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•GVHD targets organoid-forming bile duct stem cells in a TGF-β–dependent manner.•TGF-β inhibitor protects organoid-forming bile duct stem cells against GVHD and mitigates biliary dysfunction.

•We report the allo-HSCT outcome for adult hemophagocytic lymphohistiocytosis (HLH).•This study included the largest number of cord blood transplantation (CBT) for HLH.•CBT recipients had shorter overall survival than those of other donor sources.•Myeloablative conditioning (MAC) led to worse survival than reduced-intensity one.•Older age, MAC, and CBT correlated with shorter survival in multivariable analysis.

This study aimed to validate the utility of the transplant conditioning intensity (TCI) score in 1714 patients with acute myeloid leukemia (AML) undergoing allogeneic bone marrow or peripheral blood stem cell transplantation (BMT/PBSCT) and assess its applicability to 753 patients with AML undergoing umbilical cord blood transplantation (UCBT) both during first complete remission. Patients classified into a high TCI group accounted for 63% and 56% in the BMT/PBSCT and UCBT cohorts, respectively. In the BMT/PBSCT cohort, the risk of relapse was lower in patients in the high versus intermediate TCI group (P= 0.002), although non-relapse mortality (NRM) did not differ among the three TCI groups. In the UCBT cohort, both relapse and NRM did not differ among the TCI groups. Increasing cutoff points for intermediate and high TCI categories significantly improved the ability to predict relapse and NRM in the BMT/PBSCT cohort (P= 0.030 and 0.006, respectively), and relapse but not NRM in the UCBT cohort (P= 0.005 and 0.364, respectively). These findings highlight the difference in the threshold level of the TCI score for outcome discrimination between European and Japanese cohorts. The TCI scheme appears less effective for UCBT than for BMT/PBSCT.

•Calcineurin inhibitor treatment after allo-HCT suppresses differentiation of terminal Tex and induces transitory Tex.•Transitory Tex maintains responsiveness of donor T cells to PD-1 blockade and causes cGVHD after adoptive transfer into secondary recipients.

•CMV reactivation is associated with a reduced risk of relapse in both AML and ALL.•The mild antileukemia effect of CMV reactivation is independent of acute GVHD but lacks effect modification by baseline characteristics.

•In univariate analysis, the probability of overall survival was significantly greater in patients who developed grade I-II acute graft-versus-host disease (GVHD) and limited chronic GVHD among cord blood transplantation (CBT) recipients, but these effects were not significant among post-transplantation cyclophosphamide-haploidentical hematopoietic stem cell transplantation (PTCy-haplo-HCT) recipients.•In the multivariate analysis, the effect of grade I-II acute GVHD on reducing overall mortality, treatment failure, and nonrelapse mortality was significantly different between CBT recipients and PTCy-haplo-HCT recipients.•Grade I-II acute GVHD was associated with a significant improvement in overall mortality in adults with acute myeloid leukemia undergoing CBT but not those undergoing PTCy-haplo-HCT.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell–mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell–mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1–NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell–mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell–associated immunotherapies.

•Whole-genome CRISPR library screening identified CD48 as a key molecule to define susceptibility to NK-cell–mediated cytotoxicity in ATLL.•ATLL and other PTCLs had reduced CD48 expression compared with normal T cells, implicating a role of reduced CD48 in PTCL pathogenesis.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a promising treatment for adult acute lymphoblastic leukemia (ALL), an intractable hematological malignancy. The trends in allo-HCT outcomes over the past 30 years were examined to verify the efficacy of evolving treatment methods and to identify further challenges. We analyzed data from a registry database that included 8467 adult ALL patients who underwent their first allo-HCT between 1990 and 2019. The period was divided into three 10-year intervals for analysis. Five-year overall survival improved from 48.2% to 70.2% in the first complete remission (CR1), from 25.6% to 44.1% in subsequent CR, and from 10.0% to 22.7% in non-CR. Nonrelapse mortality improved over the 3 decades in each disease stage. However, the relapse rate only improved in CR1 every decade (26.3% to 15.9% in CR1, 33.4% to 32.8% in subsequent CR, and 53.6% to 54.8% in non-CR). Although there were continual improvements in adjusted survival for Philadelphia chromosome (Ph)-positive patients, the improvement was inadequate for Ph− patients with t(4;11), t(8;14), t(14;18), or hypodiploidy. Allo-HCT outcomes for adults with ALL have improved over the past 30 years. Improved outcomes in the future will require more effective prevention of relapse in patients with ALL not in CR1 and in those with high-risk chromosomal abnormalities.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a promising treatment for adult acute lymphoblastic leukemia (ALL), an intractable hematological malignancy. The trends in allo-HCT outcomes over the past 30 years were examined to verify the efficacy of evolving treatment methods and to identify further challenges. We analyzed data from a registry database that included 8467 adult ALL patients who underwent their first allo-HCT between 1990 and 2019. The period was divided into three 10-year intervals for analysis. Five-year overall survival improved from 48.2% to 70.2% in the first complete remission (CR1), from 25.6% to 44.1% in subsequent CR, and from 10.0% to 22.7% in non-CR. Nonrelapse mortality improved over the 3 decades in each disease stage. However, the relapse rate only improved in CR1 every decade (26.3% to 15.9% in CR1, 33.4% to 32.8% in subsequent CR, and 53.6% to 54.8% in non-CR). Although there were continual improvements in adjusted survival for Philadelphia chromosome (Ph)-positive patients, the improvement was inadequate for Ph−patients with t(4;11), t(8;14), t(14;18), or hypodiploidy. Allo-HCT outcomes for adults with ALL have improved over the past 30 years. Improved outcomes in the future will require more effective prevention of relapse in patients with ALL not in CR1 and in those with high-risk chromosomal abnormalities.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a potentially curative therapy for FLT3 internal tandem duplication mutant (FLT3-ITD+) acute myeloid leukemia, but relapse rate is high. A recent study showed that sorafenib, a first generation FLT3 and multikinase inhibitor, enhanced graft-versus-leukemia (GVL) effects against FLT3-ITD+leukemia via interleukin-15 (IL-15) production. However, it remains to be clarified whether this effect could be mediated by selective FLT3 inhibition. We investigated whether gilteritinib, a selective FLT3 inhibitor, could enhance GVL effects against FLT3-ITD transfected Ba/F3 leukemia (Ba/F3-FLT3-ITD) in mice. Oral administration of gilteritinib from day +5 to +14 after allo-SCT reduced expression of the co-inhibitory receptors PD-1 and TIGIT on donor CD8+T cells and enhanced IL-15 expression in Ba/F3-FLT3-ITD. Bioluminescent imaging using luciferase-transfected Ba/F3-FLT3-ITD demonstrated that gilteritinib significantly suppressed leukemia expansion after allo-SCT, whereas it did not impact the morbidity or mortality of graft-versus-host disease (GVHD), resulting in significant improvement of overall survival. In conclusion, short-term administration of gilteritinib after allo-SCT enhanced GVL effects against FLT3-ITD+leukemia without exacerbating GVHD.

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The graft-versus-leukemia (GVL) effect is one of the curative mechanisms of allogeneic hematopoietic stem cell transplantation (allo-HCT). H-Y antigens, which are encoded by Y chromosome, are important targets of the GVL effect. Thus, deletion of the Y chromosome (del[Y]) might cause the GVL effect to deteriorate in a transplantation involving a female donor and male recipient, although the clinical significance of the del(Y) group remains to be elucidated. In this study, we evaluated adult male patients who underwent allo-HCT between 2010 and 2019 in Japan. There were 155 cases in the del(Y) group and 4149 cases without del(Y) who underwent female-to-male allo-HCT. Del(Y) was significantly associated with inferior overall survival (hazard ratio [HR], 1.24; 95% confidence interval [CI], 1.00-1.53; P = .049) and an increased risk of relapse (HR, 1.40; 95% CI, 1.08-1.80; P = .0098) in multivariate analyses. There was no significant difference in nonrelapse mortality between recipients with and without del(Y) (HR, 1.08; 95% CI, 0.769-1.51; P = .67). In contrast, del(Y) was not significantly associated with any clinical outcomes in the cohort of male-to-male allo-HCT. A higher incidence of relapse might have been caused by attenuation of the GVL effect resulting from a lack of H-Y antigens. Because a GVL effect resulting from sex mismatch may not be expected in men with del(Y) who undergo allo-HCT with a female donor, additional post–allo-HCT strategies might be required to prevent disease relapse.

The graft-versus-leukemia (GVL) effect is one of the curative mechanisms of allogeneic hematopoietic stem cell transplantation (allo-HCT). H-Y antigens, which are encoded by Y chromosome, are important targets of the GVL effect. Thus, deletion of the Y chromosome (del[Y]) might cause the GVL effect to deteriorate in a transplantation involving a female donor and male recipient, although the clinical significance of the del(Y) group remains to be elucidated. In this study, we evaluated adult male patients who underwent allo-HCT between 2010 and 2019 in Japan. There were 155 cases in the del(Y) group and 4149 cases without del(Y) who underwent female-to-male allo-HCT. Del(Y) was significantly associated with inferior overall survival (hazard ratio [HR], 1.24; 95% confidence interval [CI], 1.00-1.53; P= .049) and an increased risk of relapse (HR, 1.40; 95% CI, 1.08-1.80; P= .0098) in multivariate analyses. There was no significant difference in nonrelapse mortality between recipients with and without del(Y) (HR, 1.08; 95% CI, 0.769-1.51; P= .67). In contrast, del(Y) was not significantly associated with any clinical outcomes in the cohort of male-to-male allo-HCT. A higher incidence of relapse might have been caused by attenuation of the GVL effect resulting from a lack of H-Y antigens. Because a GVL effect resulting from sex mismatch may not be expected in men with del(Y) who undergo allo-HCT with a female donor, additional post–allo-HCT strategies might be required to prevent disease relapse.

The disease-specific impact of anti-thymocyte globulin (ATG) in allogeneic hematopoietic cell transplantation (allo-HCT) has not been determined. We retrospectively assessed the impact of ATG in allo-HCT using nationwide registry data from the Japan Society for Transplantation and Cellular Therapy. We included patients who received their first allo-HCT between 2007 and 2018 for acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or malignant lymphoma (ML). In total, 8747 patients were included: 7635 patients did not receive ATG and 1112 patients received ATG as GVHD prophylaxis. The median follow-up period of surviving patients was 1457 days. There was no significant impact of pretransplant ATG on the OS or NRM rates in patients with ALL, AML, or ML. In patients with MDS, the probability of 3-year OS was 53.3% in the non-ATG group and 64.2% in the ATG group (P= 0.001). The cumulative incidence rates of relapse and NRM at 3 years were 14.2% and 30.3% (95% CI 27.2–33.3%), respectively, in the non-ATG group and 17.1% and 18.1% in the ATG group (P= 0.15 and P< 0.001). The same finding was observed in a propensity-score matched cohort. Our study suggests that the clinical benefit of ATG could vary among hematological diseases.

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.

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.

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.

F0B7GVHD damages Lgr5+gastric tissue stem cells and causes aerobic bacterial overgrowth in the duodenum.F0B7R-Spondin1 protects Lgr5+gastric tissue stem cells and ameliorates gastric GVHD.

[Introduction] Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is a curative treatment for hematological malignancies but has a risk of a chronic graft-versus-host disease (GVHD) compared to other donor sources. Post-transplant cyclophosphamide (PTCY) has been spread rapidly worldwide from HLA haploidentical-SCT (Haplo-SCT) to HLA matched-SCT. Although PTCY was theoretically thought to preserve non-alloreactive T cells which can contribute to fight infection, recent studies showed that PTCY was associated with the increased incidence of cytomegalovirus (CMV) infection. Letermovir (LTV), a novel anti-CMV agent, was useful for the prevention of CMV reactivation after SCT including Haplo-SCT using PTCY. On the other hand, low-dose antithymocyte globulin (ATG) is an alternative and useful option for GVHD prophylaxis in allo-PBSCT and often used in Japan. However, the risk of post-transplant CMV reactivation and the protective effect of LTV when using low-dose ATG remain to be elucidated.

[Introduction]Long-term in vivopersistence of chimeric antigen receptor (CAR) T-cells is associated with sustained response after CAR-T cell therapy against hematological malignancies. Ferroptosis is a novel form of programmed cell death that refers to iron-dependent regulated cell death resulting from lipid peroxidation and consequent cytomembrane rupture. The iron, lipid, amino acid, and glutathione metabolisms strictly govern the beginning and execution of ferroptosis. Recent studies demonstrated that ferroptosis limited longevity of virus-specific memory T cells (Wang Y: Nat Immunol 2022). However, the role of ferroptosis in CAR-T cells remains to be clarified. We herein explored the role of ferroptosis in CAR-T cells both in vitroand in vivo. [Methods]For CAR-T cell generation, purified T cells from naïve mice were stimulated with anti-CD3/CD28 antibodies for 48 hrs and then incubated with retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hrs, in the presence or absence of 32 µM of ferrostatin-1 (Fer-1), an inhibitor of the iron-dependent production of lipid hydroperoxide, or 10 µM of deferoxamine (DFO), an iron chelator. Flow cytometric analyses of phenotype of CAR-T cells, accumulation of reactive oxygen species (ROS), lipid peroxidation, and cytokine production were performed 96 hrs after T-cell stimulation. ROS were quantified using CellROX Green dye, and lipid peroxidation was evaluated using Lipid Peroxidation Assay Kit (abcam). [Results]First, we tested whether CAR-T manufacturing process could induce ferroptosis. We found a substantial accumulation of ROS in CAR-T cells compared to their pre-culture levels, along with a significant increase in lipid peroxidation, indicating that manufacturing process induces ferroptosis of CAR-T cells. Next, we tested whether ferroptosis inhibition by the lipid peroxidation inhibitor Fer-1, could impact the quality of CAR-T cells. When added to the culture, Fer-1 significantly reduced lipid peroxidation in CAR-T cells and significantly increased the proportion of CAR-T cells with a progenitor phenotype, TCF-1 +Tim-3 -, which is known to be associated with longevity of CAR-T cells. Importantly, ferroptosis inhibition significantly enhanced proliferative capacity and cytotoxicity against syngeneic A20 B-cell leukemia in vitro. Next, we tested whether ferroptosis inhibition by the iron chelator DFO could improve CAR-T cell functions. Similar to the effects of Fer-1, DFO mitigated lipid peroxidation of CAR-T cells in association with increased TCF-1 +Tim-3 -population, while it did not affect ROS accumulation. DFO-treated CAR-T cells demonstrated enhanced cytotoxicity against A20 cells compared with control CAR-T cells, confirming that ferroptosis inhibition improves anti-tumor effects of CAR-T cells. Finally, we examined whether ferroptosis inhibition could enhance antitumor effects of CAR-T cells in vivo. Naïve BALB/c mice were intravenously (i.v.) injected with 1.0 x 10 6luciferase-expressing A20 cells on day 0, followed by i.v. injection of 1.0 x 10 5Fer-1- or DFO-treated CAR-T cells or control CAR-T cells 7 days later. In mice injected with Fer-1-treated CAR-T cells, the numbers of CAR-T cells in peripheral blood on day 16 was significantly higher than those in mice injected with control CAR-T cells, suggesting that ferroptosis inhibition improved the persistence of CAR-T cells in vivo( Fig. 1). In vivobioluminescence imaging showed that both Fer-1-treated and DFO-treated CAR-T cells demonstrated significantly improved anti-tumor effects compared to control CAR-T cells ( Fig. 2). [Conclusions]We for the first time showed that ferroptosis limits the persistence and anti-tumor effects of CAR-T cells, while ferroptosis inhibition induces TCF-1 +Tim-3 -CAR-T cells and improves the therapeutic effect of CAR-T cell therapy.

