD. IL-35, a member of the IL-12 family of cytokines, comprising an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). It is an anti-inflammatory cytokine that suppresses the immune response through the expansion of regulatory T cells and suppression of Th17 cell development (Niedbala W, et al. European journal of immunology 2007). Rapamycin (Sirolimus; RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, has been used for the prophylaxis and treatment of several immune reactions including GVHD and solid organ rejection (Ho-Jin Shin, et al. Blood 2011). We hypothesized that IL-35 has a protective effect in aGVHD, and that its function may be increased by RAPA.Methods: We used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2)F1 (BDF1, H-2b×d) mice as recipients to create an aGVHD model (Kuroiwa T, et al. The Journal of clinical investigation 2001). Mice were divided into five groups, including a BMT control group, aGVHD control group, aGVHD treated with IL-35 group, aGVHD treated with RAPA group and aGVHD treated with IL-35 and RAPA group. Morbidity and mortality related to aGVHD were observed, and 2 weeks after BMT, tissues from the intestine and liver were stained with hematoxylin and eosin and examined by light microscopy. To detect apoptosis in intestinal sections, a modified terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method was applied. CD4+CD25+Foxp3+ regulatory T cells were measured by flow cytometry. Quantitative RT-PCR was used to measure the production of IFN-γ, TNF-α and IL-17A in the spleen and intestine of each group of mice. We also measured platelet aggregation using a turbidimetric aggregation-monitoring device. Finally, western blotting was conducted to test the signaling pathways of IL-35.Results: Mice receivingIL-35 exhibited a higher survival rate compared with GVHD mice as well as those mice receiving RAPA. When the two drugs were given together, the survival rate was much higher than that in the other groups. The aGVHD control group had the highest morbidity rate of aGVHD, and IL-35 plus RAPA could prevent the occurrence of aGVHD. Additionally, this treatment inhibited apoptosis of intestinal epithelial cells as well as donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by aGVHD. The importance of the inflammatory cytokine cascade in the pathogenesis of both clinical and experimental GVHD is now well accepted. We found that IL-35 and RAPA also markedly suppressed IFN-γ, TNF-α and IL-17A expression in the intestine and liver. Because studies by other have showed that Tregs have the ability to inhibit aGVHD, we measured Tregs in serum and found that IL-35 and RAPA treatment expanded serum Tregs. We further explored the relationship between IL-35 and platelet aggregation. Platelet aggregation was high in aGVHD mice, and the ratio of platelet aggregation was inhibited by IL-35 and RAPA. Finally, we found that the phosphorylation of STAT1 and STAT4 were inhibited in GVHD mice, and thatSTAT1 and STAT4 were phosphorylated when mice were treated with IL-35.Conclusions: IL-35 may be useful for controlling aGVHD after allo-HSCT. IL-35 suppresses inflammatory cytokines and expands anti-inflammatory cells in aGVHD. IL-35 also prevents platelet aggregation in aGVHD mice, which could be helpful in treating thrombotic complications after HSCT. These results are readily translatable to the clinic in future clinical trials. IL-35 and RAPA may have potential clinical use for the prevention or treatment of aGVHD and thrombotic complications after HSCT. DisclosuresNo relevant conflicts of interest to declare.

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.

Shift of Graft-Versus-Host-Disease Target Organ Tropism by Dietary Vitamin A

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4β7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4β7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ+CD4+ and low FoxP3+CD4+ frequencies of total donor CD4+ T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT.

