Unconventional T Cells Influence Clinical Outcome After Allogeneic Hematopoietic Cell Transplantation.

We evaluated the impact of early recovery of mucosal-associated invariant T cells (MAIT) and gamma-delta (γδ) T cells, especially Vδ2+ T cells, on the clinical outcomes of 76 patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). MAIT cells were identified at day 20-30 post-transplant using flow cytometry and defined as CD3+ TCRVα7.2+CD161+. Two subsets of Vδ2+ T cells were analyzed according to the expression of CD26. The cytotoxicity profile of MAIT and Vδ2+ T cells was analyzed according to the intracellular expression of perforin and granzyme B, and intracellular IFN-γ was evaluated after in vitro activation. CD26+Vδ2+ T cells displayed higher intracellular levels of IFN-γ, whereas CD26- Vδ2+ T were found to be more cytotoxic. Moreover, MAIT cell frequency was correlated with the frequency of Vδ2+ T cells with a better correlation observed with Vδ2+CD26+ than with the Vδ2+CD26- T cell subset. By using the composite endpoint graft-versus-host disease (GvHD)-free, relapse-free survival (GRFS) as the primary endpoint, we found that patients with a higher MAIT cell frequency at day 20-30 after allo-HCT had a significantly increased GRFS and a better overall survival (OS) and disease-free survival (DFS). Moreover, patients with a low CD69 expression by MAIT cells had an increased cumulative incidence of grade 2-4 acute GvHD (aGvHD). These results suggest that MAIT cell reconstitution may provide mitigating effects early after allo-HCT depending on their activation markers and functional status. Patients with a high frequency of Vδ2+CD26+ T cells had a significantly higher GRFS, OS and DFS, but there was no impact on cumulative incidence of grade 2-4 aGVHD, non-relapse mortality and relapse. These results revealed that the impact of Vδ2+ T cells on the success of allo-HCT may vary according to the frequency of the CD26+ subset.

Ruxolitinib for the treatment of acute graft-versus-host disease: a retrospective analysis.

Steroid-refractory acute graft-versus-host disease (aGvHD) is a serious complication after allogeneic hematopoietic stem cell transplantation, associated with significant mortality. Ruxolitinib was the first drug approved for aGvHD, based on results of the REACH2 trial; however, real-world data are limited. We retrospectively analyzed the safety and efficacy of ruxolitinib for treatment of aGvHD at our center from March 2016 to August 2022 and assessed biomarkers of risk. We identified 49 patients receiving ruxolitinib as second- (33/49), third- (11/49), fourth- (3/49), or fifth-line (2/49) treatment. Ruxolitinib was started on median day 11 (range, 7-21) after aGvHD onset; median duration of administration was 37 days (range, 20-86), with 10 patients continuing treatment at last follow-up. Median follow-up period was 501 days (range, 95-905). In the primary analysis at the 1-month assessment, overall response rate was 65%, and failure-free survival was 78%. Infectious complications ≥ CTCAE Grade III were observed in 10/49 patients within 1-month followup. Patients responding to ruxolitinib therapy required fewer steroids and exhibited lower levels of the serum biomarkers regenerating islet-derived protein 3-alpha, suppression of tumorigenicity 2, and the Mount Sinai Acute GVHD International Consortium algorithm probability. A univariate regression model revealed steroid-dependent aGvHD as a significant predictor of better response to ruxolitinib. Within 6-months follow-up, four patients experienced recurrence of underlying malignancy, and eight died due to treatment-related mortality. Overall, ruxolitinib was welltolerated and showed response in heavily pretreated patients, with results comparable to those of the REACH2 trial. Biomarkers may be useful predictors of response to ruxolitinib.

Real World Application of Recently Proposed ASTCT/CIBMTR/ eBMT/ APBMT Consensus Risk Stratification for Transplant Associated Thrombotic Microangiopathy (TA-TMA) in Children.

