Treatment Of aGVHD Research Articles

AMPK Controls Glycolytic Compensation in Gvhd-Causing T Cells

Background: The heightened metabolic requirements of alloreactive T cells following allogeneic stem cell transplantation represents a promising area for therapeutic intervention. We have shown that during the initiation of acute graft-versus-host-disease (aGVHD), murine T cells upregulate activity of AMP-activated protein kinase (AMPK), a cellular energy sensor that regulates oxidative and mitochondrial metabolism. Further, transfer of allogeneic T cells deficient in AMPK (AMPK KO) increased recipient survival and decreased disease severity while still preserving leukemia clearance. In the current studies, we demonstrate a novel role for AMPK in controlling glycolytic compensation, both in vitro and vivo, and provide a mechanism as to why cells lacking AMPK are unable to perform this vital function. Together, these data highlight the potential clinical utility in targeting metabolic decision-making to curtail T cell-mediated inflammatory responses, with significant therapeutic implications for the prevention and treatment of aGVHD. Methods: AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients and recovered on day 7 post-transplant. To delete AMPK in primary human T cells, T cells were isolated from healthy donors and electroporated with Cas 9 ribonucleoprotein complexes targeting the AMPKa1 locus. Glycolytic compensation was assessed by measuring extracellular acidification rates (ECAR) using the Agilent Seahorse analyzer and a mitochondrial stress kit. Intracellular cytokine expression for IFNgamma was assessed in day 7 murine T cells following re-stimulation with F1 splenocytes in the presence of Brefeldin A. Aldolase A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzymatic assays were run on cell lysates with Biovision-Abcam kits according to manufacturer instructions. To measure GAPDH activity, T cells were positively selected for CD45.1 from day 7 recipient spleens, followed by cell lysis and detection of enzymatic activity. Results: AMPK KO murine T cells recovered on day 7 were unable to mediate a compensatory increase in glycolysis following inhibition of the electron transport chain (Figure 1A), (-6.499±5.156 vs 29.11±7.064, p<0.0001). Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following human T cell expansion in vivo in a xenogeneic model of GVHD. Immunoprecipitation of proteins from day 7 murine T cells, using an antibody specific to phosphorylated AMPK targets, recovered lower levels of multiple glycolytic enzymes including aldolase, enolase, pyruvate kinase M, and GAPDH (Figure 1B). Functionally, murine T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation and a decrease in GAPDH activity on day 7 post-transplant. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of IFNγ upon antigenic re-stimulation with F1 splenocytes. Discussion: Our results demonstrate that in addition to influencing oxidative metabolism, AMPK also plays a role in glycolytic metabolism during GVHD in both murine and human T cells. Further, our data suggest that the more prominent lesion in human T cells post-transplant was in glycolysis , a result supported by the novel observation that a set of glycolysis-related proteins are immunoprecipitated together at much lower rates in the absence of AMPK. These findings suggest a model in which AMPK phosphorylation provides a nidus for important interactions among glycolytic enzymes and that in the absence of AMPK, these interactions are lessened, and glycolysis is consequently reduced. Important to our understanding of GVHD pathogenesis, rates of T cell glycolysis can be directly linked to IFNγ production, where reduced glycolytic activity frees GAPDH to sequester IFNγ transcripts and thereby limit IFNγ protein translation. Altogether, these results document a prominent role for AMPK in controlling T cell glycolytic activity during GVHD development and offer an additional mechanistic explanation for the decreased severity of GVHD seen following transplantation of AMPK KO T cells. Consistently similar results in human xenogeneic T cells provides a further strong rationale for development of AMPK inhibition as a clinically translatable strategy for GVHD prevention and treatment.

Palmitoleic Acid Extracted from Fish Oil Inhibit aGVHD By Immune Regulation

Objective: This study aims to explore the immunosuppressive effect of palmitoleic acid (POA), derived from fish oil extract, on lymphocytes in vitro and its potential impact on the clinical outcome and survival prognosis of rodents with acute graft-versus-host disease (aGVHD). Methods: In vitro experiments involved using CCK8 to determine the influence of various concentrations of POA on the growth and proliferation of lymphocytes. Flow cytometry was employed to assess POA's inhibitory effect on T lymphocyte activation triggered by fluorobol ester (PMA) and ionomycin (IM). For evaluating the therapeutic effect of POA on aGVHD, C57bl/c mice were used as donors and BALB/C mice as recipients. The aGVHD mouse model was established to observe the impact of POA on aGVHD treatment, survival, and prognosis. Results: In vitro experiments demonstrated that POA concentrations ranging from 250uM to 500uM inhibited the proliferation of PMA and IM-activated T lymphocytes and reduced the proportion of CD4+CD25+ cells without causing significant cytotoxicity. The aGVHD mouse model exhibited clinical symptoms like bristling, depilation, anal redness and swelling, and loose stools, along with characteristic pathological changes in the liver and small intestine. Treatment with POA significantly extended the survival period and reduced the clinical score of aGVHD in the model mice. Conclusion: Palmitoleic acid derived from fish oil shows promise as a potential treatment for acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation, warranting further research on its immunosuppressive properties. Keywords: Fish oil extract, palmitoleic acid, acute graft-versus-host disease, allogeneic hematopoietic stem cell transplantation, immunosuppression.

