ALBERT

Description: Objectives Fever during neutropenia occurs in 〉 90% and 80% of allogeneic and autologous hematopoietic stem cell transplantation (HSCT) recipients, respectively. Current guidelines recommend the prophylaxis with fluoroquinolones (FQs) in HSCT patients. Although there is evidence that antibiotic prophylaxis improve clinical outcome in patients with chemotherapy-induced neutropenia, prophylactic antibiotic therapy has not been thoroughly evaluated in HSCT recipients. Therefore, we performed a meta-analysis to evaluate the impact of systemic antibiotic prophylaxis in HSCT recipients on mortality, incidence of infection and related adverse events. Data sources We identified reports that were not restricted to those in English and not restricted to published trials through PubMed, the Cochrane Library, and references of identified studies. Review Methods We included prospective, randomized studies on systemic antibiotic prophylaxis in HSCT recipients. The outcome measures included the all-cause mortality, infection-related mortality, febrile episodes, incidence of clinically or microbiologically documented infection, bacteremia, or related adverse events. The summarized odds ratios (ORs) were calculated using the Mantel–Haenszel method and the DerSimonian–Laird method. Results Seventeen trials with 1453 patients (842 autologous and 407 allogeneic HSCT recipients) were included. The percentage of autologous and allogeneic HSCT recipients was not specified in 2 trials. Systemic antibiotic prophylaxis was compared with placebo or no prophylaxis in 10 trials and with non-absorbable antibiotic in 2 trials, respectively. Systemic antibiotics other than FQs were evaluated in five out of these 12 trials. Four trials evaluated the effect of addition of antibiotics for gram positive bacteria to FQs. Remaining 1 trial compared the two different systemic antibiotic regimens, FQs versus trimethoprim sulfamethoxazole. As a result, systemic antibiotic prophylaxis reduced the incidence of febrile episodes (OR 0.16; 95 percent confidence interval [CI], 0.09-0.30), clinically or microbiologically documented infection (OR 0.41; 95% CI 0.30-0.57) and bacteremia (OR 0.37; 95% CI 0.26-0.53) without the significant effect on all-cause mortality or infection-related mortality (OR 0.89; 95% CI 0.48-1.66, OR 1.37; 95% CI 0.50-3.76, respectively). Impact of prophylaxis with FQs on mortality was inconclusive because of small number of clinical trials evaluated. Adverse events increased in patients with systemic antibiotic prophylaxis compared to controls (OR 3.32; 95% CI 1.45-7.63). In meta-regression, percentage of allogeneic HSCT recipients was not associated with each outcome measure. With regard to the comparison between different prophylactic regimens, addition of antibiotics for gram positive bacteria to FQs decreased the incidence of bacteremia (OR 0.44; 0.24-0.80) without significant effects on all-cause mortality, infection related death and febrile episodes. There was not significant, but consistent decrease in clinically or microbiologically documented infection (OR 0.55; 95% CI 0.30-1.01). There was significant increase of adverse events in patients receiving addition of antibiotics for gram positive bacteria to FQs (OR 6.65; 95% CI 2.15-20.54). Conclusions Systemic antibiotic prophylaxis successfully reduced the incidence of infection in HSCT recipients. However, there was no significant impact on mortality. Impact of prophylaxis with FQs on mortality in HSCT recipients was inconclusive because of small number of trials evaluated. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Neutrophil recovery following allogeneic hematopoietic cell transplantation (allo-HCT) varies widely among patients. Some cases have a rapid increase in neutrophils and early engraft, while others show slow increase after neutrophils appearance and take time until engraftment. Such differences in neutrophil recovery may reflect the composition of the graft and affect the complications or prognosis after allo-SCT, though it has not been well documented in the literature. In this study, we retrospectively analyzed the influence of the slope of neutrophil recovery (N slope) following allo-HCT on post-transplant complications and prognosis. Methods: This study was a retrospective analysis of 120 patients with hematopoietic diseases who undergone first unrelated bone marrow transplantation at Jichi Medical University between January 2009 and December 2018. Patients who failed to achieve engraftment