Tryptophan catabolism is associated with acute GVHD after human allogeneic stem cell transplantation and indicates activation of indoleamine 2,3-dioxygenase

AbstractInduction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

Endothelial Stress in Steroid-Refractory Gvhd: A Burden Statins Cannot Lift

Abstract 1012Graft-versus-host disease (GvHD) is the major complication of allogeneic stem cell transplantation (alloSCT) and its therapy-resistant form causes significant morbidity and mortality. The pathomechanism of steroid resistance is currently not completely understood; however, we have recently suggested that endothelial dysfunction seems to play an important role (Luft et al., Blood 2011). The aim of this study was to validate in a large cohort of patients that steroid refractory acute GvHD is associated with endothelial stress. Secondly we assessed if this endothelial stress can be overcome by statins, which are known to have endothelial protective effects.We have therefore performed a retrospective study of 393 patients who underwent alloSCT in our institution between 2002–2010. Endothelial stress serum markers were correlated with the course of GvHD, non relapse mortality (NRM) and statins intake. Overall, 169 of 393 patients (43%) developed GVHD. Of these, 101 patients (60%) had steroid-sensitive GvHD grade 1–2, 26 patients (15%) steroid-sensitive GvHD grade 3–4 and 42 patients (25%) had steroid-refractory GvHD which was always grade 3–4 at its maximum severity. 224 patients had no signs of GvHD. In order to elucidate the pathomechanisms that contribute to steroid resistance, serum markers of endothelial stress and vascular growth (Angiopoietin-2 (ANG2), IL-8, soluble thrombomodulin (sTM), and vascular endothelial growth factor (VEGF)) were measured by ELISA and multiplex assays before and on days +50 and +100 after alloSCT. RESULTS:Landmark analyses at days +50 and +100 after alloSCT showed that NRM was dramatically high in the steroid refractory group but was equivalently low in the no GvHD-, sensitive grade 1–2 - and grade 3–4 - groups (p<0.001, Figure 1). ANG2, a mediator of endothelial cell vulnerability, measured before transplant was predictive for a steroid refractory course of GvHD (p=0.05) and higher NRM (p=0.06) under the prerequisite that patients developed grade 3–4 GvHD. Moreover, patients who developed an acute steroid refractory GvHD displayed significantly higher levels of the endothelial stress markers IL-8 (day +50: p<0.001; day +100: p<0.001) and sTM (day +50: p<0.009; day +100: p=0.003) compared to steroid-sensitive GvHD patients and patients with no GvHD. High levels of sTM, IL8 and ANG2 were significantly associated with increased NRM rates (day +50: IL8 p=0.02, sTM p=0.0008, ANG2 p=0.0001; day +100: IL8 p=0.0007, sTM p<0.0001, ANG2 p=0.05); even after multivariate adjustment for donor, conditioning intensity, disease status at alloSCT and sex mismatch. In contrast, no influence of VEGF levels on steroid-refractory GvHD. [Display omitted] When IL8, sTM, ANG2 and VEGF kinetics were compared in patients with and without concomitant statin therapy (n=81), no differences were observed in the whole patient cohort nor in the subset with steroid-refractory GVHD. Consequently, patients with or without statins had similar NRM, relapse rates and overall survival in univariate as well as multivariate analysis. CONCLUSION:This supports the hypothesis that steroid-refractory GVHD is associated with progressive microangiopathy. Statins, although reported to have protective effects on endothelial cells, were inefficient to alleviate endothelial stress in this context and, accordingly, did not change the outcome of acute GVHD patients. Disclosures:Luft:Celgene: Research Funding.

The importance of neovascularization and its inhibition for allogeneic hematopoietic stem cell transplantation

AbstractGVHD and tumor relapse are fundamental problems in allogeneic HSCT. Recent research has linked neovascularization to GVHD, tumor growth, and graft-versus-tumor (GVT) activity. Damage of the endothelium by the conditioning regimen provides the initiation stimulus for recruitment of donor-derived endothelial cells and their progenitors. During the early inflammatory phase of GVHD there is considerable neovascularization facilitating migration of inflammatory cells to target organs. In the course of GVHD, however, the vasculature itself becomes a target of alloreactive donor T cells. As a consequence, later stages of GVHD are characterized by fibrosis and rarefaction of blood vessels. Importantly, the inhibition of tumor-neovascularization by activated donor T cells that release antiangiogenic substances contributes to GVT and may be enhanced by pharmacologic inhibition of neovascularization. Furthermore, the therapeutic inhibition of neovascularization may improve immunotherapy for cancer by enhancing leukocyte infiltration in tumor tissue because of normalization of tumor vessels and stimulation of leukocyte–vessel wall interactions. These insights identify important mechanisms underlining the importance of neovascularization for allogeneic immune responses and move therapeutic approaches targeting neovascularization into the spotlight. This perspective covers current knowledge of the role of neovascularization during GVHD as well as GVT and its implications for HSCT.