Iron Dys-Homeostasis Contributed to Liver Gvhd after Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD), which results from immunological attack to recipient organs or tissues (such as the skin, liver and gut) by donor allogeneic T cells, limits the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). A correlation of high serum ferritin levels with increased mortality after allo-HSCT has been suggested by several analyses. While the role of iron dys-homeostasis in the risk of GVHD remains unclear. Whether iron dys-homeostasis contributes to the development of GVHD or iron dys-homeostasis is only a result of severe GVHD remains unknown. To assess the status of iron metabolism during GVHD following allo-HSCT, we established aGVHD mouse model by transplantation of T cell-depleted bone marrow (TCD BM) with or without splenic T cells from C57BL/6 (B6) mice to BALB/c mice after being lethally irradiated. By 28 days post-transplantation, when aGVHD stable developed, the serum iron level was significantly higher in aGVHD group compared with control. Meanwhile, ferrous-ion load in aGVHD-involved liver was also increased, while the total iron load did not differ significantly in aGVHD-involved liver compared with control. The ferrous-ion load as well as the total iron load in intestine, lung and spleen were comparable between aGVHD group and control group. To further determine the dysregulation of iron metabolism during liver aGVHD, we detected the expression levels of pivotal iron regulatory genes in liver and duodenum. The expression levels of transferrin receptor 1 ( Tfr1) and divalent metal transporter 1 ( Dmt1) were significantly upregulated in aGVHD-involved liver, which contributed to ferrous-ion overload in the liver aGVHD. Besides, Ferroportin 1 was down-regulated in aGVHD-involved duodenum, which is the result of the upregulated hepcidin ( Hamp1) in liver. Our data suggested that the increased serum iron in aGVHD was not related to iron absorption by duodenum, but may be associated with ineffectively use of iron, which was caused by the impaired marrow function after myeloablative conditioning. To explore whether ferrous-ion overload in liver would lead to induction of reactive oxygen species (ROS) via the Fenton reaction, and eventually results in liver damage during aGVHD, we detected the activity of superoxide dismutase (SOD) and the load of thiol and lipid peroxidation product, malondialdehyde (MDA). The activity of SOD and thiol load both decreased significantly in aGVHD-involved liver, while the serum MDA level increased in aGVHD group, indicating that iron dys-homeostasis may aggravate liver GVHD damage by induction of lipid peroxidation injury. Finally, to explore how iron overload affect GVHD, we fed recipient mice with high-iron diet and low-iron diet since three weeks before transplantation, respectively. The survival rates and GVHD score were significantly worse in high-iron diet group compared with normal-iron diet group. Interestingly, low-iron diet didn't show survival benefit compared with the normal-iron diet. This suggests that excessive iron deficiency is not beneficial for preventing and treating GVHD. Thus, we applied apo-transferrin (apo-Tf) administration to receipt mice, which can render the poorly soluble non-transferrin bonding iron (NTBI) into a soluble transferrin-bound form, and prevent iron from being rapidly uptake by the liver. The administration of apo-Tf alleviated the organ damage during aGVHD, and reduced the infiltration of CD4 +IFN-γ +T cells, CD4 +IL17 +T cells and CD8 +IL17 +T cells in target organs such as intestine, liver and lung. These data suggested that ferrous-ion overload leads to the increase of NTBI, which aggravate GVHD tissue damage. Our findings reveal a novel mechanism of iron dys-homeostasis during the development of liver aGVHD, which may reveal promising therapeutic targeting in the prevention and treatment of liver aGVHD.

Budesonide Added to Post-Transplantation Cyclophosphamide, Tacrolimus and Mycophenolate Mofetil for Prevention of Graft-Versus-Host Disease

Background: Topically active corticosteroids have been shown to effectively treat inflammatory bowel disease with a systemic steroid-sparing effect. However, previous studies have not demonstrated their efficacy in preventing graft-versus-host disease (GVHD) when combined with the standard GVHD prophylaxis regimen consisting of calcineurin inhibitors and methotrexate. We hypothesized that the addition of orally administered ileal-controlled-release budesonide to the post-transplant cyclophosphamide (PTCy)-based regimen could effectively prevent severe acute gastrointestinal GVHD. In this study, we present outcomes of the first 43 consecutively treated patients (pts) with the PTCy-tacrolimus (T), mycophenolate mofetil (M) and budesonide (B) (PTCy-TMB) as a GVHD prophylaxis regimen after allogeneic stem cell transplantation (alloSCT). Methods: Pts with hematologic malignancies received their first alloSCT at our institution between 05/2020-04/2023. Thirty one pts received conditioning with fludarabine (160 mg/m 2) + melphalan (100-140 mg/m 2) + TBI (2-4Gy) or thiotepa 5 mg/kg. Twelve pts received fludarabine (150mg/m 2) + cyclophosphamide (29mg/kg) and TBI 4Gy ± ATG (4.5 mg/kg). GVHD prophylaxis consisted of Cy (50 mg/kg on D+3 and +4), T (trough goal 6-10 ng/mL), M (15mg/kg TID) and budesonide (3 mg TID) starting on D+5 and continued until D+90 followed by a weekly taper if no acute GVHD (aGVHD) occurred. T was continued for at least 6 months followed by a taper, unless otherwise indicated. Results: The median age was 50 years (range 22-73), 39 pts had hematologic malignancies including AML (N=14), ALL (N=11) and NHL (N=8). Seven pts (16%) classified as high/very-high DRI. The median of HCT-CI was 4 (range 0-7). Donors were haploidentical (N=32), MSD (N=8), MUD (N=1), MMSD (1) and MMUD (N=1). PBSCs were used in 28 pts (65%). Thirty pts (70%) received RIC regimens. Median follow-up was 407 days (range 91-996 days). At day 100, the cumulative incidence (CI) of grade 2 aGVHD was 7.0% (95%CI 1.8-17.2), while none developed grade 3-4 aGVHD (Figure 1). One patient had severe chronic GVHD (bronchiolitis obliterans) with CI at 2 years of 3.2% (95%CI 0-14.0). The CI of any systemic steroid use at D+100 and D+180 was 30.2% (95%CI 17.4-44.1) and 44.8% (95%CI 28.9-59.5), respectively. Among the pts who received treatment for aGVHD, the median cumulative systemic steroid exposure (in prednisone equivalents) was 1,318 mg (IQR 623-3,775) at D+100 and 1,325 mg (IQR 525-1,640) at D+180, with a median treatment duration of 40 days (IQR 25-110). At 1 year, the OS, PFS, GRFS, relapse and NRM were 79.6% (95%CI 61.4-89.9), 69.8% (95%CI 51.6-82.3), 66.6% (95%CI 48.4-79.8), 15.3% (95%CI 6.2-28.2) and 14.9% (95%CI 5.3-29.1), respectively. The 1-year CI of newly diagnosed/worsening hyperglycemia and Cushing syndrome was 18.2% (95%CI 7.9-31.9) and 5.0% (95%CI 0.9-14.2), respectively. 3 pts (7%) required insulin treatment. The median absolute lymphocyte count (ALC) at day 30, 90 and 180 was 200/mcL, 300/mcL and 500/mcL, respectively, with no differences in the median ALC at D+180 among pts with and without GVHD. Median cell numbers of CD3 +, CD3 +/CD4 +, CD3 +/CD8 +, CD19 + and CD56 + at 1, 3, 6 months post-transplant were D+30 - 63/mcL, 18/mcL, 34/mcL, 1/mcL, 87/mcL, D+90 - 111/mcL, 64/mcL, 34/mcL, 9/mcL, 216/mcL and D+180 - 269/mcL , 78/mcL , 126/mcL , 7/mcL, 189/mcL, respectively. After 10/2022 pts were tested routinely for Reg3α and ST2 (baseline and retested if developed any signs/symptoms of aGVHD). Among 18 tested pts at baseline, the median number (range) of Reg3α and ST2 at baseline was 28 ng/mL (16-90) and 23,308 pg/mL (4,678-56,226), respectively. Three low-risk pts developed grade 1 aGVHD (N=2) and grade 2 aGVHD (N=1). Fourteen pts with GI sign/symptoms after transplant showed median number (range) of Reg3α and ST2 of 78 ng/mL (16-837) and 34,904 pg/mL (13,329-160,000), respectively. Only one pt with high-risk aGVHD algorithm developed grade 2 aGVHD (Figure 1). Conclusions: The addition of budesonide to PTCy-based GVHD prophylaxis regimen (PTCy-TMB) is feasible and appears to be effective in preventing severe GI aGVHD, without compromising transplant outcomes and immunologic reconstitution.