[Introduction]We have previously shown that T-cell exhaustion of donor alloreactive T cells plays a critical role in tolerance induction after mouse allogeneic hematopoietic cell transplantation (allo-HCT) (Asakura S: JCI 2010). Emerging evidence indicates that T-cell exhaustion is a multistep process, including precursors of exhausted T cells (pTex) with stem cell-like properties, pTex-derived transitory exhausted T cells (transitory-Tex) with potent effector-like functions, and terminally exhausted T cells (terminal-Tex) with the expression of multiple inhibitory receptors and severely impaired functions. We have demonstrated, for the first time, that calcineurin inhibitors (CNIs) inhibit tolerance induction by suppressing the terminal exhaustion of donor T cells while inducing transitory-Tex after allo-HCT in mice (Senjo H: Blood 2023). In contrast, post-transplantation cyclophosphamide (PTCy) efficiently induces tolerance with low incidence of chronic GVHD. In the current study, we evaluated the mechanisms by which PTCy could induce tolerance even with the use of CNIs. [Methods]In mouse haploidentical HCT model,B6D2F1 recipients (H-2 b/d) were lethally irradiated and transplanted with 2 × 10 6T cells together with T-cell-depleted bone marrow cells from C57BL/6 (H-2 b) donors on day 0. CSP (25 mg/kg) was administered from either day 0 (early-CSP) or day 5 (late-CSP), and PTCy (50 mg/kg) was i.p. injected on day 3 after HCT. In clinical study, peripheral blood mononuclear cells were prospectively collected from patients who underwent PTCy-based haplo-PBSCT at our institutions after approval of institutional review. [Results]We have shown that Ly6C is a specific marker for transitory-Tex in mice (Senjo H: Blood 2023). In mice, early-CSP followed by PTCy (CSP→PTCy) significantly expanded Ly6C +PD-1 +TOX lowCD8 +T cells and Ly6C +PD-1 +GZMB +CD4 +T cells on day 14, while late-CSP following PTCy (PTCy→CSP) induced T-cell differentiation towards Ly6C -PD-1 +TOX highterminally exhausted T cells. When CSP→PTCy recipients were injected with anti-PD-L1 monoclonal antibodies, transitory-Tex demonstrated vigorous proliferation, while terminal-Tex did not respond to PD-1 blockade. These findings indicate that CSP administration after PTCy promotes tolerance induction, whereas CSP administered before PTCy inhibits tolerance induction. Next, we explored the specific markers for human transitory-Tex using scRNA-seq of T cells from the patients who underwent PTCy haplo-PBSCT, in which tacrolimus (TAC) was started either from day -1 (early-TAC) or day 5 (late-TAC). UMAP analysis showed that early-TAC expanded cluster 5 (C5) out of six CD8 +clusters (C1-C6) and C8 out of three CD4 +clusters (C7-C9) (Fig.1). C5 highly expressed exhaustion markers such as Toxand Pdcd1, together with effector molecules such as Prf1, Gzmband Cx3cr1, while C8 was characterized with high expression of Gzmb, indicating that C5 and C8 are the human counterparts of transitory-Tex. Analysis of differentially expressed genes showed that Fcgr3a(CD16A) was selectively expressed in C5, while Gzmband Cd74were selectively expressed in C8. These two populations showed robust proliferative responses to TCR stimulation in vitro. ScTCR-seq demonstrated that C5 and C8 clusters showed less TCR diversity compared to other clusters, suggesting that C5 and C8 were enriched with alloreactive donor T cells that clonally expanded post-transplant. FCM confirmed that both CX3CR1 +CD16 +CD8 +T cells and GZMB +CD74 +CD4 +T cells were significantly increased in the early-TAC group compared to the late-TAC group on day 28 (Fig. 2). In the early-TAC group, 2/9 patients developed moderate and severe chronic GVHD, and only 1/9 patient was off-immunosuppressants (Fig 2). In sharp contrasts, in the late-TAC group, no patient developed chronic GVHD, and 7/9 patients were off-immunosuppressants. Importantly, two patients who developed chronic GVHD had a particularly high proportions of CX3CR1 +CD16 +CD8 +and GZMB +CD74 +CD4 +transitory-Tex on day 28 after HCT (Fig.2). [Conclusion]Our study demonstrated that early initiation of CNIs before PTCy induced transitory-Tex and inhibited tolerance induction after PTCy haplo-PBSCT. Administration of CNIs after PTCy is critical for tolerance reduction without chronic GVHD.

Background:Cytomegalovirus (CMV) infection is a major infectious complication following allogeneic hematopoietic cell transplantation (allo-HCT). Although letermovir (LMV) prophylaxis dramatically reduces the incidence of early clinically significant CMV (csCMV) infection, it remains unclear whether it has a beneficial effect on nonrelapse mortality (NRM) and overall survival (OS). Additionally, because the incidence of csCMV infection increases after completing LMV prophylaxis, the impact of late csCMV infection beyond 100 days after transplantation on posttransplant outcomes should also be verified.

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.

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.

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.

•The adjusted 5-year net survival (NS) after HCT improved over two decades•The adjusted 5-year NS improved among all heme-malignancies in all age groups•The improvement of adjusted 5-year NS was significant in CML and ALL•Both adjusted 1-year NS and adjusted NS in 1-5 years after HCT improved

Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) is a rare mesenchymal tumor which occurs in immunocompromised patients. The immune status is an important factor in the treatment of EBV-SMTs, but the efficacy of antiretroviral therapy (ART) is not elucidated in acquired immune deficiency syndrome (AIDS) related EBV-SMTs. Here, we report the first successful case of a 29-year-old man with hepatic AIDS related EBV-SMT treated with ART solely. Positron emission tomography scan was useful for the evaluation of disease status. Recent advances in ART that enables to restore patient’s immune status rapidly may change the treatment strategy in AIDS related EBV-SMT.

Since cord blood (CB) units are usually selected based on the cell dose /kg, overweight (25 kg/m2≤ body mass index (BMI) < 30 kg/m2) and obese (30 kg/m2≤ BMI) recipients tend to have difficulty in getting appropriate CB units. In general, actual body weight (ABW) is used for CB unit selection. However, ideal body weight (IBW) has been reported to be more closely correlated with successful engraftment after autologous, allogeneic bone marrow, and peripheral blood stem cell transplantation than ABW.

Background and aims

[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 6splenocytes plus 5 x 10 6bone 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 Mmp7expression, 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.

[Introduction]Cytomegalovirus (CMV) infection is a common viral infection in recipients of allogeneic hematopoietic stem cell transplantation (allo-SCT). Early CMV reactivation after allo-SCT is associated with worse non-relapse mortality (NRM) and overall survival (OS). Recently, T-cell replete HLA-haploidentical SCT using post-transplant cyclophosphamide (PTCy-haplo SCT) has been developed and spread rapidly worldwide. Rationale of this strategy is assumed to be selective and cytotoxic depletion of alloreactive T cells which are responsible for graft-versus-host disease (GVHD), while preserving non-alloreactive T cells which can contribute to fight infections. However, recent studies showed that PTCy-haplo SCT was associated with the increased incidence of CMV infection. Letermovir (LET), a novel anti-CMV agent, which inhibits the CMV DNA terminase complex, was approved for the prevention of CMV reactivation in allo-SCT recipients in 2018 in some countries including Japan based on the result of a phase 3 trial. Our facility performs LET prophylaxis in allo-SCT recipient if either donor or recipient is seropositive CMV. Although LET is effective for the prevention of CMV reactivation in allo-SCT recipients, the clinical effectiveness of LET prophylaxis in PTCy-haplo SCT is not well elucidated. Based on these things, we retrospectively evaluated the efficacy of LET prophylaxis in PTCy-haplo SCT.

Arai: ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Kyowa Kirin CO., LTD.: Honoraria, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Elsai Co Ltd: Research Funding; Abbott Japan LLC: Honoraria; Nippon Shinyaku Co. Ltd: Honoraria, Research Funding; Otsuka Pharmaceuticals Co. Ltd: Research Funding; Novartis Pharma KK: Honoraria; Takeda Pharmaceuticals Co Ltd: Honoraria, Research Funding; Shionogi & Co Ltd: Research Funding; Asahi Kasri Pharma Corporation: Research Funding; Sanofi: Honoraria; Pfizer japan: Honoraria; Astellas Pharma Inc.: Honoraria. Yamamoto: Bristol-Myers Squibb Company: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; NIPPON SINYAKU CO., LTD: Honoraria; Novartis Pharma: Honoraria; ONO PHARMACEUTICAL CO.: Honoraria; Otsuka Pharmaceutical: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria. Nakamae: Astellas Pharma Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Simon-Kucher & Partners: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; Novartis: Honoraria, Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb　Company: Honoraria, Research Funding; Alexion: Research Funding; PPD-SNBL K.K: Research Funding; CMIC HOLDINGS Co., Ltd: Research Funding. Ichinohe: Repertoire Genesis Inc.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria; Celgene: Honoraria; Zenyaku Kogyo Co.: Research Funding; Takara Bio Inc.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Nippon Shinyaku Co: Research Funding; Ono Pharmaceutical Co.: Honoraria, Research Funding; Kyowa Kirin Co.: Honoraria, Research Funding; FUJIFILM Wako Chemicals.: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Eisai Co.: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co.: Honoraria; AbbVie Pharma: Research Funding; Astellas Pharma: Honoraria, Research Funding. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria; Meiji Seika Pharma Co, Ltd.: Honoraria.

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.

Nakagawa: AbbVie GK: Research Funding; Takeda Pharmaceutical Company: Research Funding. Teshima: Gentium/Jazz Pharmaceuticals: Consultancy; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Pfizer Inc.: Honoraria; Nippon Shinyaku Co., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Fuji pharma 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; TEIJIN PHARMA Limited: Research Funding; Astellas Pharma Inc.: Research Funding; Bristol Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Other; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Sanofi S.A.: Research Funding.

•In this updated Japanese study, bone marrow transplantation (BMT) recipients still have shown superior survival to peripheral blood stem cell transplantation (PBSCT) recipients from matched related donors (MRDs).•The PBSCT without anti-thymocyte globulin (ATG) cohort showed a higher incidence of grade III–IV, stage 2–4 gut, high-risk, and steroid-refractory acute graft-versus-host disease (GVHD) than BMT.•Prophylactic ATG has the potential to reduce severe acute GVHD of PBSCT recipients from MRDs.

Goblet cells play a protective role against GVHD after allogeneic stem cell transplantation in a Lypd8-dependent manner.