BACKGROUND: Consensus diagnostic and risk stratification of transplant associated thrombotic microangiopathy (TA-TMA) was recently achieved from international transplantation groups (Schoettler et al, TCT, 2023). While the diagnostic criteria proposed have been applied to multiple pediatric cohorts, there are scant data applying the novel risk stratification approach in children with TA-TMA. METHODS: In this retrospective cohort study, all children undergoing an allogeneic HCT or autologous HCT for neuroblastoma were prospectively screened for TA-TMA, diagnosed and risk-stratified using Jodele criteria from August 2019- October 2023. Our institutional practice during the study period was treat all Jodele intermediate and high-risk patients (IR, HR) with eculizumab. Harmonization risk stratification criteria were retrospectively applied. All survival analyses were calculated from the day of TA-TMA diagnosis. To identify which specific harmonization high-risk feature(s) were the most important predictors for NRM, full and reduced logistic regression models were tested. The lowest BIC and optimal Mallows' CP statistic were used to identify the best subset. SAS 9.4 (Cary, NC) was used to complete the analysis. RESULTS: Fifty-two children were diagnosed with TA-TMA during the study period a median of 37.5 days post HCT (range 3 to 735). Using Jodele risk stratification, 11 (21%) were SR, 21 (40%) intermediate risk, and 20 (39%) high- risk (HR). Forty (77%) were treated with eculizumab. There were no statistically significant differences in NRM among Jodele risk groups, though overall survival was significantly different. Using the harmonized stratification, 49 (94%) of children were stratified as HR and 3 as SR, there were no statistically significant differences in NRM or OS between groups. Eight (15.4%) children were classified as SR using Jodele risk stratification but re-stratified as HR using the harmonization criteria. One (12.5%) died in the setting of severe GVHD and the remaining 7 patients are alive at last follow up. In a best subset model, LDH >2x ULN (OR 6.52, 95% CI 0.96-44.3, p=0.05), grade 2-4 acute GVHD at the time of TA-TMA diagnosis (OR 15.4, 95% CI 2.14- 110.68, p=0.01), and multi-organ dysfunction at the time of TA-TMA (OR 21.5, 95% CI 2.96-156.37, p=0.002) were significantly associated with NRM; elevated sC5b-9, rUPCR, and viral infections were not significantly associated with NRM. Using these best fit criteria, 14 patients were classified as SR and 38 as HR; NRM was significantly higher and OS significantly lower. DISCUSSION: In this cohort of children with TA-TMA retrospective application of harmonization criteria resulted in more patients stratified as HR than previously described Jodele criteria. The intention of the harmonization criteria was to identify those at highest risk of poor outcomes; while all harmonization SR patients survived, this risk stratification was very sensitive. Previous criticisms of harmonization risk stratification include limited access to sC5b-9 testing- these data suggest that concurrent MOD, acute GVHD, and LDH >2X ULN are the most important predicators of NRM in this cohort, supporting the use of harmonization risk stratification even in the absence of available sC5b-9 testing. Additional studies are needed to validate these findings.

Recent infection with SARS-CoV-2 in donors was associated with a higher incidence of acute graft-versus-host disease in recipients undergoing allogeneic haematopoietic stem cell transplantation.

The global pandemic has resulted in the common occurrence of SARS-CoV-2 infection in the population. In the post-pandemic era, it is imperative to understand the influence of donor SARS-CoV-2 infection on outcomes after allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed allo-HSCTs from donors with mild SARS-CoV-2 infection or early recovery stage (ERS) (group 1, n = 65) and late recovery stage (group 2, n = 120). Additionally, we included allo-HSCT from donors without prior SARS-CoV-2 infection as group 0 (n = 194). Transplants from donors with different SARS-CoV-2 infection status had comparable primary engraftment and survival rates. However, group 1 had higher incidences of acute graft-versus-host disease (aGvHD), grade II-IV (41.5% vs. 28.1% in group 0 [p = 0.014] and 30.6% in group 2 [p = 0.067]) and grade III-IV (22.2% vs. 9.6% [p = 0.004] in group 0 and 12.2% in group 2 [p = 0.049]). Conversely, the risk of aGvHD in group 2 was similar to that in group 0 (p > 0.5). Multivariable analysis identified group 1 associated with grade II-IV (hazard ratio [HR] 2.307, p = 0.010) and grade III-IV (HR 2.962, p = 0.001) aGvHD, which yielded no significant risk factors for survival. In conclusion, we preliminarily demonstrated donors in the active infection state or ERS of mild SARS-CoV-2 infection were associated with higher incidences of aGvHD in transplants from related donors.