Real-World Data Suggest Effectiveness of Allogeneic Mesenchymal Stromal (MSC-FFM) Cells in Ruxolitinib-Refractory Acute Graft-Versus-Host Disease

Introduction: Acute graft-versus-host disease (aGvHD) remains the leading cause of treatment-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Current first-line treatment of aGvHD is with high-dose corticosteroids, the standard for steroid-refractory aGvHD (SR-aGvHD) is the Janus kinase (JAK) inhibitor ruxolitinib. Outcomes for patients with ruxolitinib-refractory or -intolerant aGvHD (RR-aGvHD) are poor. Thus in a recent real-world study of adult patients with RR-aGvHD (Abedin BJH 2021) median survival was 28 days (21 days for ruxolitinib refractoriness, 50 days for ruxolitinib intolerance), the probability of overall survival (OS) at 6, 12, and 24 months were about 20%, 16%, and 10%, respectively. We assessed real-world outcomes of patients with RR-aGvHD treated with the random-donor allogeneic MSC preparation “MSC-FFM,” available via Hospital Exemption in Germany. Patients: Between December 2017 and February 2023, 156 patients, including 33 children and adolescents (<18 years of age), received MSC-FFM for RR-aGvHD. Thirty-two German sites contributed 139 patients, while the remaining 17 patients came from seven transplant centers in France, Hungary, Norway, Sweden, and Switzerland. The adult cohort (n=123) consisted of 41% females, with an age range of 19 to 79 years (median 55 years). Except for one patient, all had been diagnosed with malignant diseases (missing information, n=3). Among the adult patients with malignant diseases, 88% (n=105) received HSCT for acute myeloid leukemia (AML), advanced myelodysplastic syndrome, myeloproliferative neoplasm or lymphoma, and 12% (n=15) for acute lymphoblastic leukemia (ALL). In the pediatric cohort (n=33), 48% of patients were females, with an age range of 0 to 17 years (median 9 years). Overall, 58% of pediatric patients (n=19) had malignant diseases as the indication for allogeneic HSCT, 42% (n=8) having ALL and 32% (n=6) with AML. The remaining 42% of pediatric patients (n=14) had non-malignant diseases as the indication for HSCT. Of the adult patients, 6 (4.9%) had grade II, 52 (42.3%) had grade III, and 63 (51.2%) had grade IV aGvHD, with no specification reported for 2 patients (1.6%). In children, 2 (6.1%) had grade II, 10 (30.3%) had grade III, and 21 (63.6%) had grade IV Treatment: MSC-FFM is manufactured from pooled bone marrow mononuclear cells from eight HLA-disparate healthy donors. MSCs are selected by plastic adherence, expanded until the end of passage 3, then frozen in saline-albumin with DMSO. The recommended dosing for MSC-FFM is 1-2 million cells/kg body weight (BW), for four weekly doses. The median dose administered was 1.18 million cells/kg BW, with a median number of four doses and a median inter-dose interval of 7 days. Results: Safety: Tolerability of MSC-FFM was good, with only five adverse drug reactions reported in three adult patients (chills, BK virus cystitis, increase in C-reactive protein [reported twice in one patient], nausea) and did not result in cessation of MSC treatment or dose reductions. Response: The overall response rate at day +28 was 49% (95%-CI: 41-58%), with 45% (36-55%) in adults and 64% (45-80%) in children. Most responses were durable, resulting in an overall response rate of 49% (41-57%) for both adults and children at 2 months, and 40% (31-48%) at 6 months. Survival: Overall survival at 6, 12, and 24 months was 47% (38-56%), 35% (27-44%), and 29% (21-38%) for adults, and 59% (40-74%), 42% (24-58%), and 37% (19-54%) for children, respectively (Table 1). Median overall survival was 5.8 months. These outcomes compare favourably to published overall survival estimates for adult RR-aGvHD patients not treated with MSC-FFM (Abedin BJH, see above). Summary: The unique MSC-preparation MSC-FFM appears to be effective against RR-aGvHD.

Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR=0.07 (0.01-0.35); P<0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA+MTX [HR=0.19 (0.03-1.1), P<0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P<0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

Novel developments in the prophylaxis and treatment of acute GVHD

AbstractAcute graft-versus-host disease (aGVHD) is a major life-threatening complication after allogeneic hematopoietic cell transplant. Traditional standard prophylaxis for aGVHD has included a calcineurin inhibitor plus an antimetabolite, whereas treatment has relied mainly on corticosteroids, followed by multiple nonstandard second-line options. In the past decade, this basic framework has been reshaped by approval of antithymocyte globulin products, the emergence of posttransplant cyclophosphamide, and recent pivotal trials studying abatacept and vedolizumab for GVHD prophylaxis, whereas ruxolitinib was approved for corticosteroid-refractory aGVHD treatment. Because of this progress, routine acute GVHD prophylaxis and treatment practices are starting to shift, and results of ongoing trials are eagerly awaited. Here, we review recent developments in aGVHD prevention and therapy, along with ongoing and future planned clinical trials in this space, outlining what future goals should be and the limitations of current clinical trial designs and end points.

Pathophysiology of gastrointestinal acute graft-versus-host disease and the potential role of glucagon-like peptide 2.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after allogeneic haematopoietic cell transplantation, with gastrointestinal (GI) tract involvement (GI aGVHD) being one of the leading causes of morbidity and mortality. Whilst systemic steroids are the standard first-line treatment for aGVHD, approximately 50% of patients become steroid refractory (SR), which is associated with poor outcomes. Existing options for SR-GVHD are limited, and there is a significant unmet need for new non-immunosuppressive treatment approaches in patients with GI aGVHD. Here, we review newer concepts in the pathogenesis of GI aGVHD and present the evidence for the role of glucagon-like peptide 2 (GLP-2) in maintaining and protecting GI epithelial cells, including the enterocytes, intestinal stem cells and Paneth cells, which are direct targets of aGVHD. Finally, we discuss the therapeutic rationale for GLP-2 treatment as a tissue regeneration approach and the potential use of the novel GLP-2 analogue apraglutide as an adjunctive treatment for GI aGVHD.

Human umbilical cord mesenchymal stem cells ameliorate acute graft-versus-host disease by elevating phytosphingosine

Acute graft-versus-host disease (aGVHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (allo-HSCT) and even leads to death after HSCT. Human umbilical cord mesenchymal stem cells (HUCMSCs) are effective in aGVHD treatment and have mild side effects, but the underlying mechanisms remain unclear. Phytosphingosine (PHS) is known to prevent loss of moisture from the skin; regulate epidermal cell growth, differentiation, and apoptosis; and exert bactericidal and anti-inflammatory effects. In this study, our results revealed the efficacy of HUCMSCs in alleviating aGVHD in a murine model, with striking changes in metabolism and significantly elevated PHS levels due to sphingolipid metabolism. In vitro, PHS reduced CD4+ T cell proliferation, enhanced apoptosis and reduced T helper 1 (Th1) cell differentiation. Transcriptional analysis of donor CD4+ T cells treated with PHS revealed significant decreases in transcripts regulating proinflammatory pathways, such as NF-κB. In vivo, the administration of PHS significantly ameliorated aGVHD development. Collectively, these beneficial effects indicate proof-of-concept that sphingolipid metabolites could be a safe and effective means to prevent aGVHD in the clinic.

Clinical analysis of 76 patients with severe aplastic anemia treated with haploid hematopoietic stem cell transplantation

Objective: The purpose of this study is to determine the efficacy of haploidentical donor hematopoietic stem cell transplantation in the treatment of severe aplastic anemia. Methods: The clinical data of 76 patients with severe aplastic anemia (SAA) patients who underwent haplo-HSCT from December 2014 to October 2020 were selectively analyzed. There were 50 males and 26 females with a median age of 16 (3-52) years old. There were 49 SAA-Ⅰ patients, 18 SAA-Ⅱ patients, and 9 patients with hepatitis-associated aplastic anemia. There were 15 cases of bone marrow put together with peripheral blood stem cell transplantation and 61 cases of peripheral blood stem-cell transplantation. Conditioning regimens were Cyclophosphamide (CY) + Fludarabine (Flu) + ATG for 46 patients and Busulfan (Bu) + CY+Flu+ATG for 30 patients. Results: Three patients died during the myelosuppressive phase following transplantation, and 73 patients had a median time of neutrophil engraftment of 12 (9-21) days; in addition to 3 patients who died early, 8 patients did not obtain platelet reconstruction after transplantation, and 65 patients had platelet engraftment with a medium time of 14 (9-90) d. The incidence of primary graft failure was 10.9% and the incidence of secondary graft failure was 5.5%. The incidence of Ⅱ-Ⅳ acute graft-versus-host disease (aGVHD) was 38.4%, the incidence of Ⅲ-Ⅳ aGVHD was 16.4%, the incidence of chronic graft anti-host disease (cGVHD) was 35.8%, and the incidence of extensive cGVHD was 22.4%. The medium follow-up time was 19.5 (1-75) months, the prospective overall survival (OS) for 2 years was (78.6±5.0) %, the failure-free survival (FFS) was (75.9±5.1) %, and the transplant-related mortality was (20.2±4.9) %. Multi-factor analysis revealed that the patient older than 35 years old, Ⅲ/Ⅳ aGVHD, HCT-CI≥3, the pre-transplant ferritin ≥1 500 μg/L, the number of neutrophils >1×10(9)/L at the time of onset were risk factors affecting OS (P=0.008, 0.008, 0.014, 0.004, 0.027) . Patients with graft failure had lower OS and FFS than other patients (P<0.001) . Conclusion: Haplo-HSCT is an effective method for treating SAA in children, adolescents, and young patients, and the occurrence of severe aGVHD and severe infection, as well as graft failure, are the main causes of survival rate. The prevention and treatment of severe aGVHD and infection are essential to improve efficacy.