Blockade of osteopontin reduces alloreactive CD8+ T cell–mediated graft-versus-host disease

AbstractGraft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation, is caused by alloreactive donor T cells that trigger host tissue damage. The inflammatory environment inside recipients is critical for GVHD pathogenesis, but the underpinning mechanisms remain elusive. Using mouse model of human GVHD, we demonstrate osteopontin (OPN), a potent proinflammatory cytokine, plays an important role in regulating activation, migration, and survival of alloreactive T cells during GVHD. OPN was significantly elevated after irradiation and persisted throughout the course of GVHD. Blockade of OPN attenuated GVHD with reduced accumulation of donor T cells in recipient organs. Amelioration was the result of migration and survival suppression caused by anti-OPN treatment on donor-derived T cells for 2 reasons. First, OPN promoted the migration and infiltration of naive and alloreactive CD8+ T cells into host organs. Second, it also facilitated activation and viability of donor-derived CD8+ T cells via synergizing with T-cell receptor/CD3 signaling. Finally, anti-OPN treatment retained graft-versus-leukemia effect of alloreactive CD8+ T cells. This study demonstrates, to our knowledge for the first time, the critical effect of OPN in the initiation and persistence of CD8+ T cell-mediated GVHD and validates OPN as a potential target in GVHD prevention.

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.

Stability of Foxp3 Expression Is a Critical Factor In the Ability of Regulatory T Cells to Mitigate Graft Versus Host Disease