[Effect of CD8+ CD28- T Cells on Acute Graft-Versus-Host Disease after Haploidentical Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the effect of CD8+ CD28- T cells on acute graft-versus-host disease(aGVHD) after haploidentical hematopoietic stem cell transplantation(haplo-HSCT). METHODS: The relationship between absolute count of CD8+ CD28- T cells and aGVHD in 60 patients with malignant hematological diseases was retrospectively analyzed after haplo-HSCT, and the differences in the incidence rate of chronic graft-versus host disease(cGVHD), infection and prognosis between different CD8+ CD28- T absolute cells count groups were compared. RESULTS: aGVHD occurred in 40 of 60 patients after haplo-HSCT, with an incidence rate of 66.67%. The median occurrence time of aGVHD was 32.5(20-100) days. At 30 days after the transplantation, the absolute count of CD8+ CD28- T cells of aGVHD group was significantly lower than that of non-aGVHD group (P =0.03). Thus the absolute count of CD8+ CD28- T cells at 30 days after transplantation can be used to predict the occurrence of aGVHD to some extent. At 30 days after transplantation, the incidence rate of aGVHD in the low cell count group (CD8+ CD28- T cells absolute count < 0.06/µl) was significantly higher than that in the high cell count group (CD8+ CD28- T cells absolute count ≥0.06/µl,P =0.011). Multivariate Cox regression analysis further confirmed that the absolute count of CD8+ CD28-T cells at 30 days after transplantation was an independent risk factor for aGVHD, and the risk of aGVHD in the low cell count group was 2.222 times higher than that in the high cell count group (P =0.015). The incidence of cGVHD, fungal infection, EBV infection and CMV infection were not significantly different between the two groups with different CD8+ CD28- T cells absolute count. The overall survival, non-recurrent mortality and relapse rates were not significantly different between different CD8+ CD28- T cells absolute count groups. CONCLUSION: Patients with delayed CD8+ CD28- T cells reconstitution after haplo-HSCT are more likely to develop aGVHD, and the absolute count of CD8+ CD28- T cells can be used to predict the incidence of aGVHD to some extent. The absolute count of CD8+ CD28- T cells after haplo-HSCT was not associated with cGVHD, fungal infection, EBV infection, and CMV infection, and was also not significantly associated with the prognosis after transplantation.

Basiliximab Treatment for Patients With Steroid-Refractory Acute Graft-Versus-Host Disease Following Matched Sibling Donor Hematopoietic Stem Cell Transplantation.

Basiliximab is an important treatment for steroid-refractory acute graft-versus-host disease (SR-aGVHD). We performed this retrospective study to evaluate the efficacy and safety of basiliximab treatment in SR-aGVHD patients following matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) (n = 63). Overall response rate (ORR) was 63.5% and 54% at any time and at day 28 after basiliximab treatment. Grade III-IV aGVHD before basiliximab treatment predicted a poor ORR after basiliximab treatment. The rates of virus, bacteria, and fungi infections were 54%, 23.8%, and 3.1%, respectively. With a median follow-up of 730 (range, 67-3,042) days, the 1-year probability of overall survival and disease-free survival after basiliximab treatment were 58.6% (95% confidence interval [CI] = 47.6%-72.2%) and 55.4% (95% CI = 44.3%-69.2%), respectively. The 3-year cumulative incidence of relapse and non-relapse mortality after basiliximab treatment were 18.9% (95% CI = 8.3%-29.5%) and 33.8% (95% CI = 21.8%-45.7%), respectively. Comorbidities burden before allo-HSCT, severity of aGVHD and liver aGVHD before basiliximab treatment showed negative influences on survival. Thus, basiliximab was safe and effective treatment for SR-aGVHD following MSD-HSCT.

