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Increasing evidence suggests that the gut microbiome may modulate the efficacy of cancer immunotherapy. In a B cell-lymphoma patient cohort from five centers in Germany and the United States (Germany, N=66; US, N=106, Total, N=172), we demonstrate that wide-spectrum antibiotics treatment (‘high-risk antibiotics’) prior to CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is associated with adverse outcomes, but this effect is likely confounded by an increased pre-treatment tumor burden and systemic inflammation in high-risk antibiotics-pre-treated patients. To resolve this confounding effect and gain insights into antibiotics-masked microbiome signals impacting CAR-T efficacy, we focused on the high-risk antibiotics non-exposed patient population. Indeed, in these patients, significant correlations were uncovered between pre-CAR-T-infusion Bifidobacterium longum and microbiome-encoded peptidoglycan biosynthesis, and CAR-T treatment-associated 6-months survival or lymphoma progression. Furthermore, predictive pre-CAR-T treatment microbiome-based machine-learning algorithms trained on the high-risk antibiotics non-exposed German cohort and validated by the respective US cohort robustly segregated long-term responders from non-responders. Bacteroides, Ruminococcus, Eubacterium and Akkermansia were most important in determining CAR-T responsiveness, with Akkermansia also associating with pre-infusion peripheral T cell levels in these patients. Collectively, we uncover conserved microbiome features across clinical and geographical variations, which may enable cross-cohort microbiome-based predictions of outcomes in CAR-T cell immunotherapy.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1 −/− mice, and adoptive transfer of wild-type ( WT ) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant ( n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila , a species of mucin-degrading bacteria ( P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

Acute graft-versus-host disease (aGVHD) limits the therapeutic benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and requires immunosuppressive prophylaxis that compromises antitumor and antipathogen immunity. OX40 is a costimulatory receptor upregulated on circulating T cells in aGVHD and plays a central role in driving the expansion of alloreactive T cells. Here, we show that OX40 is also upregulated on T cells infiltrating GVHD target organs in a rhesus macaque model, supporting the hypothesis that targeted ablation of OX40+ T cells will mitigate GVHD pathogenesis. We thus created an OX40-specific cytotoxic receptor that, when expressed on human T cells, enables selective elimination of OX40+ T cells. Because OX40 is primarily upregulated on CD4+ T cells upon activation, engineered OX40-specific T cells mediated potent cytotoxicity against activated CD4+ T cells and suppressed alloreactive T-cell expansion in a mixed lymphocyte reaction model. OX40 targeting did not inhibit antiviral activity of memory T cells specific to Epstein-Barr virus, cytomegalovirus, and adenoviral antigens. Systemic administration of OX40-targeting T cells fully protected mice from fatal xenogeneic GVHD mediated by human peripheral blood mononuclear cells. Furthermore, combining OX40 targeting with a leukemia-specific chimeric antigen receptor in a single T cell product provides simultaneous protection against leukemia and aGVHD in a mouse xenograft model of residual disease posttransplant. These results underscore the central role of OX40+ T cells in mediating aGVHD pathogenesis and support the feasibility of a bifunctional engineered T-cell product derived from the stem cell donor to suppress both disease relapse and aGVHD following allo-HSCT.

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is commonly performed to treat a variety of benign and malignant hematological diseases. Acute graft-versus-host disease (GVHD) is a major life-threatening complication that often occurs following allo-HSCT. Recently, improvements in methods to characterize the microbiota have led to a greater appreciation for how frequently and profoundly an alteration in microbial composition, or dysbiosis, can occur in allo-HSCT recipients to better decipher the complex interplay of between microbiota and allo-HSCT outcomes. AREAS COVERED: This article reviews the current knowledge of the microbiota’s impact on allo-HSCT outcomes, including effects of microbiota-derived metabolites, and crosstalk between commensals and the allogeneic immune response. This article also summarizes the effects of HSCT and transplant-related procedures on microbiota, and recent developments in interventional strategies. EXPERT OPINION: A growing body of literature indicate that the composition of the intestinal microbiota can function as a predictive biomarker for the risk and severity of acute GVHD, as well as overall survival, in patients undergoing allo-HSCT. Mechanisms underpinning this associations, however, are not well-understood, and clinical strategies that modulate the microbiome to improve outcomes have yet to be fully developed. There is an unmet need to determine mechanisms to improve the efficacy of allo-HSCT.

Background Pegfilgrastim has equivalent efficacy to daily granulocyte colony-stimulating factor (G-CSF) in enhancing neutrophil recovery after chemotherapy, but data on its use for peripheral blood stem cell (PBSC) mobilization are limited. We evaluated the safety and efficacy of CD34+ PBSC mobilization by low-dose (3.6 mg) pegfilgrastim after chemotherapy in patients with malignant lymphoma. Study design and methods Twenty patients with malignant lymphoma were enrolled in this study. Cytotoxic chemotherapy was started on day 1, and 3.6 mg of pegfilgrastim was subcutaneously administered on day 7. CD34+ cells were counted in the peripheral blood daily from days 11 to 14 using a flow cytometric analysis. Results In 19 of the 20 patients (95%), the CD34+ cell counts in the peripheral blood exceeded 10 × 106/L, with a mean value of 20.3 on day 11, 38.0 on day 12, 40.3 on day 13, and 40.1 on day 14. Older age was associated with lower maximum CD34+ cell mobilization. The most frequent adverse events associated with pegfilgrastim were back pain, nausea, appetite loss, and lactate dehydrogenase elevation. Conclusion Our data indicated that a single dose of 3.6 mg pegfilgrastim on day 7 after chemotherapy safely and effectively mobilized CD34+ cells.

Aim: To measure histological villous atrophy and to clarify the diagnostic accuracy of endoscopic villous atrophy in gastrointestinal graft-versus-host disease. Methods: Data for patients who underwent upper and/or lower endoscopic examinations after hematopoietic stem cell transplantation were retrospectively collected. In study 1, group A included 56 patients in whom GI-GVHD was histologically confirmed and group B included 60 patients in whom GI-GVHD was not histologically confirmed. Group C included 59 patients before HSCT. The lengths of villi and crypts in the duodenum and terminal ileum were histologically measured. In study 2, the diagnostic accuracies of villous atrophy of the duodenum and of the terminal ileum using magnifying endoscopy were evaluated. Results: In study 1, the lengths of villi and the villi/crypt (V/C) ratios of the duodenum and terminal ileum in group A were significantly smaller than those in the other groups (p

Myeloid differentiation factor 88 (MyD88) signaling has a crucial role in activation of both innate and adoptive immunity. MyD88 transduces signals via Toll-like receptor and interleukin-1 receptor superfamily to the NFκB pathway and inflammasome by forming a molecular complex with interleukin-1 receptor-associated kinase 4. The MyD88/interleukin-1 receptor-associated kinase 4 pathway plays an important role, not only in innate immunity, but also T-cell immunity; however, its role in donor T cells on the pathophysiology of graft-versus-host disease (GvHD) remains to be elucidated. We addressed this issue by using MyD88-deficient T cells in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-SCT). While MyD88-deficient and wild-type T cells proliferated equivalently after transplantation, MyD88-deficient T cells demonstrated impaired survival and differentiation toward Th1, Tc1, and Th17, and induced less severe GvHD compared to wild-type T cells. Administration of interleukin-1 receptor-associated kinase 4 inhibitor PF-06650833 significantly ameliorated GvHD after allo-SCT. These results thus demonstrate that donor T-cell MyD88/interleukin-1 receptor-associated kinase 4 pathway is a novel therapeutic target against GvHD after allo-SCT.