or did not receive granulocyte-colony stimulating factor were excluded. The N slope was defined as the increase of neutrophil counts from the last day of lowest neutrophil count after transplantation to the date of neutrophil engraftment. We evaluated the predictive value of N slope for acute graft-versus-host disease (GVHD) using the area under the receiver operating characteristic (ROC) curve and determined the cut-off value to maximize the sum of the sensitivity and specificity. Results: The median N slope was 205.5 /µL/day (range 26.4-7574). An ROC analysis showed that a cut-off value of N slope for grade II-IV acute GVHD was 207.5 /µL/day (sensitivity 0.73, specificity 0.6, AUC = 0.67, 95 % confidence interval (CI) = 0.59-0.79). Then, we classified patients into the low (n = 59) and high (n = 61) N slope groups according to the cut-off value of 200 /µL/day. The high N slope group correlated with older patients, RIC regimen, higher infused CD34+ cells ≥ 1.5 × 106/kg, and former transplantation between 2009 and 2013 compared with the low N slope group (p = 0.028, p = 0.003, p = 0.036 and p = 0.025, respectively). Cumulative incidence of grade II-IV acute GVHD at day 100 was significantly higher in the high N slope group than the low N slope group (44.3 % vs. 16.9%, p = 0.0007). With regard to the risk factors for grade II-IV acute GVHD, male, donor age ≥ 40 years, N slope ≥ 200 /µL/day and former transplantation between 2009 and 2013 were significant in univariate analysis. In multivariate analysis, donor age ≥ 40 years, N slope ≥ 200 /µL/day and former transplantation were identified as significant independent risk factors (hazard ratio (HR) 3.6, 95%CI 1.7-7.7, p = 0.001; HR 2.8, 95%CI 1.5-5.3, p = 0.002; HR 3.0; 95%CI 1.5-6.1, p = 0.02;, respectively). Cumulative incidence of grade III-IV acute GVHD at day 100 and chronic GVHD at 2 years did not differ in the high and low N slope groups (10.8 % vs. 5.1 %, p = 0.328; 63.3 % vs. 50.1 %, p = 0.12, respectively). In addition, there was no difference in relapse, non-relapse mortality and overall survival between the two groups (p = 0.76, p = 0.32, p = 0.65, respectively). Conclusion: The current study showed that a steeper slope of neutrophil recovery following HCT was associated with increased acute GVHD, though it did not affect relapse, NRM and OS. Early diagnosis and therapeutic intervention for acute GVHD may improve the outcome of patients with rapid neutrophil recovery. Disclosures Kanda: Chugai: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Ono: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Ono: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Takara-bio: Consultancy, Honoraria; CSL Behring: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; CSL Behring: Research Funding; Mochida: Consultancy, Honoraria; Eisai: Consultancy, Honoraria, Research Funding; Taisho-Toyama: Research Funding; Otsuka: Research Funding; MSD: Research Funding; Takara-bio: Consultancy, Honoraria; Shionogi: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Nippon-Shinyaku: Research Funding; MSD: Research Funding; Asahi-Kasei: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Tanabe Mitsubishi: Research Funding; Celgene: Consultancy, Research Funding; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Pfizer: Research Funding; Alexion: Consultancy, Honoraria; Sanofi: Research Funding; Celgene: Consultancy, Research Funding; Otsuka: Research Funding; Taiho: Research Funding; Novartis: Research Funding; Taiho: Research Funding; Taisho-Toyama: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Tanabe Mitsubishi: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Description: Background Following activation by recognition of foreign antigens, human T-cells alter their metabolic pathways to meet the increasing energetic demands for efficient immune response. Like cancer cells, alloreactive T-cells show a preference for aerobic glycolysis rather than oxidative phosphorylation, which is referred to as "Warburg effect". Until recently, it has been thought that extracellular fatty acid (FA) uptake and β-oxidation are severely reduced in alloreactive T-cells; however, some studies have indicated that lipid metabolism is rather increased in alloreactive mouse T-cells, and that metabolic pathway of FA can be a promising target for GVHD. To determine the role of lipid metabolism in human alloreactive T-cells after hematopoietic stem cell transplantation, we investigated the metabolic changes in human T-cells in vivo using human-into-mouse