A Case Report of Refractory Graft-Versus-Host Disease Colitis Managed With Robotic Total Abdominal Colectomy.

Graft-versus-host disease (GvHD) is a common complication following hematopoietic stem cell transplant (HSCT) and has protean manifestations. It results from the activation of transplanted T lymphocytes against the HLA antigens of recipient cells, resulting in tissue destruction. The most commonly involved sites of acute GvHD are the skin and gut, with high mortality reported in the latter. Historically, surgery for gut GvHD has been reserved for those with frank perforations or uncontrolled hemorrhage. Here, we present a case of steroid and ruxolitinib refractory colonic GvHD in a 41-year-old female, which was ultimately managed with robotic-assisted total abdominal colectomy with resolution of enteric symptoms. This case highlights the role of surgical management in gut GvHD in patients who are refractory to the growing arsenal of immunomodulating agents. Given the rarity of surgical intervention in this population, more data are needed to minimize morbidity in this setting.

Model-Based Antithymocyte Globulin in αßhaplo-Hematopoietic Stem Cell Transplantation Facilitates Engraftment, Expedites T Cell Recovery, and Mitigates the Risk of Acute Graft-versus-Host Disease.

In αß T-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αßhaplo-HSCT) recipients, antithymocyte globulin (ATG; Thymoglobulin) is used for preventing graft rejection and graft-versus-host disease (GVHD). The optimal dosing remains to be established, however. Here we present the first comparative analysis of 3 different ATG dosing strategies and their impact on immune reconstitution and GVHD. Our study aimed to evaluate the effects of 3 distinct dosing strategies of ATG on engraftment success, αß+ and γδ+ T cell immune reconstitution, and the incidence and severity of acute GVHD in recipients of αßhaplo-HSCT. This comparative analysis included 3 cohorts of pediatric patients with malignant (n = 36) or nonmalignant (n = 8) disease. Cohorts 1 and 2 were given fixed ATG doses, whereas cohort 3 received doses via a new nomogram, based on absolute lymphocyte count (ALC) and body weight (BW). Cohort 3 showed a 0% incidence of day 100 grade II-IV acute GVHD, compared to 48% in cohort 1 and 27% in cohort 2. Furthermore, cohort 3 (the ALC/BW-based cohort) had a significant increase in CD4+ and CD8+ naïve T cells by day 90 (P = .04 and .03, respectively). Additionally, we found that the reconstitution and maturation of γδ+ T cells post-HSCT was not impacted across all 3 cohorts. Cumulative ATG exposure in all cohorts was lower than previously reported in T cell-replete settings, with a lower pre-HSCT exposure (<40 AU*day/mL) correlating with engraftment failure (P = .007). Conversely, a post-HSCT ATG exposure of 10 to 15 AU*day/mL was optimal for improving day 100 CD4+ (P = .058) and CD8+ (P = .03) immune reconstitution without increasing the risk of relapse or nonrelapse mortality. This study represents the first comparative analysis of ATG exposure in αßhaplo-HSCT recipients. Our findings indicate that (1) a 1- to 2-fold ATG to ATLG bioequivalence is more effective than previously established standards, and (2) ATG exposure post-HSCT does not adversely affect γδ+ T cell immune reconstitution. Furthermore, a model-based ATG dosing strategy effectively reduces graft rejection and day 100 acute GVHD while also promoting early CD4+/CD8+ immune reconstitution. These insights suggest that further optimization, including more distal administration of higher ATG doses within an ALC/BW-based strategy, will yield even greater improvements in outcomes.