A 25-year-old male with a medical history of stress polycythemia was admitted to a previous hospital for leukocytosis, anemia, and thrombocytopenia. Bone marrow examination revealed left-shifted myeloid hyperplasia without increased blasts and normal male karyotype. No mutations of JAK2, V617F, and colony-stimulating factor 3 receptor gene (CSF3R) were detected. Fluorescence in-situ hybridization for BCR-ABL1 and FIP1L1-PDGFRA were negative. Based on these findings, a diagnosis of an unclassifiable myeloproliferative neoplasm was made, and he was started on hydroxyurea treatment. He was referred to our hospital in April 2016 for transfusion dependence. Bone marrow examination performed at our hospital revealed granulocytic dysplasia and CSF3R T618I was detected. After induction therapy, CSF3R T618I became undetectable, and he went on to undergo allogeneic stem cell transplantation in October 2016. He has been in remission for4 years posttransplantation. CSF3R T618I is one of the genes responsible for chronic neutrophilic leukemia and atypical chronic myeloid leukemia, suggesting its involvement in the pathogenesis of this case.

Adapted from the haploidentical hematopoietic stem cell transplantation (HCT) literature, post-transplantation cyclophosphamide (PTCy) is being used increasingly with HLA-matched donors, generally with a calcineurin inhibitor, such as tacrolimus (Tac), and with or without mycophenolate mofetil (MMF). Owing to its immunosuppressive and potentially antitumor and antimicrobial properties, MMF is an attractive drug; the benefit gained when it is used with PTCy/Tac remains unclear, however. To assess this, we compared PTCy/Tac (n = 242) and PTCy/Tac/MMF (n = 144) regimens in recipients of HLA-matched donor transplantation. In multivariate analysis, the PTCy/Tac/MMF group had a significantly higher risk of grade II-IV acute graft-versus-host disease (aGVHD) (hazard ratio [HR], 2.1; 95% confidence interval [CI], 1.6 to 2.8; P.001), and steroid-refractory/dependent aGVHD (HR, 4.8; 95% CI, 2.4 to 9.6; P.001), yet a significantly lower risk of relapse (HR, .5; 95% CI, .3 to .9; P = .009) and better progression-free survival (PFS) (HR, .7; 95% CI, .5 to .9; P = .04). There were no differences in the risk of grade III-IV aGVHD, chronic graft-versus-host disease (cGVHD), nonrelapse mortality, or overall survival. MMF was associated with prolonged neutrophil engraftment by 2 days and an elevated risk of bacterial infection. In an exploratory stool microbiome analysis (n = 16), we noted a higher relative abundance of β-glucuronidase-producing bacteria in the MMF group, which may have a role in the pathogenesis of MMF-related GVHD. Our data suggest that the addition of MMF to PTCy/Tac for HLA-matched donor HCT does not provide any advantage for GVHD prevention. Further studies are needed to decipher this mechanism and understand its role with PTCy-based prophylaxis.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that block immune inhibitory pathways. Administration of ICIs augments T cell-mediated immune responses against tumor, resulting in improved overall survival in cancer patients. It has emerged that the intestinal microbiome can modulate responses to ICIs via the host immune system and that the use of antibiotics can lead to reduced efficacy of ICIs. Recently, reports that fecal microbiota transplantation can lead to ICI therapy responses in patients previously refractory to therapy suggest that targeting the microbiome may be a viable strategy to reprogram the tumor microenvironment and augment ICI therapy. Intestinal microbial metabolites may also be linked to response rates to ICIs. In addition to response rates, certain toxicities that can arise during ICI therapy have also been found to be associated with the intestinal microbiome, including in particular colitis. A key mechanistic question is how certain microbes can enhance anti-tumor responses or, alternatively, predispose to ICI-associated colitis. Evidence has emerged that the intestinal microbiome can modulate outcomes to ICI therapies via two major mechanisms, including those that are antigen-specific and those that are antigen-independent. Antigen-specific mechanisms occur when epitopes are shared between microbial and tumor antigens that could enhance, or, alternatively, reduce anti-tumor immune responses via cross-reactive adaptive immune cells. Antigen-independent mechanisms include modulation of responses to ICIs by engaging innate and/or adaptive immune cells. To establish microbiome-based biomarkers of outcomes and specifically modulate the intestinal microbiome to enhance efficacy of ICIs in cancer immunotherapy, further prospective interventional studies will be required.

The diversity of the human microbiome heralds the difference of the impact that gut microbial metabolites exert on allogenic graft-versus-host (GVH) disease (GVHD), even though short-chain fatty acids and indole were demonstrated to reduce its severity. In this study, we dissected the role of choline-metabolized trimethylamine N-oxide (TMAO) in the GVHD process. Either TMAO or a high-choline diet enhanced the allogenic GVH reaction, whereas the analog of choline, 3,3-dimethyl-1-butanol reversed TMAO-induced GVHD severity. Interestingly, TMAO-induced alloreactive T-cell proliferation and differentiation into T-helper (Th) subtypes was seen in GVHD mice but not in in vitro cultures. We thus investigated the role of macrophage polarization, which was absent from the in vitro culture system. F4/80(+)CD11b(+)CD16/32(+) M1 macrophage and signature genes, IL-1β, IL-6, TNF-α, CXCL9, and CXCL10, were increased in TMAO-induced GVHD tissues and in TMAO-cultured bone marrow–derived macrophages (BMDMs). Inhibition of the NLRP3 inflammasome reversed TMAO-stimulated M1 features, indicating that NLRP3 is the key proteolytic activator involved in the macrophage’s response to TMAO stimulation. Consistently, mitochondrial reactive oxygen species and enhanced NF-κB nuclear relocalization were investigated in TMAO-stimulated BMDMs. In vivo depletion of NLRP3 in GVHD recipients not only blocked M1 polarization but also reversed GVHD severity in the presence of TMAO treatment. In conclusion, our data revealed that TMAO-induced GVHD progression resulted from Th1 and Th17 differentiation, which is mediated by the polarized M1 macrophage requiring NLRP3 inflammasome activation. It provides the link among the host choline diet, microbial metabolites, and GVH reaction, shedding light on alleviating GVHD by controlling choline intake.