xenogeneic GVHD models. Methods NOG mice received 250cGy of total body irradiation (TBI) and were subsequently injected intravenously with human pan T-cells. All mice developed severe GVHD and died within 2 weeks, while mice that received TBI only survived without any symptoms of GVHD. Cells were harvested from GVHD target organs of mice at day 9 after transplantation. For the measurement of glucose and fatty acid (FA) uptake by flow cytometry, cells were stained with fluorescent-labeled deoxyglucose analogue (2-NBDG) and long-chain fatty acid analogue (BODIPY 500/510 C12), respectively. PCR array and extracellular flux analysis were performed according to manufacturer's instructions. Results Glucose uptake, determined by flow cytometry, was significantly increased in human T-cells obtained from GVHD mice. Extracellular FA uptake was also increased in human T-cells in GVHD mice, and was associated with cell proliferation rate. Effector memory T-cells followed by central memory T-cells showed a higher FA uptake than did naive T-cells. These findings were similarly observed in both human CD4+ and CD8+ T-cells. Robust T-cell proliferation was observed even in MHC class I/II deficient (MHC−/−) NOG mice after transplantation, although to a lesser extent than MHC+/+ NOG mice, in a process known as homeostatic proliferation. Extracellular uptake of FA as well as glucose in T-cells was significantly decreased in MHC−/− NOG mice. Of note, even when compared among only fully proliferated T-cells between MHC+/+ and MHC−/− NOG mice, FA uptake was still significantly decreased in MHC−/− NOG mice, suggesting that the recognition of host MHC molecules by allogeneic T-cells accelerate this process. To compare the ability of human naive and memory T-cells to incorporate extracellular FA, we isolated human naive (CD45RA high) and memory (CD45RA low) T-cells and separately injected into NOG mice. Although it has been shown that memory T-cells exhibit different effector functions, the FA uptake in memory T-cells was comparable to that in naive T-cells. This suggests that memory T-cells can also alter their lipid metabolism following encounter with alloantigens. Finally, we assessed the expression of genes associated with lipid metabolism in human T-cells obtained from GVHD mice. Quantitative real-time PCR analysis detected up-regulation of mRNAs encoding the enzymes involved in FA transport including carnitine palmitoyltransferase (CPT1B), fatty acid binding protein (FABP1-4, FABP6, and FABP7), and β-oxidation pathway including acyl-CoA synthase (ACSBG2) and acyl-CoA dehydrogenase (ACAD9-11, ACADS, and ACADL) when compared with T-cells in MHC−/− NOG mice. Similarly, the expression of genes encoding the enzymes in triacylglycerol metabolism such as glycerol kinase (GK, GK2) and lipoprotein lipase (LPL) was up-regulated in GVHD mice. Furthermore, the expression of genes associated with mevalonate pathways such as HMG-CoA synthase (HMGCS1, HMGCS2), was also upregulated. These observations suggest that T-cells activated by alloantigens in vivo promote lipid hydrolysis, mitochondrial FA transport, and β-oxidation, resulting in greater utilization of free FA. Conclusion Human alloreactive T-cells increased extracellular uptake of FA as well as glucose, and intracellular lipid metabolism in response to alloantigens (summarized in the graphical abstract). Therapeutic effects of specific inhibition of lipid metabolic pathways by pharmacological inhibitors including etomoxir are now being investigated in this model. Figure. Figure. Disclosures Fujiwara: Shire: Consultancy; Pfizer: Consultancy; Chugai: Consultancy; Kirin: Consultancy; Kyowa-Hakko: Consultancy; Astellas: Consultancy. Ohmine:Kyowa Hakko Kirin: Speakers Bureau; Takara Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Celgene Corporation: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Alexion Pharmaceuticals: Speakers Bureau; Ono Pharmaceutical: Consultancy. Muroi:Japanese Red Cross Society: Speakers Bureau; Dickinson and Company: Speakers Bureau; Becton: Speakers Bureau; JCR: Speakers Bureau. Kanda:Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Nippon-Shinyaku: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

Description: To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions. The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery. We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model. CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone. Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding.