Ruxolitinib in steroid-refractory acute graft-vs-host disease: Japanese subgroup analysis of the randomized REACH2 trial.

Acute graft-versus-host disease (aGvHD) is a major complication after allogeneic hematopoietic stem cell transplantation in Japan and other countries. Nearly one-third of patients do not respond to standard systemic steroid therapy and no standard second-line treatment has been established in Japan. We report efficacy and safety findings of ruxolitinib versus best available therapy (BAT) from a subgroup analysis of the international, phase 3 REACH2 study in Japanese patients with steroid-refractory aGvHD. The primary endpoint was overall response rate (ORR) at day 28. Overall, 9 patients received ruxolitinib and 21 received BAT. The ORR at day 28 (88.9% vs 52.4%) and durable ORR at day 56 (66.7% vs 28.6%) were higher with ruxolitinib versus BAT. The estimated cumulative incidence of loss of response at 6 months was 12.5% with ruxolitinib and 18.2% with BAT. The median failure-free survival was longer with ruxolitinib versus BAT (2.73 vs 1.25 months). The most common adverse events up to day 28 in the ruxolitinib and BAT groups were anemia (55.6% vs 19.0%) and thrombocytopenia (44.4% vs 4.8%, respectively). Ruxolitinib showed better efficacy outcomes and a consistent safety profile compared with BAT in the Japanese subgroup, and the findings were consistent with overall study results.

Multiplexed Gastrointestinal PCR Panels for the Evaluation of Diarrhea in Hematopoietic Stem Cell Transplantation Recipients.

Multiplexed gastrointestinal PCR panels (MGPPs) are frequently used to aid the diagnosis and management of diarrhea in hematopoietic stem cell transplantation (HCT) recipients. Many issues related to the optimal use of MGPPs in HCT patients remain to be clarified. We aimed to better define MGPP diagnostic and therapeutic stewardship in HCT recipients, including indications for and benefits of testing, optimal timing of tests, and interpretation of results. We retrieved 463 consecutive MGPPs ordered on 651 consecutive first HCT (312 allogeneic, 339 autologous) performed at our institution between June 2015 and June 2023. One hundred and sixteen of the 463 MGPPs (25%) identified at least 1 diarrheagenic pathogen, and 12 (3%) identified more than 1 diarrheagenic pathogen. A positive result was more likely if the test was ordered within 48 hours of a hospital admission (41%; 32 of 78) or as an outpatient (41%; 46 of 111) compared with evaluation of hospital-onset diarrhea (14%; 38 of 274). Among the positive results, the most frequent pathogens identified included Clostridioides difficile (64%), diarrheagenic Escherichia coli (20%), norovirus (9%), and adenovirus 40/41 (5%). Thirty-eight percent of the positive C. difficile MGPP determinations were associated with a positive test for toxin. In our allogeneic HCT cohort, 3% of MGPPs for hospital-onset diarrhea yielded an organism other than C. difficile. Fifty-six percent of positive and 14% of all submitted tests resulted in a change in treatment. For organisms other than C. difficile, only 1% of all tests and 5% of positive tests resulted in initiation of therapy. For patients at risk for acute graft-versus-host disease (aGVHD), a positive or negative MGPP result was not predictive of a new diagnosis of aGVHD in proximity to diarrhea onset. These results suggest that MGPP testing is most useful when performed at hospital admission or on an outpatient basis. Because MGPPs are sensitive and do not distinguish between colonization and causes of diarrhea, caution is needed when interpreting results, especially for toxin-negative C. difficile and diarrheagenic gram-negative organisms.

Leveraging machine learning for predicting acute graft-versus-host disease grades in allogeneic hematopoietic cell transplantation for T-cell prolymphocytic leukaemia.