Chronic graft-versus-host disease (GVHD) profoundly affects the quality of life of long-term survivors of allogeneic hematopoietic stem cell transplantation (SCT). The eyes are frequently involved, and dry eye syndrome is the most common manifestation of ocular chronic GVHD. We explored the role of heat shock protein 47 (HSP47) in ocular GVHD and developed a novel antifibrotic topical therapy using vitamin A–coupled liposomes containing HSP47 small interfering RNA (siRNA) against HSP47 (VA-lip HSP47). In a mouse model of chronic GVHD, infiltration of HSP47+ fibroblasts and massive fibrosis surrounding the lacrimal ducts were observed after allogeneic SCT, leading to impaired tear secretion. After ocular instillation, VA-lip HSP47 was distributed to the lacrimal glands, knocked down HSP47 expression in fibroblasts, reduced collagen deposition, and restored tear secretion after allogeneic SCT. Ocular instillation of VA-lip HSP47 also ameliorated established lacrimal gland fibrosis and dry eye syndrome. VA-lip HSP47 eye drops are a promising prophylactic and therapeutic option against dry eye syndrome in chronic GVHD.

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic hematopoietic stem cell transplantation (HSCT). Intestinal goblet cells form the mucus layers, which spatially segregate gut microbiota from host tissues. Although it is well known that goblet cell loss is one of the histologic features of GVHD, effects of their loss in pathophysiology of GVHD remain to be elucidated. In mouse models of allogeneic HSCT, goblet cells in the colon were significantly reduced, resulting in disruption of the inner mucus layer of the colon and increased bacterial translocation into colonic mucosa. Pretransplant administration of interleukin-25 (IL-25), a growth factor for goblet cells, protected goblet cells against GVHD, prevented bacterial translocation, reduced plasma concentrations of interferon-γ (IFN-γ) and IL-6, and ameliorated GVHD. The protective role of IL-25 was dependent on Lypd8, an antimicrobial molecule produced by enterocytes in the colon that suppresses motility of flagellated bacteria. In clinical colon biopsies, low numbers of goblet cells were significantly associated with severe intestinal GVHD, increased transplant-related mortality, and poor survival after HSCT. Goblet cell loss is associated with poor transplant outcome, and administration of IL-25 represents an adjunct therapeutic strategy for GVHD by protecting goblet cells.

Sinusoidal obstruction syndrome (SOS)/hepatic veno-occlusive disease (VOD) is a well-documented complication after hematopoietic stem cell transplantation (HSCT). Transabdominal ultrasonography (US) enables the visualization of blood flow abnormalities and is therefore useful for the diagnosis of SOS/VOD. We herein prospectively evaluated accuracy of a novel US diagnostic scoring system of SOS/VOD based on US findings. We carried out US in 106 patients on day 14 and when SOS/VOD was suspected after allogeneic HSCT. Among 106 patients, 10 patients (9.4%) were diagnosed as SOS/VOD by Baltimore or Seattle criteria. According to univariate analysis of 17 US findings (US-17 screening), we established a novel scoring system (HokUS-10) consisting of 10 parameters, such as gallbladder wall thickening, ascites, and blood flow signal in the paraumbilical vein. The sensitivity and specificity were 100% and 95.8%, respectively. Diagnostic performance of the HokUS-10 was significantly better than US-17 screening. In 4 of 10 patients US detection of SOS/VOD preceded to clinical diagnosis. The HokUS-10 scoring system is useful in the diagnosis of SOS/VOD; however, our results should be validated in other cohorts.

Chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (SCT) is characterized by multiorgan fibrosis and profoundly affects the quality of life of transplant survivors. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in collagen synthesis in myofibroblasts. We explored the role of HSP47 in the fibrotic process of cutaneous chronic GVHD in mice. Immunohistochemical analysis showed massive fibrosis with elevated amounts of collagen deposits and accumulation of F4/80+ macrophages, as well as myofibroblasts expressing HSP47 and retinol-binding protein 1 in the skin after allogeneic SCT. Repeated injection of anti-colony-stimulating factor (CSF-1) receptor-blocking antibodies significantly reduced HSP47+ myofibroblasts in the skin, indicating a macrophage-dependent accumulation of myofibroblasts. Vitamin A-coupled liposomes carrying HSP47 small interfering RNA (siRNA) (VA-lip HSP47) delivered HSP47 siRNA to cells expressing vitamin A receptors and knocked down their HSP47 in vitro. Intravenously injected VA-lip HSP47 were specifically distributed to skin fibrotic lesions and did not affect collagen synthesis in healthy skin. VA-lip HSP47 knocked down HSP47 expression in myofibroblasts and significantly reduced collagen deposition without inducing systemic immunosuppression. It also abrogated fibrosis in the salivary glands. These results highlight a cascade of fibrosis in chronic GVHD; macrophage production of transforming growth factor β mediates fibroblast differentiation to HSP47+ myofibroblasts that produce collagen. VA-lip HSP47 represent a novel strategy to modulate fibrosis in chronic GVHD by targeting HSP47+ myofibroblasts without inducing immunosuppression.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are at risk for graft-versus-host disease (GVHD). The gastrointestinal tract has been identified to be a primary target of allogeneic donor T cells in allo-HSCT. The intestinal microbiota is known to interact with the host immune system and has been found to be an important modulator of GVHD. Broad-spectrum antibiotics such as carbapenems are often used in allo-HSCT patients to treat infections but have been found to increase the risk for intestinal GVHD, possibly via bystander depletion of beneficial commensal bacteria. However, whether loss of beneficial commensal bacteria is mechanistically sufficient to aggravate intestinal GVHD is not known. To examine this further, we utilized a mouse model of GVHD to study the effects of meropenem, a commonly used carbapenem in allo-HSCT patients. Lethally irradiated B6D2F1 (H-2 b/d) mice were intravenously injected with 5 × 10 6 bone marrow cells and 5 × 10 6 splenocytes from major histocompatibility complex-mismatched B6 (H-2 b) or syngeneic donors on day 0. Meropenem was additionally administered to some allo-HSCT recipient mice in the drinking water from days 3 to 15 after allo-HSCT. We found that meropenem treatment aggravated GVHD primarily in the colon of mice, and 16S ribosomal RNA gene sequencing analysis demonstrated that many bacterial genera were depleted in meropenem-treated mice, including Blautia and Lachnoclostridium that belong to the class Clostridia. Simultaneously, the abundance of the genus Bacteroides was increased in meropenem-treated mice, which we also observed in allo-HSCT patients at our institution following meropenem treatment. Interestingly, when meropenem-treated mice were additionally treated orally with decontaminating antibiotics, GVHD severity and survival were ameliorated, suggesting that meropenem could induce worsened GVHD via expansion of pro-inflammatory bacteria as well as depletion of beneficial bacteria. We explored which species of Bacteroides was the most expanded by meropenem treatment and identified Bacteroides thetaiotaomicron (BT) as significantly increased species. Reintroduction of BT restored aggravated GVHD in mice treated with decontaminating antibiotics. BT is a gram-negative obligate anaerobe with a broad ability to degrade dietary polysaccharides as well as host mucin glycans. In meropenem-treated allogeneic mice, we observed significantly thinner colonic mucus, increased myeloid-cell infiltration into colonic tissue, and increased bacterial translocation into mesenteric lymph nodes. Decontamination, in contrast, led to preservation of the colonic mucus layer in meropenem-treated allogeneic mice. BT, in the presence of multiple suitable carbohydrate substrates, has been found to preferentially consume certain carbohydrates first and only after depleting these will it then upregulate utilization genes targeting other available polysaccharides. Host mucin glycans are particularly low on the metabolic hierarchy for BT. RNA sequencing of stool samples from meropenem-treated allogeneic mice demonstrated that BT upregulated expression of β-galactosidase, sialidase and α-L-fucosidase, all of which participate in the degradation of host mucin glycans. Carbohydrate mass spectrometry profiling of colonic luminal contents showed decreased levels of polysaccharides comprised of xylose. Interestingly, xylose supplementation in meropenem-treated allogeneic mice significantly prevented thinning of the colonic mucus layer, indicating that providing an alternative carbohydrate source was sufficient to suppress mucus-degrading behavior in BT (Figure). In conclusion, expansion of BT in meropenem-treated allogeneic mice is strongly associated with GVHD-related mortality. Broad-spectrum antibiotics such as meropenem are useful for treating infections in allo-HSCT, but can also lead to an altered intestinal environment with changes in levels of microbial-derived metabolites or decreased nutrients. This, in turn, can lead to expansion and altered behavior of commensal bacteria such as BT to target intestinal mucus, which can contribute to increased severity of GVHD. Specific nutritional supplementation strategies such as providing xylose may be helpful to combat changes to the intestinal environment in allo-HSCT patients with antibiotic-mediated microbiome injury. Figure 1 Figure 1. Disclosures Jenq: MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; LisCure: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seres: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Kaleido: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prolacta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Microbiome DX: Consultancy; Karius: Consultancy.