AbstractAbstract 731Graft versus host disease (GVHD) is a proinflammatory T cell-mediated syndrome that is the major complication of allogeneic bone marrow transplantation (BMT). During the course of GVHD, there is a progressive loss of regulatory T cells (Tregs), leading to an imbalance between the effector and regulatory arms of the immune system. Tregs have been subdivided into two distinct subsets, termed natural and induced, which have overlapping yet unique characteristics. While the role of natural regulatory T cells (nTregs) in GVHD biology has been extensively examined, the role of induced regulatory T cells (iTregs) remains largely unknown. An attractive aspect of the latter cell population is that they can be differentiated in vitro from conventional T cells and expanded in large numbers making them a potential source for regulatory T cell therapy in vivo. To determine whether in vitro-expanded iTregs were able to suppress alloreactive donor T cell responses and to compare the efficacy of these cells relative to nTregs, studies were performed using an MHC-incompatible murine BMT model (B6[H−2b]−Balb/c[H−2d]). In initial studies, purified CD4+ Foxp3EGFP– T cells obtained from B6 Foxp3EGFP reporter mice were cultured with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and TGF-b. After three days in culture, approximately 60–70% of cells were Foxp3+, expressed GITR, CD25, and CD103, and were equally suppressive to nTregs in mixed lymphocyte cultures. To determine if iTregs were suppressive in vivo, lethally irradiated Balb/c mice were transplanted with either B6 BM alone, B6 BM and spleen cells, or B6 BM/spleen cells and in vitro-expanded iTregs. In contrast to in vitro results, adoptive transfer of iTregs failed to protect mice from lethal GVHD even when administered at high Treg: effector T cell ratios (5:1) and were much less effective than equivalent doses of nTregs at abrogating GVHD pathology. iTregs also had no additive effect when co-administered with nTregs. Notably, we observed that whereas transferred nTregs persisted for up to 60 days in transplanted animals, iTregs were undetectable after only 14 days in liver, lung, colon and spleen, indicating that reduced in vivo survival was a potential explanation for the lack of protection. Further examination, however, revealed that the inability to detect iTregs was primarily attributable to the loss of Foxp3 expression and the subsequent in vivo reversion of these cells to a proinflammatory phenotype characterized by the secretion of interferon-gamma. In prior studies (Chen et al, Blood, 2009), we demonstrated that blockade of IL-6 signaling augmented reconstitution of nTregs and reduced overall GVHD severity. To determine whether inhibition of IL-6 could stabilize Foxp3 expression and prevent phenotypic reversion of iTregs, lethally irradiated Balb/c recipients were transplanted with B6 BM and spleen cells along with in vitro-differentiated iTregs and then treated with either isotype control or anti-IL-6R-specific antibody. Analysis of cells obtained from spleen, liver, lung and colon revealed that blockade of IL-6 signaling did not prevent loss of Foxp3 expression or reversion of iTregs to a Th1 cytokine phenotype. While Tregs can be converted from conventional T cells in vitro, they can also be generated in vivo during inflammatory syndromes. We therefore examined whether in vivo induction of iTregs occurred during GVHD and the extent to which blockade of IL-6 signaling affected iTreg expansion and overall GVHD protection. To address this question, lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4+ Foxp3EGFP– T cells, and then treated with either anti-IL-6R or control antibody. We observed that in vivo conversion of Tregs was negligible in control animals (<1%), but that administration of anti-IL-6R antibody significantly increased the relative and absolute number of iTregs in GVHD target tissues with a commensurate reduction in overall pathological damage. Thus, blockade of IL-6 signaling was able to enhance reconstitution of iTregs in vivo, but had no discernible affect on adoptively transferred iTregs. In summary, these studies demonstrate that the stability of Foxp3 expression is a critical factor in the maintenance of transplantation tolerance and that instability of expression limits the utility of adoptively transferred iTregs as a source of cellular therapy for the abrogation of GVHD. Disclosures:No relevant conflicts of interest to declare.

Graft-Versus-Host Disease Alters Intestinal Microbial Ecology by Inhibiting Production of Enteric Defensins

AbstractAbstract 244Bacterial infection is a serious complication of bone marrow transplantation (BMT). Intestinal GVHD, in particular, significantly enhances the risk for Gram-negative septicemia. The majority of the intestinal microbiome of the hosts consists of non-cultivable obligate anaerobes, while the Gram-negative bacteria such as Escherichia coli (E. coli) make up a small proportion of the microflora. Thus, it remains unclear why Gram-negative septicemia is dominant in intestinal GVHD, while the role of systemic immunosuppression and use of antibiotics is well-appreciated. We evaluated gut flora changes in the course of GVHD in mouse models of BMT without giving antibiotic or immunosuppresive drugs. Lethally irradiated B6D2F1 (H-2b/d) or B6C3F1 (H-2b/k) mice were injected with 5 × 106 T-cell depleted BM alone (non-GVHD controls) or with 2 × 106 T cells (GVHD group) from MHC-mismatched B6 (H-2b) donors on day 0. Intestinal microflora was identified by using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene libraries constructed from each sample of gut contents. It consisted of approximately 80% of obligate anaerobe, and 20% of anaerobe such as Lactobacilli and Clostridia with very few E. coli before BMT. After BMT, this diversity of gut flora was preserved in non-GVHD controls (Table). In contrast, GVHD mice showed a marked increase of E. coli with a significant decrease in the members of obligate anaerobe. This was associated with dissemination of E. coli to the mesenteric lymph nodes (mLNs) and liver, with elevated serum levels of lipopolysaccharide (LPS). Such a loss of diversity of gut flora with a flora shift towards E. coli was significantly associated with morbidity and mortality of GVHD. Numbers of T-RFLP peaks that indicate diversity of intestinal flora were inversely correlated with GVHD clinical scores (p<0.001). A degree of E. coli proportion has significant correlation with GVHD clinical scores (p<0.001) and GVHD mortality (80% in high E. coli group vs. 0% in low E. coli group). We then investigated the underlying mechanisms of the disruption of intestinal ecology in GVHD. Paneth cell derived α-defensins are essential regulators of intestinal microbial ecology. We therefore hypothesized that Paneth cell damage in GVHD inhibited production of enteric defensins and disrupted intestinal ecology. Immunohistochemistry for lysozyme that marks Paneth cells showed significant reduction of Paneth cells in GVHD and quantitative real-time PCR analysis showed dramatically reduced expression of enteric defensins including Defa21, Defa1, Defa4, Defa5, and Defcr-rs1 (Table). These results suggest that Paneth cell injury in GVHD could lead to lower expression of enteric defensins and a shift of gut flora from commensal microorganisms towards widespread prevelance of gram-negative bacteria in the intestinal microbiome, and the subsequent high risk for the development of life threatening Gram-negative septicemia. LPS derived from Gram-negative bacteria plays an important role in amplifying systemic GVHD. Thus, such an alteration of intestinal ecology may be related to exaggeration of systemic GVHD. These results thus uncover the previously unrecognized role of the intrinsic antimicrobial peptides and the crosstalk between hosts and the intestinal microbes in the pathogenesis of GVHD and infection after allogeneic BMT. Disclosures:No relevant conflicts of interest to declare.