Orphan diseases, exemplified by T-cell prolymphocytic leukemia, present inherent challenges due to limited data availability and complexities in effective care. This study delves into harnessing the potential of machine learning to enhance care strategies for orphan diseases, specifically focusing on allogeneic hematopoietic cell transplantation (allo-HCT) in T-cell prolymphocytic leukemia. The investigation evaluates how varying numbers of variables impact model performance, considering the rarity of the disease. Utilizing data from the Center for International Blood and Marrow Transplant Research, the study scrutinizes outcomes following allo-HCT for T-cell prolymphocytic leukemia. Diverse machine learning models were developed to forecast acute graft-versus-host disease (aGvHD) occurrence and its distinct grades post-allo-HCT. Assessment of model performance relied on balanced accuracy, F1 score, and ROC AUC metrics. The findings highlight the Linear Discriminant Analysis (LDA) classifier achieving the highest testing balanced accuracy of 0.58 in predicting aGvHD. However, challenges arose in its performance during multi-class classification tasks. While affirming the potential of machine learning in enhancing care for orphan diseases, the study underscores the impact of limited data and disease rarity on model performance.

Research advances on short-chain fatty acids in gastrointestinal acute graft-versus-host disease.

Gastrointestinal acute graft-versus-host disease (GI-aGVHD) is a severe early complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). It has been shown that the intestinal microbiota plays a critical role in this process. As metabolites of the intestinal microbiota, short-chain fatty acids (SCFAs) are vital for maintaining the host-microbiota symbiotic equilibrium. This article provides an overview of the protective effect of SCFAs in the gastrointestinal tract, emphasizes their association with GI-aGVHD, and explores relevant research progress in prevention and treatment research.

Research advances on short-chain fatty acids in gastrointestinal acute graft-versus-host disease Gastrointestinal acute graft-versus-host disease (GI-aGVHD) is a severe early complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). It has been shown that the intestinal microbiota plays a critical role in this process. As metabolites of the intestinal microbiota, short-chain fatty acids (SCFAs) are vital for maintaining the host-microbiota symbiotic equilibrium. This article provides an overview of the protective effect of SCFAs in the gastrointestinal tract, emphasizes their association with GI-aGVHD and explores relevant research progress in prevention and treatment research.

Decreasing the steroid rapidly may help to improve the clinical outcomes of patients with intestinal steroid-refractory acute graft-versus-host disease receiving basiliximab treatment.

Intestinal steroid refractory acute graft-versus-host disease (SR-aGVHD) is the major cause of mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). This retrospective cohort study aimed to identify the relationship between different steroid decreasing velocity and therapeutic response in patients with intestinal SR-aGVHD receiving basiliximab treatment, and also aimed to propose a reasonable steroid decreasing regimen for these patients. The median time for steroid dose decreasing to the 50% of initial dose and decreasing to the low-dose steroid for patients achieving ORR was 5 days and 12 days, respectively, which was both shorter than patients without achieving ORR. The ORR, NRM and survival in rapid and medium steroid decreasing group were all better than slow group. The cumulative incidence of ORR at any time was 90.4%, 78.1% and 62.3%, respectively, in rapid, medium, and slow group. The cumulative incidence of NRM at 1 year after basiliximab treatment was 18.7% (95% CI 11.3%-26.1%), 22.8% (95% CI 14.2%-31.4%) and 32.8% (95% CI 24.1%-41.5%), respectively, in rapid, medium, and slow group. The probability of OS at 1 year after basiliximab treatment was 76.9% (95% CI 68.9%-84.9%), 72.7% (95% CI 63.7%-81.7%), and 62.3% (95% CI 53.5%-71.1%), respectively, in rapid, medium, and slow group. Hence, it was helpful to decrease steroid to the 50% of initial dose ≤ 5 days and to the low-dose steroid ≤ 12 days after basiliximab treatment for intestinal SR-aGVHD patients, which may also be the reasonable steroid decrease protocol for these patients.

ST2 and Reg3α: Can they predict aGvHD, steroid refractoriness and transplant-related mortality in pediatric patients after HSCT?