[Introduction] Graft-versus-host disease (GVHD) is a potentially life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). We and others have shown that GVHD targets adult tissue stem cells in the gut and skin, while a role of liver tissue stem cells in hepatic GVHD remains to be clarified. Biliary epithelial cells (BECs) are primary targets in hepatic GVHD and a single BEC stem cell gives rise to multipotent liver organoids (Cao W: Gastroenterology 2017). We studied the fate and role of BEC stem cells in experimental hepatic GVHD. [Methods] B6D2F1 recipients were lethally irradiated and transplanted with 5 x 10 6 splenocytes plus 5 x 10 6 bone marrow cells from allogeneic (B6) or syngeneic (B6D2F1) donors on day 0. Liver organoids were generated from the bile ducts isolated from the liver right lobe. [Results] Flowcytometric analyses of the liver demonstrated donor T cell infiltration in the liver within the first week after allo-HCT, followed by massive infiltration of monocytes and macrophages. Hepatic GVHD was characterized by apoptosis of BEC (Figure 1A), elevated expression of Mmp7, a biomarker of biliary injury (Figure 1B), and elevation of plasma levels of total bilirubin (Figure 1C). To evaluate fate of BEC stem cells in hepatic GVHD, we enumerated BEC-derived organoids generated from the right lobe of the liver isolated after allo-HCT. The organoid-forming BEC stem cells were profoundly reduced at later time point after allo-HCT (Figure 1D), while they persisted in syngeneic controls, indicating that GVHD targets BEC stem cells. Next, we explored the mechanism of BEC stem cell injury. Among the cytokines elevated in the liver after allo-HCT, we found that TGF-β , not IFN-γ or TNF-α, inhibited generation of liver organoids (Figure 1E). Furthermore, we found that liver infiltrating mononuclear cells isolated from the allogeneic livers, not from syngeneic livers, suppressed growth of liver organoids. This effect was abrogated by the addition of a TGF-β inhibitor, SB-431542 in culture. Among these cells, monocyte-derived macrophages, not Kupffer cells, demonstrated enhanced production of TGF-β after allo-HCT (Figure 1F), suggesting that TGF-β from inflammatory macrophages damaged BEC stem cells. Based on these findings, we next tested if TGF-β inhibition could protect BEC stem cells and ameliorate hepatic GVHD after allo-HCT. Admnistration of SB-431542 from day +14 to day +28 after allo-HCT significantly increased organoid-forming BEC stem cells, suppressed BEC apoptosis and Mmp7 expression, and mitigated jaundice on day +28 after allogeneic HCT, indicating that TGF-β inhibition is a novel therapeutic strategy against hepatic GVHD (Figure 1, G-J). [Conclusion] Our results for the first time demonstrated that hepatic GVHD targets BEC stem cells via a TGF-β-dependent manner. Mmp7 and organoid-forming capacity could be the biomarkers for hepatic GVHD. BEC stem-cell protection by TGF-β inhibition is a promising novel therapeutic strategy against hepatic GVHD. Figures1: (A) Proportion of cleaved caspase 3 (cCaspase3) + cells among the biliary epithelial cells (BECs) on day +28. (B) Total RNA extracted from the liver on day +28 was subjected to Q-PCR targeting Mmp7. (C) Plasma levels of total bilirubin at indicated time points. (D) The numbers of organoid derived from the right lobe of the liver at indicated time point are shown. (E) Absolute numbers of TGF-β producing Kupffer cells and macrophages (Mφ) in the liver on day +28 are shown. (F) Liver organoids were enumerated after incubation in the presence or absence of TGF-β for 4 days. (G-J) Allogeneic recipients were intraperitoneally injected with 5 mg/kg SB-431542 daily from day +14 to day +28 after allogeneic HCT, and recipient mice were sacrificed on day +28. Numbers of organoids derived from the right lobe of the livers (G), the proportion of cCaspase3 + BECs (H), relative expression of Mmp7 in the liver (I), and plasma levels of total bilirubin (J) are shown. *; p < .05, **; p < .01, ***; p < .005. Figure 1 Figure 1. Disclosures Teshima: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Gentium/Jazz Pharmaceuticals: Consultancy; Bristol Myers Squibb: Honoraria; Sanofi S.A.: Research Funding; TEIJIN PHARMA Limited: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fuji pharma CO.,Ltd: Research Funding; Pfizer Inc.: Honoraria; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Astellas Pharma Inc.: Research Funding; Janssen Pharmaceutical K.K.: Other.

Hayase et al. show that R-Spondin1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spondin1 or recombinant α-defensin prevents dysbiosis mediated by graft-versus-host disease, representing a novel approach to restore intestinal ecosystems and homeostasis., The intestinal microbial ecosystem is actively regulated by Paneth cell–derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host–microbiota cross talk toward therapeutic benefits.

Posttransplant high-dose cyclophosphamide (PTCY) has been increasingly used as graft-versus-host disease (GVHD) prophylaxis after HLA-haploidentical or matched hematopoietic stem cell transplantation (SCT). However, PTCY alone is insufficient and requires additional immunosuppressants such as calcineurin inhibitors. In the current study, we evaluated effects of a novel GVHD prophylaxis with PTCY in combination with short-term KRP203, a selective agonist of sphingosine-1-phosphate receptor 1 that regulates egress of lymphocytes from the secondary lymphoid organs (SLOs) in mice. Short-term oral administration of KRP203 alone induced apoptosis of donor T cells in the SLOs and ameliorated GVHD. Administration of KRP203 significantly preserved graft-versus-leukemia effects compared to cyclosporin. A combination of KRP203 on days 0 to +4 and PTCY on day +3 synergistically suppressed donor T-cell migration into the intestine and skin, and ameliorated GVHD more potently than PTCY alone. A combination of short-term KRP203 and PTCY is a promising novel calcineurin-free GVHD prophylaxis in HLA-haploidentical SCT.

Myeloid differentiation factor 88 (MyD88) signaling has a crucial role in activation of both innate and adoptive immunity. MyD88 transduces signals via Toll-like receptor and interleukin-1 receptor superfamily to the NFκB pathway and inflammasome by forming a molecular complex with interleukin-1 receptor-associated kinase 4. The MyD88/interleukin-1 receptor-associated kinase 4 pathway plays an important role, not only in innate immunity, but also T-cell immunity; however, its role in donor T cells on the pathophysiology of graft-versus-host disease (GvHD) remains to be elucidated. We addressed this issue by using MyD88-deficient T cells in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-SCT). While MyD88-deficient and wild-type T cells proliferated equivalently after transplantation, MyD88-deficient T cells demonstrated impaired survival and differentiation toward Th1, Tc1, and Th17, and induced less severe GvHD compared to wild-type T cells. Administration of interleukin-1 receptor-associated kinase 4 inhibitor PF-06650833 significantly ameliorated GvHD after allo-SCT. These results thus demonstrate that donor T-cell MyD88/interleukin-1 receptor-associated kinase 4 pathway is a novel therapeutic target against GvHD after allo-SCT.

The R-Spondin (R-Spo) family regulates WNT signaling and stimulates the proliferation and differentiation of intestinal stem cells (ISCs). R-Spo plays a critical role in maintaining intestinal homeostasis, but endogenous producers of R-Spo in the intestine remain to be investigated. We found that R-Spo3 was the major R-Spo family member produced in the intestine and it was predominantly produced by CD45−CD90+CD31+ lymphatic endothelial cells (LECs) in the lamina propria of the intestinal mucosa. Transcriptome analysis demonstrated that LECs highly expressed R-Spo receptor, Lgr5, suggesting an autocrine stimulatory loop in LECs. LECs were significantly reduced in number, and their R-Spo3 production was impaired in intestinal graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. The impaired production of R-Spo3 in the intestine may be a novel mechanism of delayed tissue repair and defective mucosal defense in intestinal GVHD. We demonstrate a novel role of intestinal LECs in producing R-Spondin3 to maintain intestinal homeostasis.

The association between Wilms tumor 1 (WT1) expression, genetic abnormalities and homozygous single polymorphism (SNP) in WT1 gene was evaluated in 252 acute myelogenous leukemia (AML) patients. WT1 expression correlated with prognostic genetic abnormalities. Homozygous WT1 SNP rs16754 was associated with lower expression of WT1. WT1 expression had no prognostic impact in any cytogenetic group or SNP status. Background: The prognostic impact of WT1 expression at diagnosis of acute myelogenous leukemia (AML) has been controversial. The aim of this study was to determine the correlations of WT1 expression at diagnosis of AML with established prognostic alterations. Patients and Methods: We analyzed diagnostic bone marrow samples from 252 patients. WT1 expression, SNP in WT1 gene (rs16754), and Flt3-ITD mutation were analyzed for all patients. NPM1 mutation and CEBPA double mutation were analyzed for cytogenetically normal (CN)-AML. KIT mutation was analyzed for core-binding factor (CBF)-AML. Results: Within the cytogenetically favorable prognosis group, WT1 expression in AML with inv(16) or t(15;17) was significantly higher than that in AML with t(8;21). In cases with CN-AML, Flt3-ITD and NPM1 mutations were both correlated with higher expression of WT1, whereas CEBPA double mutation was related to lower WT1 expression. The existence of both Flt3- ITD and NPM1 mutations showed synergistically higher expression of WT1 in CN-AML. SNP in WT1 gene (rs16754) was significantly associated with lower expression of WT1. WT1 levels were not prognostic factors in the total cohort, in any cytogenetic group nor SNP status. Conclusion: Since WT1 expression correlated to known prognostic factors, the prognostic impact of WT1 levels might be misunderstood depending on the distribution of collaborative mutations in each cohort. We conclude that the prognostic significance of WT1 at diagnosis of AML is weak compared to other established prognostic factors.

Benign precursors of B lymphocytes, termed hematogones, are observed in the regenerative state of hematopoiesis following chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous studies have demonstrated that expansion of hematogones correlates with better clinical outcomes after allo-HSCT. We retrospectively analyzed the association between hematogones and clinical outcomes in 309 consecutive patients who underwent allo-HSCT, which is the largest population-based cohort reported so far. The incidence of hematogones was significantly higher in complete remission (CR) patients at the time of transplantation than in non-CR patients, after myeloablative conditioning than after reduced-intensity conditioning, with tacrolimus-based graft-versus-host disease (GVHD) prophylaxis than with cyclosporine-based prophylaxis, and with disease other than malignant lymphoma (all P.05). Patients with hematogones developed less acute GVHD and infections than did those without them (P.05). Emergence of hematogones was associated with superior GVHD-free relapse-free survival and lower nonrelapse mortality, and was an independent prognostic factor for overall survival, irrespective of donor sources.

Although graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), cytomegalovirus (CMV) reactivation also occurs in patients after allo-HSCT and these conditions often clinically overlap. The aim of this study was to determine reliable endoscopic findings of CMV colitis in patients with gastrointestinal graft-versus-host-disease (GI-GVHD). Patients after allo-HSCT who were histologically confirmed to have GI-GVHD with or without CMV colitis and patients with an immunosuppressive condition were retrospectively analyzed. We divided the patients into three groups: GI-GVHD with CMV colitis (group A), GI-GVHD without CMV colitis (group B), and CMV colitis without undergoing allo-HSCT (group C). From medical records, the involved colorectal areas and endoscopic findings according to the groups were compared. A total of 70 patients were divided into three groups (group A: n = 19, group B: n = 28, group C: n = 23). Mucosal injuries in groups A and C frequently occurred in the cecum including ileocecal valves. On the other hand, there were no abnormal lesions on ileocecal valves in group B. Furthermore, ulcer lesions were more frequently observed in groups A and C than in group B (p

Graft-versus-host disease (GVHD) is the major complication after allogeneic stem cell transplantation (SCT). Emerging evidence indicates that GVHD leads to injury of intestinal stem cells. However, it remains to be investigated whether skin stem cells could be targeted in skin GVHD. Lgr5+ hair follicle stem cells (HFSCs) contribute to folliculogenesis and have a multipotent capacity to regenerate all epithelial cells in repair. We studied the fate of Lgr5+ HFSCs after SCT and explored the novel treatment to protect Lgr5+ HFSCs against GVHD using murine models of SCT. We found that GVHD reduced Lgr5+ HFSCs in association with impaired hair regeneration and wound healing in the skin after SCT. Topical corticosteroids, a standard of care for a wide range of skin disorders including GVHD, damaged HFSCs and failed to improve skin homeostasis, despite of their anti-inflammatory effects. In contrast, JAK1/2 inhibitor ruxolitinib significantly ameliorated skin GVHD, protected Lgr5+ HFSCs, and restored hair regeneration and wound healing after SCT. We, for the first time, found that GVHD targets Lgr5+ HFSCs and that topical ruxolitinib represents a novel strategy to protect skin stem cells and maintain skin homeostasis in GVHD.

Allogeneic hematopoietic stem cell transplantation(allo-SCT)is curative therapy for various hematological diseases. Graft-versus-host disease(GVHD)and infection remain the main problems in allo-SCT. Gastrointestinal tract is targeted by GVHD. In intestinal GVHD, intestinal stem cells and Paneth cells reside at the base of crypts are damaged by donor T cells. These damage leads to disruption of intestinal mucosal barrier and intestinal dysbiosis, resulting in more exaggerated GVHD. Recently, we and others have reported that R-Spondin1 and interleukin-22 as factors regenerated intestinal tissue homeostasis in murine model of bone marrow transplantation. It is important to understand biology of various cells targeted by intestinal GVHD and develop novel methods to protect them from GVHD damage.

Introduction Chronic graft-versus-host disease (GVHD) is characterized by multiorgan fibrosis and affects the quality of life of transplant survivors. Ocular manifestations are found in 40-60% of GVHD patients, and the most common symptom is dry eye syndrome associated with lacrimal gland fibrosis. We previously demonstrated heat shock protein 47 (HSP47), a collagen specific molecular chaperone, plays a critical role in excessive collagen synthesis in chronic GVHD, and systemic administration of vitamin A-coupled liposomes carrying HSP47 siRNA (VA-lip HSP47) ameliorates skin and salivary gland fibrosis in chronic GVHD (Yamakawa, et al, Blood 2018). In the current study, we evaluated the role of HSP47 in ocular GVHD and tested if ocular instillation of VA-lip HSP47 could ameliorate dry eye syndrome in a mouse model of chronic GVHD. Method BALB/c mice received 6-Gy total body irradiation followed by intravenous injection with 8 × 106 bone marrow cells and 20 × 106 splenocytes from minor histocompatibility antigen-mismatched B10.D2 or syngeneic BALB/c donors on day 0. Instillation of 2µl VA-lip HSP47 eye drops was performed daily after bone marrow transplantation (BMT). Allogeneic control recipients were daily treated with ocular instillation of VA-lip containing scrambled siRNA after BMT. Exocrine function of the lacrimal gland was evaluated as wet cotton length after inserting cotton threads under the lower eyelids for 15 seconds. Results Histological studies demonstrated massive fibrosis and accumulation of HSP47+ activated fibroblasts in the lacrimal glands 21 days after allogeneic BMT. The amounts of collagen deposit were significantly elevated in association with reduced tear production in allogeneic mice compared to syngeneic controls on day +35 (Fig. 1). Next, we tested if HSP47 could be a therapeutic target in ocular GVHD by using VA-lip HSP47 eye drops. First, fluorescent-labeled VA-lip HSP47 eye drops were daily administrated unilaterally from day +29 to +33 after allogeneic BMT to test if VA-lip HSP47 could reach to lacrimal glands after eye instillation. We found VA-lip HSP47 particles were distributed in the lacrimal glands on treated side but not on the contralateral side. Ocular instillation of VA-lip HSP47 daily on days +1 to +34 after allogeneic BMT depleted HSP47+ fibroblasts, reduced the amounts of collagen deposit, and ameliorated fibrosis in the lacrimal glands, in association with preserved lacrimal excretory function (Fig. 1). Finally, we found that ocular instillation of VA-lip HSP47 started on day +21 after allogeneic BMT ameliorated established lacrimal gland fibrosis and dry eye syndrome in chronic GVHD (Fig. 2). Conclusions Ocular instillation of VA-lip HSP47 is a promising prophylactic and therapeutic treatment against lacrimal gland fibrosis and dry eye syndrome in chronic GVHD.

[Introduction] Emerging evidences suggest that perturbations in the gut microbiota are associated with graft-versus-host disease (GVHD), and dominance of Enterobacteriaceae is related to poor prognosis after allogeneic hematopoietic stem cell transplantation (SCT) (Taur Y, Blood. 2014; 124:1174-1182). We recently reported that degree of goblet-cell loss was significantly corelated with poor prognosis in 90 patients who underwent SCT in our institute (Ara, et al. 2018 Tandem BMT meeting #220). Goblet cells play a critical role in forming the mucus layer that constitutes not only physical but also chemical barrier, by retaining antimicrobial peptides, against invading microbes from gut lumen. In the current study, we explored the mechanism by which goblet cells protect recipients against GVHD, especially focusing on the role of antimicrobial peptide Lypd8 that is produced by colon epithelial cells and specifically suppresses motilities and biotranslocation of flagellated bacteria including harmful Enterobacteriaceae. [Methods] Mice were lethally irradiated and injected with 5 × 106 bone marrow cells and 7.5 × 106 splenocytes from allogeneic or syngeneic donors on day 0. Recipient mice were intraperitoneally injected with 0.3 mg recombinant mouse IL-25 (rmIL-25) or vehicle from day -6 to 0. [Results] In the B6 → B6D2F1 model, goblet cells in the colon were significantly decreased in association with severe GVHD (Figure A). Fluorescent in situ hybridization (FISH) using the universal bacterial probe EUB338 showed bacterial translocation to the colonic mucosa after SCT. Quantitative PCR targeting bacterial 16S rRNA confirmed that bacterial load in the lamina propria was significantly increased in allogeneic mice compared to syngeneic controls and naive mice (Figure B). Immunofluorescent staining showed Lypd8 at the border of the inner mucus layer and colonic epithelial cells was reduced in allogeneic mice. To evaluate role of Lypd8 in GVHD, lethally irradiated B6-Lypd8-/- mice and wild type (WT) B6 controls were transplanted from BALB/c mice. Both FISH and quantitative PCR showed an increased bacterial translocation into the colonic mucosa in Lypd8-deficient recipients compared to WT recipients (Figure C), indicating that Lypd8 plays a protective role against bacterial translocation into the colonic mucosa. In association with enhanced bacterial translocation, significantly more donor T cells were infiltrated into the gut and liver in Lypd8-/- recipients compared to WT controls (Figure D). Strikingly, GVHD was significantly more severe with shorter survival in B6-Lypd8-/- mice compared to WT recipients (Figure E). GVHD exacerbation in Lypd8-/- mice was reproduced when Lypd8-/- recipients were co-housed with WT recipients for 4 weeks before SCT, excluding the virulent microbiota in Lypd8-/- mice as the potential mechanism of GVHD exacerbation in these mice. Finally, we tested if the goblet-cell protection could attenuate GVHD. Pre-transplant administration of IL-25 mitigated goblet-cell loss and bacterial translocation, reduced plasma levels of IFN-g and IL-6, and ameliorated GVHD mortality. Protective effects of pre-transplant IL-25 was abrogated when Lypd8-/- mice were used as recipients, suggesting that goblet cells protect recipients against GVHD via a Lypd8-dependent manner. [Conclusion] Our results demonstrated that goblet cells suppress bacterial translocation into the colon mucosa and play a protective role against GVHD via a Lypd8-dependent manner. Since increase in flagellated bacteria in the gut could be associated with GVHD exacerbation, goblet cells and Lypd8 could be potentially prophylactic and therapeutic targets for GVHD. Figures: (A) The amount of goblet cells in the colon of allogeneic or syngeneic recipients, or naïve mice are shown. (B) Colon samples were harvested from syngeneic and allogeneic recipients on day +7 after SCT. DNA was extracted from colon samples after removing epithelial cells by incubating with EDTA and subjected to quantitative PCR with 16S rRNA specific primers. (C-E) WT or Lypd8-/- B6 mice were lethally irradiated and transplanted from allogeneic BALB/c mice. Bacterial load in the colon lamina propria (C) and absolute numbers of donor T cells in the liver and colon (D) on day +5, and survival curves (E) after SCT are shown. *; p Disclosures No relevant conflicts of interest to declare.

Intestinal microbiota plays an important role in the regulation of allogeneic immune reaction after allogeneic hematopoietic stem cell transplantation (allo-SCT). Intestinal graft-vs-host disease (GVHD) is one of the major causes of mortality after allo-SCT and often complicated with intestinal dysbiosis. Recent studies suggest that antibiotic-induced dysbiosis is a risk factor for intestinal GVHD. We retrospectively evaluated the impacts of antibiotic use on the incidence of intestinal GVHD occurring before day 100 after allo-SCT. Among 213 patients who underwent allo-SCT, 200 patients achieving engraftment were analyzed. Antibiotics were classified into carbapenem, quinolone, penicillin, cephem, and glycopeptide. Among 128 patients who developed acute GVHD, intestinal GVHD developed in 36 patients. Patients with intestinal GVHD received significantly longer administration of carbapenem and glycopeptide compared to those without it in periengraftment period. In multivariate analysis, use of carbapenem for greater than 7 days was associated with an increased risk of intestinal GVHD. However, use of antibiotics for greater than 7 days was not associated with poor overall survival and high nonrelapse mortality. Long use of carbapenem in periengraftment period may be a risk for intestinal GVHD. Prospective studies are required to validate our findings.

Primary bone lymphoma (PBL) comprises less than 1% of all malignant lymphomas. Because few studies of PBL have been conducted in Japan, the characteristics of Japanese patients with PBL have not been fully elucidated. We retrospectively analyzed 17 patients diagnosed with PBL at our institution between 2001 and 2011. Median patient age was 60 years. Eleven patients had diffuse large B-cell lymphoma and 2 patients had T-cell lymphoma histology. The spine was the most frequently involved site at the time of presentation. There were 11 patients with stage IV disease and 11 patients with high or high-intermediate risk according to the International Prognostic Index (IPI). Thirteen patients achieved complete response (CR) after initial treatment. At a median follow-up of 31 months, the 3-year overall survival (OS) and progression free survival were 63.5 and 49.9%, respectively. Localized disease, low or low-intermediate IPI, and CR after initial treatment were associated with a good outcome in patients with PBL and significantly associated with a better OS. Spine involvement and T/NK-cell phenotype are more frequent in Japanese than in Caucasian patients with PBL.

A 22-year-old woman presented with high fever, chest tightness and cough in January 20XX. Since CT scans revealed an anterior mediastinal mass, percutaneous needle biopsies of the mass were performed and she was diagnosed with T-cell lymphoblastic lymphoma (T-LBL). After the immunophenotype of lymphocytes in her pleural effusion had been identified, she received CHOP therapy because her dyspnea worsened, and induction therapy for acute lymphoblastic leukemia was subsequently performed after confirmation of her diagnosis as T-LBL. During this induction therapy, she developed paralytic ileus. One week thereafter, she suddenly exhibited visual disturbance, headache and nausea. Her cerebrospinal fluid was normal. Magnetic resonance imaging showed symmetrical high signal intensities on T2-weighted and fluid-attenuated inversion recovery images, and low signal intensities on T1-weighted images in the cortical and subcortical white matter of the posterior parietal and occipital lobes. Based on these findings, she was diagnosed as having posterior reversible encephalopathy syndrome (PRES). During chemotherapy for hematologic malignancies, some patients with PRES reportedly develop paralytic ileus or tumor lysis syndrome. PRES should be considered in patients with neurological abnormalities following such complications during chemotherapy.

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. Figure 1 VA-Lip HSP47 knocked down Hsp47 in myofibroblasts Figure 1. VA-Lip HSP47 knocked down Hsp47 in myofibroblasts Figure 2 Administration of VA-Lip ameliorated skin fibrosis in chronic GVHD Figure 2. Administration of VA-Lip ameliorated skin fibrosis in chronic GVHD Disclosures No relevant conflicts of interest to declare.