Soluble Fas Levels in Sera of Bone Marrow Transplantation Recipients Are Increased During Acute Graft-Versus-Host Disease But Not During Infections

AbstractGraft-versus-host disease (GVHD) and infections are two major complications of allogeneic bone marrow transplantation (BMT). In the course of GVHD, one of the pathways that activated cytotoxic T cells use to execute their killing mechanisms is the Fas/Fas ligand pathway. This killing mechanism might be accompanied by the release of soluble Fas (sFas) in the circulation. To examine the association of serum sFas levels and post-BMT complications, we have analyzed sFas levels in sera of bone marrow recipients with and without GVHD. Postallogeneic BMT sFas levels were significantly increased during clinically relevant acute GVHD (aGVHD; P = .002). However, during infections sFas levels tended to decrease (P = .088). Yet, the simultaneous occurrence of GVHD and infections resulted in extreme high sFas levels. These results suggested that sFas release may be correlated with the amount of tissue damage, because aGVHD induces more damage than infections. The presence of significantly increased sFas levels during aGVHD provides new insights into the GVHD pathogenesis.

Enhanced graft-versus-host disease in older recipient mice following allogeneic bone marrow transplantation.

The incidence and severity of GVHD following bone marrow transplantation increases with recipient age. The role of recipient age on the development of GVHD was analyzed in a semi-allogeneic (C57BL/6-->(C57BL/6 x DBA/2)F1) murine GVHD system. Young adult (2 months) and old (12-14 months) recipient mice were lethally irradiated and reconstituted with young adult T cell-depleted bone marrow (ATBM) or ATBM and young spleen cells. A significantly higher percentage of old vs young recipients developed lethal GVHD. Furthermore, while pre-transplant conditioning with irradiation was not required to observe increased mortality in old recipients, irradiation predisposed the older animals for a more severe course of GVHD, suggesting that GVHD occurred in old compared to young animals in the absence of pre-transplant conditioning but was exacerbated by irradiation. Histologically, the immunological responses in the GVHD target organs were more severe in the old GVHD animals. In support of this observation, increased spontaneous proliferation was observed using lymphoid cells isolated from old vs young GVHD mice. These findings demonstrate that old recipients develop a more severe course of GVHD following BMT, and may present a unique opportunity to study age-related factors in the generation of GVHD.