Description: Background After hematopoietic stem cell transplantation, donor T-cells home to secondary lymphoid organs and recognize alloantigens within MHC molecules presented by host APCs. Following activation, donor T-cells acquire effector functions and then migrate into host organs along the chemokine gradients. Animal models targeting chemokine signals for prevention or treatment of GVHD have shown promising results; however, there have been significant inconsistencies among studies probably due to differences in species and conditioning regimens. The aim of this study is to evaluate the role of chemokines and their receptors, CCR5 (receptor of CCL3-5) and CXCR3 (receptor of CXCL9-10), in human T-cell homing and the development of GVHD using xenogeneic GVHD mouse model. Methods NOG mice received 250cGy of total body irradiation (TBI) if not otherwise specified, and were subsequently injected intravenously with human pan T-cells. All mice developed severe GVHD and died within 2 weeks, while the mice that received TBI only survived without any symptoms of GVHD. Peripheral blood was collected from mice at a certain interval for chemokine measurement. To assess the expression of chemokine receptors and genes associated with T-cell homing, cells were harvested from GVHD target organs of mice at day 9. For CCR5 blockage, mice were treated with 31 mg/kg maraviroc once daily by oral gavage after transplantation. Results Extensive infiltration of human T-cells and tissue destruction were observed in lungs and liver, but less severely in colon of GVHD mice. Consistent with this, quantitative real-time PCR analysis for five chemokine-related genes detected up-regulation of murine CXCL9 and CXCL10 in lungs, CCL4 in lungs and liver, but no up-regulation in colon. Similarly, the multiplex analysis of nine chemokines in plasma showed a marked increase in murine CCL4, CXCL9, and CXCL10 in GVHD mice. These observations suggest that the increased expression of CCL4, CXCL9, and CXCL10 on individual organs and following their systemic release play a critical role in the homing of allogeneic T-cells. Quantitative real-time PCR analysis of 84 genes associated with chemokines and chemokine receptors in human T-cells obtained from GVHD target organs revealed down-regulation of 36 genes, most of which are critical for T-cell homing into lymph nodes, such as CCL21 (-6.73-fold) and its receptor, CCR7 (-51.6-fold), and up-regulation of 16 genes such as CCL3 (225.5-fold), CCL4 (25.2-fold), CCR1 (11.4-fold), CCR5 (3.94-fold), and CXCL10 (2.88-fold). Focusing on chemokine receptors on human T-cells, flow cytometric analysis showed significantly higher expression of CCR5 on CD4+ and CD8+ T-cells, and CXCR3 on CD4+ T-cells in GVHD mice, whereas CXCR3 on CD8+ T-cells was strongly expressed even in resting state. Tissue damages were less apparent in GVHD mice that received human T-cells only compared with irradiated GVHD mice. Consistent with this, not only a total number but also the proliferation rate of human T-cells was decreased in non-irradiated GVHD mice. Also, non-irradiated GVHD mice showed significantly decreased plasma CCL4 and CXCL10 levels in plasma, and lower expression of CCR5 on CD4+ and CD8+ T-cells, and CXCR3 on CD4+ T-cells. The same was observed, to a significantly greater extent, in MHC class I/II deficient mice, suggesting that recognition of host MHC molecules by T-cells are critical for both host and donor chemokine signals. Taken together, TBI promotes host chemokine secretion and chemokine receptor expression on donor T-cells, leading to faster recruitment of donor T-cells into host organs and their proliferation. Contrary to the previous reports, CCR5 inhibitor treatment failed to attenuate GVHD and to improve the survival of mice. Although none of chemokine ligands but CCL4 was up-regulated on the liver, the number of infiltrated T-cells and tissue destruction were almost equivalent compared to the control. These observations indicate that compensatory chemokine pathways involving alternative receptors for CCL3-5, such as CCR1 and CCR2 on effector T-cells may overcome CCR5 blockage. Conclusion This study firstly provides a comprehensive picture of human T-cell homing through CCR5 and CXCR3 signaling in xenogeneic GVHD models. Our data supports the development of novel preventive and therapeutic strategies targeting chemokine signaling for GVHD. Disclosures Fujiwara: Shire: Consultancy; Pfizer: Consultancy; Chugai: Consultancy; Kirin: Consultancy; Kyowa-Hakko: Consultancy; Astellas: Consultancy. Ohmine:Kyowa Hakko Kirin: Speakers Bureau; Takara Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Celgene Corporation: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Alexion Pharmaceuticals: Speakers Bureau; Ono Pharmaceutical: Consultancy. Muroi:Japanese Red Cross Society: Speakers Bureau; Dickinson and Company: Speakers Bureau; Becton: Speakers Bureau; JCR: Speakers Bureau. Kanda:Taisho-Toyama: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Tanabe-Mitsubishi: Research Funding; CSL Behring: Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; MSD: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Nippon-Shinyaku: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

Description: Recently, a growing body of evidence has suggested that adiponectin, which is secreted by adipose tissues, plays a critical role in obesity-related and autoimmune diseases. We compared the concentrations of adiponectin among 26 normal subjects and 34 allogeneic stem cell transplantation recipients. The concentrations of adiponectin were significantly higher in recipients with chronic graft-versus-host disease (cGVHD) than those in subjects without cGVHD (21.7 ± 11.0 vs 9.1 ± 6.1 μg/mL in females, P 〈 .001; and 10.1 ± 6.8 vs 4.3 ± 2.9 μg/mL in males, P = .003). Multivariate analysis revealed that a higher concentration of adiponectin was associated with female sex (β-coefficient 8.2, P 〈 .0001) and the severity of cGVHD (β-coefficient 6.6, 12.7, and 15.6, P 〈 .01, each for mild, moderate, and severe cGVHD, respectively). In addition, adiponectin levels increased as cGVHD progressed, decreased as cGVHD improved, and did not change with stable cGVHD. In conclusion, adiponectin was associated with the severity of cGVHD and might play a role in the pathophysiology of cGVHD.