USP11 plays a critical role in the onset and progression of acute graft-versus-host disease：Novel target for precision therapeutics

Acute graft-versus-host disease (aGvHD) is considered a result of “cytokine storm.” Targeted therapeutic interventions on cytokines via ubiquitination regulatory pathways may provide a potential approach for aGvHD treatment. Ubiquitin-specific peptidase 11 (USP11) has been reported to play key roles in a variety of physiopathological processes by regulating the stability and function of several vital protein molecules. However, its role in aGvHD remains unclear. In this study, we identified USP11 was associated with aGvHD in patients. In the aGvHD mouse model, the colon and liver were more seriously affected in recipient mice who received USP11 wt bone marrow (BM) cells and eased after the donor was treated with a USP11 inhibitor or received USP11 ko BM cells. In mouse models, IL-6 was identified as a major effecter in accelerating aGvHD induced by USP11. In the cell model, IL-6 mRNA transcript was affected by USP11. In addition, USP11 also inhibited IL-6 degradation by affecting IL-6 ubiquitination. Furthermore, the positive correlation between USP11 and IL-6 was confirmed in the GvHD patients’ samples. Collectively, all results indicated that USP11 played a critical role in the onset and progression of aGvHD. USP11 might be a potential target for aGvHD treatment.

EPC infusion ameliorates acute graft-versus-host disease-related endothelial injury after allogeneic bone marrow transplantation.

Graft-versus-host disease (GVHD) damages vascular endothelium. Endothelial progenitor cell (EPC) can differentiate to endothelial cell and promote angiogenesis, but its role in endothelial damage in GVHD is unclear. In this study, we intend to assess whether EPC infusion promotes the repair of endothelial injury in GVHD mouse model. Male BALB/c mice were randomly divided into 5 groups: control group, total body irradiation group (TBI group), allogeneic bone marrow transplantation group (Allo-BMT group), acute graft versus host disease group (GVHD group), EPC infusion group (GVHD+EPC group) followed by analysis of mice survival, acute GVHD (aGVHD) score, T cell infiltration by immunofluorescence, as well as continuity of vascular endothelium in liver. Compared with Allo-BMT group, the clinical and pathological score of aGVHD mice were higher. On day 21 after transplantation, a large number of mononuclear cell infiltrations were seen in the target tissues of aGVHD mice and mice died within 30 days. In addition, aGVHD group also presented increased subendothelial infiltration of CD3+ T cells in the liver, decreased VE-cadherin expression and elevated major histocompatibility complex (MHC) II molecule expression in the endothelium. Moreover, expression of MHC-II molecule increased in endothelial cell after irradiation injury and LPS stimulation, indicating abnormally activated endothelial cell with antigen-presenting function. Interestingly, infusion of EPC reduced the clinical and pathological score of aGVHD, decreased infiltration of mononuclear cells, improved survival as well as upregulated VE-cadherin and downregulated MHC-II molecule. EPC infusion can mobilize to affected endothelium to decrease the infiltration of T cells and pathological endothelial activation contributing to ameliorating the damage of endothelium. EPC infusion combined with bone marrow transplantation might be a perspective strategy for the prevention and treatment of aGVHD.

Efficacy and Safety of MSCs Plus Basiliximab for the Treatment of Steroid-Resistant aGVHD after Haploidentical Stem Cell Transplantation: A Multicenter, Randomized, Open-Label Trial

Efficacy and Safety of MSCs Plus Basiliximab for the Treatment of Steroid-Resistant aGVHD after Haploidentical Stem Cell Transplantation: A Multicenter, Randomized, Open-Label Trial

Phase I Study of Baricitinib Gvhd Prophylaxis in HLA-Matched, Peripheral Blood Allogeneic Hematopoietic Cell Transplant

Acute graft versus host disease (aGVHD) is a leading cause of non-relapse mortality after allogeneic peripheral blood hematopoietic cell transplant (PB-HCT). JAK inhibition has been shown to be an effective treatment for steroid refractory aGVHD. We performed a single arm, open label, phase I trial to evaluate the safety of a best-in-class balanced JAK1/2 inhibitor, baricitinib (Bari), for the prevention of aGVHD after HLA-matched, allogeneic PB-HCT (Choi et al. Leukemia2018; Kim et al. Leukemia, 2022). The study enrolled 24 subjects into two dosing cohorts (2mg and 4mg daily) from 2019 - 2022. The data cut-off for this analysis was June 1, 2022. The primary objectives were to determine the incidence of graft failure at 28 days post HCT and the cumulative incidence of grade III-IV acute GVHD by day 100. The secondary objective was to determine day 180 treatment related mortality. Subjects were treated with Bari 2mg or 4mg (n = 12/cohort) oral daily from days -3 to +100, followed by a taper. All subjects received standard of care GVHD prophylaxis with tacrolimus and mini-methotrexate +/- thymoglobulin (n=19) or post-transplant cyclophosphamide (pTCy) plus mycophenolate mofetil and tacrolimus (n=5). At data cut, the median follow up was 320 days (63-368 days). The median age was 57 (range 28-72), and diagnoses included AML (16), MDS (6), and ALL (2). All subjects with AML were required to be in CR/CRi and 8 were MRD positive by flow cytometry at the time of transplant. Ten recipients had PB-HCT from sibling donors and 14 had PB-HCT matched unrelated donors. Conditioning included myeloablative regimens (MAC, n=19) or reduced intensity (RIC, n=5). GVHD prophylaxis included tacrolimus /methotrexate for all subjects in the 2mg cohort and pTCy was used in 5 / 12 subjects in 4mg dosing cohort. Thymoglobulin was allowed for MUD recipients (n=5). No events of primary graft failure were observed. The median ANC engraftment was 12.5 days (range 12-21). The median platelet engraftment was 20 days (range 13-52). Treatment related mortality at day 180 was 8% (2/24). The cumulative incidence of grade III/IV aGVHD at day 100 was 4% (1/24, occurring in the 4mg cohort), while the overall incidence of grade III/IV aGVHD at any time was 12.5% (3/24) and two of these cases occurred after Bari discontinuation. The one case of grade III aGVHD prior to day 100 was a steroid-responsive lower GI GVHD that occurred at day 21 in the 4mg cohort and did not recur with taper of immune suppression and Bari. Grade II - IV aGVHD occurred in 33% (8/24, 4 in the 2mg cohort and 4 in the 4mg cohort). Two cases were steroid refractory (one in 2mg and one in 4mg cohorts). All cases of grade II aGVHD were responsive to steroids. We found no association between GVHD prophylaxis backbone regimen and incidence of aGVHD. 11 cases of chronic GVHD (3 mild and 8 moderate severity) have been reported. Of the moderate cGVHD cases, 4 developed in the 2mg cohort and 4 in the 4mg cohort. No unexpected grade 3/4 adverse events (AE) were observed. Thromboembolic disease was an AE of special interest. Four DVTs were observed: 1 in the 2mg cohort vs. 3 in 4mg cohort. All subjects in the 4mg cohort had pulmonary embolism occurring while off Bari, one occurring at 10 months post-transplant, one at day 141 and another at day 152. Of the evaluable cases at day 100, 14/15 (93%) receiving MAC vs. 4/5 (80%) receiving RIC conditioning achieved >95% bone marrow donor engraftment, whereas, 11/16 (69%) receiving MAC vs. 4/4 (100%) receiving RIC had mixed peripheral blood CD3 T-cell chimerism. Of those with mixed CD3 chimerism at day 100, 12 remain in remission and 3 have relapsed. The incidence of disease relapse at 1 year was 21%, with 2 relapses occurring in the 2mg cohort and 3 in the 4mg cohort. Of the 8 cases with MRD positivity at transplant, 2 relapsed within 3 months of transplant and both had AML. The 1-year overall survival was 70%. The one-year refined GVHD relapse free survival (GRFS) was 33%. In conclusion, Baricitinib is safe after HLA-matched, allogeneic PB-HCT with no episodes of primary graft failure and low rates of grade III/IV acute GVHD while on therapy. A follow up efficacy study to evaluate extended duration of the 2mg dose of Bari focused on AML patients receiving HLA matched transplants and myeloablative conditioning is planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pooled Fecal Allogenic Microbiotherapy for Refractory Gastrointestinal Acute Graft-Versus-Host Disease: Results from the Early Access Program in France