BACKGROUND/AIM: There are several complications of hematopoietic stem cell transplantation. Without any doubt, most important of these is aGvHD that increases transplant-related mortality. The aim of this study is to investigate whether ST-2 and Reg3α levels measured at an early stage in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation can be individual biomarkers identifying future GvHD and predicting treatment response. MATERIALS AND METHODS: From January 2019 to January 2021, 27 patients undergoing hematopoietic stem cell transplantation for primary immunodeficiency or hematopoietic diseases formed the study group. During their follow-up, the patients were classified into two groups as those developing and those not developing aGvHD. Nineteen healthy volunteers from a similar age group who needed their blood samples drawn for other reasons and who did not have any history of chronic disease, infection or medication use formed the control group. Blood samples of patients scheduled to have allogeneic HSCT were obtained before the administration of the preparative regimen, on Day +7 post-transplant and on the day of diagnosis if they developed aGvHD. Serum samples were stored at -20ºC until the day of processing. ST2 and Reg3α levels were measured using the ELISA method. RESULTS: For patients who developed aGvHD (n = 13), ST2 levels obtained before the transplantation, on Day +7 post-transplant and on the day of aGvHD diagnosis (in patients developing GvHD) were significantly higher compared to the healthy Control Group (p-value <0.05). As regards to the samples obtained on the same days, ST2 levels did not differ significantly among patients who developed and those who did not develop GvHD (n = 14; p-value >0.05). ST2 levels of samples obtained on the days that acute skin and gastrointestinal tract GvHD developed did not differ significantly between these two groups (p-value >0.05). Reg3α levels of the pre-transplant samples, on Day +7 after the transplantation and on the day of aGvHD diagnosis did not show any difference between any of the groups (p-value >0.05). As only two patients died after transplantation, thus correlation of ST2 and Reg3α levels with transplant-related mortality could not be proven. CONCLUSION: The results of this study suggest that ST2 and Reg3α levels are neither diagnostic nor prognostic or predictive biomarkers of aGvHD, steroid resistance or transplant-related mortality in pediatric patients. This study can be regarded as a pilot study because of the small patient population; more research involving a larger patient population is required.

[Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease].

OBJECTIVE: To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). METHODS: Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). RESULTS: We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. CONCLUSION: By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Assessment of Risk Factors for Acute Kidney Injury with Machine Learning Tools in Children Undergoing Hematopoietic Stem Cell Transplantation.

Background: Although acute kidney injury (AKI) is a common complication in patients undergoing hematopoietic stem cell transplantation (HSCT), its prophylaxis remains a clinical challenge. Attempts at prevention or early diagnosis focus on various methods for the identification of factors influencing the incidence of AKI. Our aim was to test the artificial intelligence (AI) potential in the construction of a model defining parameters predicting AKI development. Methods: The analysis covered the clinical data of children followed up for 6 months after HSCT. Kidney function was assessed before conditioning therapy, 24 h after HSCT, 1, 2, 3, 4, and 8 weeks after transplantation, and, finally, 3 and 6 months post-transplant. The type of donor, conditioning protocol, and complications were incorporated into the model. Results: A random forest classifier (RFC) labeled the 93 patients according to presence or absence of AKI. The RFC model revealed that the values of the estimated glomerular filtration rate (eGFR) before and just after HSCT, as well as methotrexate use, acute graft versus host disease (GvHD), and viral infection occurrence, were the major determinants of AKI incidence within the 6-month post-transplant observation period. Conclusions: Artificial intelligence seems a promising tool in predicting the potential risk of developing AKI, even before HSCT or just after the procedure.

The history of haploidentical stem cell transplantation: a trip from the bench to the bedside.

Allogeneic bone marrow transplantation is a curative intervention for both neoplastic and non-malignant conditions. However, not all patients have an HLA-matched donor. Therefore, the development of an approach that expand the donor pool was of paramount relevance. The development of post-transplantation cyclophosphamide as graft versus host disease prophylaxis allows the safe use of haploidentical donors, solving the donor availability problem to the vast majority of patients in need. The present paper reviews the history of the development of haploidentical transplantation at Johns Hopkins University, from the bench to the bedside.