A 76-year-old woman presented with vaginal bleeding and discharge in September 2009. She was admitted to our hospital because a tumor of 5 cm in diameter was found in the vagina in a nearby clinic. She was diagnosed with primary vaginal diffuse large B-cell lymphoma (DLBCL) on biopsy of the tumor because CT, MRI and FDG-PET showed no area of lymphomatous involvement other than the vagina and direct involvement into the bladder. She achieved complete response (CR) after chemotherapy followed by localized radiation therapy, but she had a relapse in the central nervous system (CNS) two months after CR. A study of 57 reported cases of primary vaginal lymphoma suggested that the most common histologic type was DLBCL, and most of patients were in a localized stage and responded well to combination of chemotherapy and radiation therapy. To date, two cases of primary vaginal lymphoma with a relapse in the CNS have been reported. We presumed that direct involvement into the bladder of vaginal lymphoma contributed to the relapse in the CNS in this case.

Introduction: Graft-versus-host disease (GVHD) and infections are the major obstacles to perform allogeneic hematopoietic stem cell transplantation (HSCT). We previously demonstrated that intestinal stem cells (ISCs) and their niche Paneth cells were targeted in GVHD. The intestinal microbial ecology is regulated by Paneth cells through secretion of antimicrobial molecules. Loss of Paneth cells by GVHD frequently leads to intestinal dysbiosis (Eriguchi et al, Blood, 2012). R-spondin1 (R-Spo1) is a growth factor of ISCs and protects ISCs from the damages by conditioning and GVHD (Takashima et al, J Exp Med. 2011). However, the effects of R-Spo1 on Paneth cell biology and intestinal microbial ecology have never been demonstrated. Method: Naïve B6D2F1 (H-2b/d) mice were intravenously injected with R-Spo1 (200 μg/day) or diluent for 6 days. In HSCT models, lethally irradiated B6D2F1 mice were transplanted with 5 × 106 splenocytes plus 5 × 106 bone marrow cells from allogeneic B6 (H-2b) or syngeneic B6D2F1 donors on day 0. Mice were injected with R-Spo1 or diluent from day -3 to -1 and day +1 to +3 after HSCT. On days +4 and +7, small intestine was harvested to evaluate histology and fecal samples were collected to measure fecal levels of alpha-defensin and to determine composition of microbiota by 16S rRNA gene sequencing. Result: In naïve mice, R-Spo1 administration significantly increased Paneth cells in the small intestine (Table). Fate mapping analysis using LGR5-creERT2 × Rosa26Tomato mice to visualize LGR5+ ISCs and their progenies revealed that R-Spo1 did not expand pre-existing Paneth cells but stimulated de novo differentiation of Paneth cells from ISCs. R-Spo1-induced Paneth cells produced antimicrobial peptide alpha-defensin and its activator matrix metalloproteinase-7, leading to a significant increase in fecal levels of alpha-defensin (Table). Next, we evaluated the effects of R-Spo1 on Paneth cells and intestinal microbial ecology in a HSCT model. The numbers of Paneth cells in the small intestine and fecal levels of alpha-defensin were significantly reduced in allogeneic controls compared to those in syngeneic controls on day +7 (Table), resulting in dysbiosis with a significant outgrowth of the family Enterobacteriaceae, including the genus Escherichia, and the family Bacteroidaceae in allogeneic animals (Figure). R-Spo1 protected Paneth cells from GVHD, resulting in significantly more Paneth cells persisting on days +4 and +7 compared to allogeneic controls (Table). Importantly, R-Spo1 restored the fecal levels of alpha-defensin in allogeneic recipients (Table), resulted in preservation of intestinal commensals (Figure). R-Spo1 reduced GVHD mortality as previously reported, but this survival benefit mediated by R-Spo1 was abrogated when mice were treated with broad-spectrum antibiotics including ampicillin, streptomycin, vancomycin and metronidazole, suggesting that R-Spo1-mediated GVHD suppression depended on intestinal commensals. Conclusion: We demonstrated for the first time that R-Spo1 is a growth factor of Paneth cells. R-Spo1 administration in allogeneic recipients restored fecal levels of alpha-defensin, prevented intestinal dysbiosis, and improved the outcome of HSCT in the presence of intestinal commensals. Brief administration of R-Spo1 represents a novel pharmacological approach to modify intestinal microbial ecology. Such strategies to target the intestinal microbiota may have benefits in the treatments of inflammatory bowel diseases other than GVHD. Table. No. of Paneth cells / crypt Fecal levels of alpha-defensin4(ng / g feces) Naïve B6D2F1 Control R-Spo1 3.6 ± 0.2 10.2 ± 0.7* 46.3 ± 5.3 105.3 ± 28.8* Transplant (day4) Allo Allo+R-Spo1 2.2 ± 0.1 3.9 ± 0.2* 25.9 ± 4.0 108.2 ± 12.6* Transplant (day7) Syn Allo Allo+R-Spo1 3.1 ± 0.1 0.7 ± 0.1 1.3 ± 0.2* 18.9 ± 3.8 7.1 ± 2.9 25.6 ± 4.2* Data are shown as mean ± SE. NA; not assessed, *P Figure 1. Figure 1. R-Spo1 prevents dysbiosis in allogeneic recipients: Lethallyirradiated B6D2F1 mice were transplanted from syngeneic B6D2F1 or allogeneic B6 mice. A group of allogeneic recipients were treated with R-Spo1 from day -3 to -1 and day +1 to +3 after HSCT. 16S rRNA sequence analysis was performed to assess the intestinal microbiota on day +7 after HSCT. Disclosures No relevant conflicts of interest to declare.

Histiocytic sarcoma (HS) is a rare but aggressive malignant neoplasm of histiocytic lineage with a poor prognosis. Immunohistochemically, the neoplastic cells are positive for CD163, CD68, and lysozyme, and negative for B and T cell markers. However, molecular studies on the origin of the neoplastic cells remain inconclusive. A 54-year-old woman was admitted to our hospital because of painful swelling of the left knee. Examination revealed generalized lymphadenopathy and splenomegaly. HS was diagnosed according to morphologic and immunohistochemical features observed on biopsy of the left inguinal lymph node. The tumor demonstrated a clonal immunoglobulin heavy chain gene rearrangement and a clonal cytogenetic abnormality including t(14;18) which was confirmed by fluorescence in situ hybridization analysis showing the IgH/BCL2 fusion gene. The neoplastic cells were negative for PAX5, a B cell associated transcription factor, and positive for CEBPβ, a transcription factor mediating macrophage and myeloid differentiation. Positron emission tomography showed disseminated areas of increased 18F-fluorodeoxyglucose uptake in multiple lymph nodes, the liver, spleen, both lungs, both kidneys, and many bony sites. The patient received localized irradiation therapy followed by chemotherapy, she failed to respond and died of the disease progression. The case findings suggest lineage promiscuity or plasticity related to the pathogenesis of HS.