Introduction Acute graft-versus-host disease (aGvHD) is a major source of mortality following allogeneic hematopoietic cell transplantation (allo-HCT). Standard 1st line therapy for the treatment of aGvHD involves corticosteroids (CS). However, more than 50% of patients are refractory to CS (SR-aGvHD) and the associated mortality rate is of up to 80%. Recently, Ruxolitinib has been approved as 2nd line treatment for SR-aGvHD. However, patients with severe gastrointestinal (GI) aGvHD seem less likely to respond to Ruxolitinib and have poor outcomes with limited therapeutic options. In this context, fecal microbiotherapy has shown promising results in several pilot studies in patients with refractory GI-aGvHD. Here we report clinical outcomes from 81 patients diagnosed with SR or dependent (SD) GI-aGvHD treated with the pooled allogeneic microbiotherapy MaaT013 as part of the Early Access Program (EAP) in France. Methods 81 patients with SR/SD GI-aGvHD (Classical n=62, late onset n=12, overlap syndrome n=7) were treated with MaaT013 as part of an EAP in France. These patients had failed 1 to 6 prior systemic GvHD treatment lines (median 2; 66/81 received Ruxolitinib). Most patients had grade III-IV aGvHD (11% grade II, 51% grade III, 38% grade IV). For each patient, a total of 3 MaaT013 administrations were planned every 7 +/- 2 days. Each dose comprised 30g of feces in 150 mL of diluent, from 4 to 8 healthy donors, administered by enema (except for 1 patient by nasogastric tube). Treatment response was calculated among all treated patients based on aGvHD staging and grading at day 28 (D28) at the time of the EAP request. Results At D28, the GI-overall response rate (ORR) was 56%: 30 complete response (CR, 37%), 11 very good response rate (VGPR, 14%), 4 partial response (PR, 5%). The GI-ORR was higher in patients with lower grade aGvHD (89% in grade II, 66% in grade III, 32% in grade IV) and higher in SD versus SR patients (92% versus 49%). Including skin and liver symptoms (n=78), response rate was 49%, including 24 CR, 11 VGPR and 3 PR and inversely correlated with aGvHD initial grade (88% in grade II, 55% in grade III, 30% in grade IV). Overall survival (OS) was 51% at 6 months (M6) and 39% at M12. The median follow-up among surviving patients was 355 days (range, 53-731). OS was significantly higher in patients achieving at least GI-PR at D28 (Responder, R; n=45) compared to patients in treatment failure (Non-responder, NR; n=35): 69% versus 28% at M6, 59% versus 14% at M12, respectively. Median survival duration in R was 451 days versus 32 days in NR. Interestingly, aGvHD response was improved in the subgroup of 31 patients previously treated with Ruxolitinib as 2nd line and MaaT013 as 3rd line (65% D28 GI-ORR, OS M6 55% and 49% M12 and 74% versus 15% at M6 and 74% versus 0% at M12 for R and NR respectively). MaaT013 displayed a good overall safety profile in the EAP population: 20 pharmacovigilance cases were reported in 18 patients: sepsis in 11 patients, C. difficile colitis in 2, E. coli osteoarthritis in 1, G. silvicola in stools from 1, P. aeruginosa sinusitis in 1, appearance of air bubbles in the mesorectum in 1, respiratory distress in 1. No pathogen transmission was reported. In 2 patients, non-pathogenic commensal bacteria isolated following infectious events were detected in the administered MaaT013 batch. Causality could not be formally excluded in these cases. The overall incidence of bacteremia (14%) remains low, compared to an incidence of 31% to 74% in bloodstream infections reported in patients with GI-aGvHD. This suggests that fecal microbiotherapy may have a protective effect on bacterial translocation, but this needs to be confirmed in further clinical trials. 47 deaths have been reported; the cause of which was GvHD in 21 patients, severe infection in 13, relapse of underlying malignancy in 6, COVID-19 in 3, hemorrhage during surgery in 1, neurological complications post allo-HCT in 1, and cardiac arrest in 2 patients. No causality link with MaaT013 administration has been identified. Conclusion Overall, EAP clinical data showed that MaaT013 was safe and effective for the treatment of SR/SD-GI-aGvHD especially in patients having previously received ruxolitinib. Interestingly, GI-response to aGvHD correlates with increased OS, suggesting a strong favorable benefit-risk profile for MaaT013. A Phase 3 trial is currently ongoing to confirm these results in ruxolitinib-refractory patients (NCT04769895). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Neihulizumab (ALTB-168) in Patients with Steroid-Refractory Acute Graft-Versus-Host-Disease (SR-aGVHD) or Treatment-Refractory Acute Graft-Versus-Host-Disease (TR-aGVHD)

Background: Neihulizumab represents a novel immune checkpoint agonistic antibody that binds to human CD162 (PSGL-1), leading to downregulation of activated T-cells. Results of early-phase trials of neihulizumab have suggested a benefit in patients with auto-immune conditions, including psoriasis, psoriatic arthritis and ulcerative colitis. Here, we report preliminary results of a Phase I study investigating the use of neihulizumab for SR-aGVHD or TR-aGVHD (ClinicalTrials.gov Identifier: NCT03327857). Aims: The aims of this study were to establish the pharmacokinetics, receptor occupancy (RO), safety and efficacy of neihulizumab in patients with SR-aGVHD or TR-aGVHD. Methods: SR-aGVHD was defined as aGVHD that (1) progressed after 3 days of treatment with methylprednisolone (MP) 2 mg/kg/day equivalent, or (2) persisted after 7 days of treatment with MP 2 mg/kg/day equivalent, or (3) progressed to involve a new organ after treatment with MP 1 mg/kg/day equivalent for skin and upper gastrointestinal aGVHD, or (4) recurred during or after steroid taper. TR-aGVHD was defined as having received one prior systemic treatment for aGVHD in addition to corticosteroid. Participants with demonstrable skin SR-aGVHD were enrolled and received a single dose of neihulizumab in Cohort 1 at either 3 mg/kg (Dose Level 1 [DL 1]) or 6 mg/kg (Dose Level 2 [DL 2]). With the completion of Cohort 1 and without observation of dose limiting toxicities, participants with any grade SR-aGVHD were enrolled for multiple dose treatment of neihulizumab (Cohort 2) with first dose at 6 mg/kg followed by weekly doses of 4 mg/kg for 3 weeks (6-4-4-4 regimen). Results: A total of 37 participants were enrolled with 13 participants in Cohort 1 and 24 participants in Cohort 2. In Cohort 1, all 13 participants had SR-aGVHD with erythematous manifestation of cutaneous aGVHD. In Cohort 2, 12 participants had SR-aGVHD and 12 had TR-aGVHD. Notably, 11 TR-aGVHD participants received ruxolitinib prior to neihulizumab treatment. A summary of study demographics and baseline disease characteristics is shown in Table 1. In Cohort 1, at 4 hours after the end of the neihulizumab infusion, the median RO was 91% for both DL 1 and DL 2. At Day 8, median RO decreased to 69% (range 23-80%) in DL 1 but was maintained at 91% (range 90-102%) in DL 2. In Cohort 1, most frequently observed Grade 3-4 adverse events (AE, at least 5%) were lymphocyte count decreased, platelet count decreased, hyponatremia, hyperglycemia and white blood cell decreased. Day 28 overall response rate (ORR) was 86% in DL 1 and 50% in DL 2, including 1 complete response (CR). Details are described in Table 2. In Cohort 2, 13 participants (54%) received all four doses of neihulizumab and reached the Day 28 aGVHD evaluation, 3 (13%) received three doses, and 8 received one or two doses. In this 6-4-4-4 dosing regimen, at 1 hour after the end of the first dose of neihulizumab infusion (6 mg/kg), the median RO was 93%. For subsequent weekly infusions (4 mg/kg) for three weeks, the median RO was maintained above 80% (range 84-93%) until Day 35. Day 28 ORR was 33% among SR-aGVHD participants, including 17% CR, and 67% among TR-aGVHD participants, including 25% CR (Table 2). The most frequently observed Grade 3-4 AEs (at least 5%) were anemia, platelet count decreased, hypocalcemia, lymphocyte count decreased, hypokalemia, hypoxia, ileus, sepsis and white blood cell decreased. Among the 24 participants in Cohort 2, 5 (42%) SR-aGVHD and 4 (33%) TR-aGVHD participants died during the study period (Day 180). The median survival was 122.5 and 124 days (by 26th July 2022) for SR-aGVHD and TR-aGVHD, respectively. Summary/Conclusion: Neihulizumab appeared to be well-tolerated in participants with SR-aGVHD or TR-aGVHD. With the current 6-4-4-4 dosing regimen (6 mg/kg followed by 4 mg/kg weekly doses for 3 weeks), RO was maintained at least 80% throughout most of the treatment period. The Day 28 ORR was 69% in Cohort 1 and 50% in Cohort 2. The higher ORR in Cohort 1 is likely due to the participants with predominantly steroid refractory skin aGVHD. Of note, the ORR in participants with TR-aGVHD, of which 11 of 12 were receiving combination ruxolitinib therapy, appeared to be higher than in those with SR-aGVHD (67% vs 33%). These promising results support further investigation of neihulizumab as an agent in ruxolitinib-refractory SR-GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extracorporeal photopheresis (ECP) improves overall survival in the treatment of steroid refractory acute graft-versus-host disease (SR aGvHD).

Steroid refractory acute graft-versus-host disease (SR aGvHD) is a major limitation of successful allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) has been used to treat SR aGvHD effectively and with low treatment related toxicity. In this study, we retrospectively analyzed the outcomes of 103 Steroid Refractory aGvHD (SR aGvHD) patients to identify factors associated with improved outcomes including the use of ECP. A total of 79 patients received ECP as part of their SR aGVHD treatment compared to 24 patients who did not. Both groups had similar aGVHD grade and maximum organ stage at onset of aGVHD and treatment initiation. Patients in the group that received ECP had better OS (p = 0.01), DFS (p = 0.008), lower relapse (p = 0.05) and similar NRM compared to the group that did not receive ECP. Patients that received ECP treatment also had shorter hospital stays in the first 180 days after onset of SR aGvHD (20 vs. 38 days, p = 0.03). Multivariable analysis for OS indicated patient CMV status (CMV+ versus CMV-, HR 2.34, CI 1.16-4.69), regimen intensity (Myelo vs. non-Myeloablative, HR 0.39, CI 0.20-0.75), and the use of ECP (ECP vs. no ECP, HR 0.39, CI 0.20-0.75) were associated with OS. In summary, the use of ECP in the treatment of SR aGvHD results in improved overall survival secondary to lower relapse rates compared to other therapeutic modalities that do not incorporate ECP.

Oxidative stress indicators during the course of acute graft versus host disease

Aim: This prospective study aimed to observe the changes in oxidative stress indicators, including total anti-oxidant status (TAS), total oxidant status (TOS), paraoxanase-1 (PON1), total thiol (TT), native thiol (NT), disulphide (DS) and nitric oxide (NO) levels from sequential blood samples obtained during a de-novo episode of acute graft versus host disease (aGvHD) and evaluate their association with disease severity and the risk of steroid resistant disease.&#x0D; Material and Method: Sequential patients who underwent an allogeneic stem cell transplantation (ASCT) in our unit and subsequently developed a de-novo episode of aGvHD between January 2022 and May 2022 were included in case they gave informed consent. All patients were started high dose (2 mg/kg/day) methylprednisolone as institutional standard first-line treatment of aGvHD as soon as the clinical diagnosis is evident. All episodes were confirmed simultaneously with gastrointestinal (GI) endoscopy and/or skin biopsies. TAS, TOS, PON1, TT, NT, DS and NO were studied from blood samples collected on days 0, +3 and +7 of steroid treatment. Demographic characteristics, diagnoses, donor type, GvHD prophylaxis, stage and grade of aGvHD, performance status (PS), the presence of cytomegalovirus (CMV) reactivation and response to steroid therapy were also noted.&#x0D; Results: A total of 15 cases was included. The median age was 49 (23-77). Males constituted 60.0% (n=9). The most frequent diagnosis and donor type were acute leukemia (53.3%, n=8) and matched related donor (46.7%, n=7), respectively. High grade aGvHD with Glucksberg grading and International Bone Marrow Transplant Registry severity index (IBMTR-SI) included 53,3% (n=8) and 86.7% (n=10) of cases, respectively. Non-responders (20.0%, n=3) significantly had advanced stage GI involvement, higher grade of aGvHD with Glucksberg grading and IBMTR-SI, and lower PS (p=0.005, p=0.04, p=0.006, and p=0.02, respectively). The changes in TAS, TOS, PON1, TT, NT, DS and NO levels on days 0, +3 and +7 of steroid treatment were not significant. Median PON1 levels on days 0, +3 and +7 of steroid treatment were significantly lower among non-responders (p

HUC-MSCs Attenuate Acute Graft-Versus-Host Disease through Chi3l1 Repression of Th17 Differentiation.

Mesenchymal stem cells (MSCs) have already demonstrated definitive evidence of their clinical benefits in acute graft-versus-host disease (aGvHD) and other inflammatory diseases. However, the comprehensive mechanism of MSCs' immunomodulation properties has not been elucidated. To reveal their potential immunosuppressive molecules, we used RNA sequencing to analyze gene expression in different tissue-derived MSCs, including human bone marrow, umbilical cord, amniotic membrane, and placenta, and found that chitinase-3-like protein 1 (Chi3l1) was highly expressed in human umbilical cord mesenchymal stem cells (hUC-MSCs). We found that hUC-MSCs treated with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) exhibited increased expression of Chi3l1 and concurrently repressed T-helper 17 cell (Th17) differentiation through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, Chi3l1 knockdown hUC-MSCs exhibited impaired therapeutic efficacy in aGvHD mice with an increased inflammatory response by promoting Th17 cell differentiation, including an increase in IL-17A in the spleen, intestine, and serum. Collectively, these results reveal a new immunosuppressive molecule, Chi3l1, in hUC-MSCs in the treatment of aGvHD that regulates Th17 differentiation and inhibits STAT3 activation. These novel insights into the mechanisms of hUC-MSC immunoregulation may lead to the consistent production of hUC-MSCs with strong immunosuppressive functions and thus improved clinical utility.

Extracorporeal photopheresis versus standard treatment for acute graft-versus-host disease after haematopoietic stem cell transplantation in children and adolescents.

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after haematopoietic stem cell transplantation (HSCT), occurring in 8% to 85% of paediatric recipients. Currently, the therapeutic mainstay for aGvHD is treatment with corticosteroids. However, there is no established standard treatment for steroid-refractory aGvHD. Extracorporeal photopheresis (ECP) is a type of immunomodulatory method amongst different therapeutic options that involves ex vivo collection of peripheral mononuclear cells, exposure to the photoactive agent 8-methoxypsoralen and ultraviolet-A radiation, and reinfusion of these treated blood cells to the patient. The mechanisms of action of ECP are not completely understood. This is the second update of a Cochrane Review first published in 2014 and updated in 2015. To evaluate the effectiveness and safety of ECP for the management of aGvHD in children and adolescents after HSCT. We searched the Cochrane Register of Controlled Trials (CENTRAL), MEDLINE (PubMed) and Embase (Ovid) databases from their inception to 25 January 2021. We searched the reference lists of potentially relevant studies without any language restrictions. We searched five conference proceedings and nine clinical trial registries on 9 November 2020 and 12 November 2020, respectively. We sought to include randomised controlled trials (RCTs) comparing ECP with or without standard treatment versus standard treatment alone in children and adolescents with aGvHD after HSCT. Two review authors independently performed the study selection. We resolved disagreement in the selection of trials by consultation with a third review author. We identified no additional studies in the 2021 review update, so there are still no studies that meet the criteria for inclusion in this review. The efficacy of ECP in the treatment of aGvHD in children and adolescents after HSCT is unknown, and its use should be restricted to within the context of RCTs. Such studies should address a comparison of ECP alone or in combination with standard treatment versus standard treatment alone. The 2021 review update brought about no additions to these conclusions.