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Serum cytokine profiles during engraftment syndrome and acute graft-versus-host disease in adult patients after hematopoietic stem cell transplantation.

BACKGROUND: The underlying biology of engraftment syndrome (ES) following allogeneic hematopoietic stem cell transplantation (HSCT) is not fully elucidated, and the extent of its overlap with acute graft-versus-host disease (aGvHD) remains unclear. In order to establish potential indicator to distinguish ES more accurately, we conducted a retrospective analysis of cytokine levels during HSCT. METHODS: A total of 121 consecutive adult patients who underwent HSCT were enrolled in this study. Blood samples for interleukin (IL)-2, IL-2R, IL-4, IL-5, IL-6, IL-8, IL-10, IL-1ß, IL-12p70, interferon (IFN)-γ, IFN-α, tumor necrosis factor alpha (TNF-α) and C-reactive protein CRP were regularly assessed after transplantation and during transplantation related adverse events. Additionally, the balance of naïve, central memory and effector memory of CD4+ and CD8+ was analyzed around 30 and 60 days after stem cell infusion, respectively. RESULTS: Thirty (24.79 %) and 33 (27.27 %) patients were diagnosed with ES and aGvHD, respectively. ES was characterized by a significant increase in level of IL-5, IL-6, IL-8 and sIL-2R, while aGvHD was associated with a significant upregulation of IL-6, IL-5, IL-10 and sIL-2R in the patients from grade I to grade IV. Notably, patients got much higher levels of IL-6, IL-5 and sIL-2R when developed to ES than to aGvHD. Moreover, a pronounced shift from naïve to memory cells, both in CD4+ and CD8+ subsets, was found in ES patients. CONCLUSIONS: These findings suggest that cytokine profiles could serve as potential indicators for detecting and differentiating ES and aGvHD, enabling timely clinical intervention. Prospective clinical trials involving larger, independent patient cohorts are required to validate these observations.

Efficacy of off-the-shelf bone marrow mesenchymal stem cells for pediatric steroid-refractory acute graft-versus-host disease.

Introduction: Temcell is a mesenchymal stem cell (MSC) product approved for steroid-refractory acute graft-versus-host disease (SR-aGVHD) in Japan. However, reports regarding Temcell's efficacy in pediatric patients have been scarce, and the appropriate use of MSC therapy against pediatric SR-aGVHD also remains to be determined. Patients and Methods: We retrospectively assessed a cohort of pediatric patients treated with Temcell for SR-aGVHD following allogeneic hematopoietic transplantation. MSCs were infused intravenously at a dose of 2 × 106 cells/kg according to the manufacturer's instructions. Results: Twelve patients received eighteen cycles of MSC therapy (median age, 10.3 [1.7-17.8] years), with four receiving additional cycles (one cycle: n = 3, three cycles: n = 1). The severity of aGVHD before MSC therapy was grade I-II in three patients and grade III-IV in nine patients (gut stage 3-4, n= 7; liver stage 3-4; n =2). The median number of immunosuppressive therapy regimens received prior to MSC administration was two (range: 1-5). The first MSC cycle displayed the best overall response rate of 83%, including six patients with a complete response (CR) and with a 49% reduction in the mean daily dose of prednisone after eight weeks. The median time to first response was 3.5 days (range: 2-15 days). Two of the four patients who were re-administered MSCs for recurrent or persistent GVHD achieved a CR. The three-year overall survival rate was 69.4%, while the three-year failure free survival (FFS) rate was 22.2%, with a median FFS of 4.9 months. There were no observable side effects of MSC therapy. Conclusions: MSC therapy appears to be an effective and safe treatment for pediatric SR-aGVHD, with a steroid-sparing effect and satisfactory efficacy upon re-administration. Further studies are needed to determine its appropriate combination with additional treatments and the optimal use of re-administration of MSCs.