Description: Background Dimethyl fumarate (DMF), a fumaric acid derivative, is currently used worldwide as a therapeutic agent for autoimmune diseases, such as multiple sclerosis and psoriasis. As an activator of Nrf-2, DMF protects cells from oxidative stress by inducing anti-oxidant enzymes. In addition, a recent report in Science has shown that DMF catalytically inactivates GAPDH, thereby reduces glycolytic activity, and results in immune modulation in activated CD4+ T-cells. We have previously shown that DMF and its metabolite monomethyl fumarate (MMF) significantly inhibit 3H-thymidine uptake in activated T-cells. DMF also decreased the expression of proliferation marker Ki-67 and intracellular IFN-γ of activated T-cells in a dose dependent manner. These findings prompted us to investigate whether DMF can be used for the treatment of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation. In the current study, we investigated whether, and if so, how DMF inhibits human T-cell immune response and suppress acute GVHD in vivo using a xenogeneic GVHD mouse model. Methods To induce acute GVHD, human peripheral blood mononuclear cells (hPBMCs) were intravenously injected into sublethally irradiated (250 cGy) NOG mice. We allocated the mice into two groups; DMF treatment and non-treatment (control mice). Mice in the DMF group were administered DMF orally (100 mg/kg) for consecutive 7 days (day -3 to +3), and compared with the control mice treated with the same volume of vehicle. Results First, we observed that DMF treatment prolonged the survival of mice (Figure 1). Supporting the result, histopathological analysis showed that the number of hPBMCs infiltrated in the lungs and liver was decreased in the DMF group. Next, to identify the alteration of donor human cell populations after DMF treatment, hPBMCs were retrieved from the lungs on day 9 after transplantation and were analyzed by flow cytometry. Consistent with the histological findings, the absolute number of hPBMCs (hCD45+), and also T-cells (hCD45+hCD3+), in the lungs was significantly lower in the DMF group compared with the control (p 〈 0.01) (Figure 2). Notably, the number of CD4+ T-cells, but not CD8+ T-cells, was decreased by the DMF treatment. The proportion of regulatory T-cells (Tregs) (hCD45+CD4+CD25+Foxp3+) was elevated in the DMF group, and this finding is consistent with existing reports that DMF may increase the proportion of Tregs. Furthermore, the expression level of PD-1 on hCD4+ T-cells was significantly lower in the DMF group. These results suggest that DMF treatment mainly regulates cell proliferation and functional differentiation of donor human CD4+ T-cells, leading to reduced severity of GVHD. Given that GAPDH and aerobic glycolysis have been shown as potential targets of DMF, we then measured glycolytic activity in human T-cells obtained from mice during GVHD. Extracellular acidification rate, an indicator of glycolytic activity, was monitored under basal conditions followed by sequential treatment with glucose, oligomycin, and 2-deoxy-D-glucose (a competitive inhibitor of glucose). Glycolytic activity after the addition of glucose was significantly lower in the T-cells of DMF group than in those of the control group (Figure 3). DMF treatment also led to a significant reduction in glycolytic capacity and glycolytic reserve. Furthermore, the oxygen consumption rate, an indicator of oxidative phosphorylation, was decreased in the DMF group, indicating that DMF disrupts mitochondrial energy production in T-cells, either directly or indirectly. Similar results were obtained from CD4+ T-cells. These results suggest that DMF treatment can negatively regulate aerobic glycolysis in alloreactive T-cells, leading to the mitigation of GVHD. Conclusion Oral administration of DMF ameliorates GVHD and prolongs the survival of mice by reducing donor CD4+ T-cell proliferation, while the number of Tregs is maintained. Our data suggests that DMF treatment drives donor T-cells into a metabolically inactive state by inhibiting aerobic glycolysis. This investigation provides pre-clinical data to use oral DMF as a prophylactic agent for acute GVHD. Disclosures Kanda: Daiichi Sankyo: Honoraria; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Takeda Pharmaceuticals: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Celgene: Honoraria; Otsuka: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Janssen: Honoraria; Astellas Pharma: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mochida Pharmaceutical: Honoraria; Mundipharma: Honoraria; Sanofi: Honoraria, Research Funding; Meiji Seika Kaisha: Honoraria; Bristol-Myers Squibb: Honoraria; Shionogi: Research Funding; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding.