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Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the best curative treatment modality for many malignant hematologic disorders. In the absence of a matched related donor, matched unrelated donors (MUDs) and haploidentical donors are the most important stem cell sources. In this registry-based retrospective study, we compared the outcomes of allo-HSCTs from 10/10 MUDs with antithymocyte globulin (ATG)-based regimens (n = 7050) vs haploidentical transplants (Haplo-Tx) using posttransplant cyclophosphamide (PT-CY Haplo; n = 487) in adult patients with hematologic malignancies between 2010 and 2020. Cox proportional hazard-and competing risks regression models were formed to compare the outcomes. Overall survival (OS), Disease-free survival (DFS), and graft-versus-host disease (GVHD)-free and relapse-free survival (GRFS) were superior for 10/10 MUDs (OS [hazard ratio (HR), 1.27; 95% confidence interval (CI), 1.10-1.47; P = .001]; DFS [HR, 1.17; CI, 1.02-1.34; P = .022]; GRFS [HR, 1.34; CI, 1.19-1.50; P < .001]). The risk of acute GVHD (aGVHD) grade 2 to 4, aGVHD grade 3 to 4, and chronic GVHD (cGVHD) was higher in the PT-CY Haplo group than the 10/10 MUD group (aGVHD grade 2-4 [HR, 1.46; CI, 1.25-1.71; P < .001]; aGVHD grade 3-4 [HR, 1.74; CI, 1.37- 2.20; P < .001]; cGVHD [HR, 1.30; CI, 1.11-1.51; P = .001]). A lower incidence of relapse was observed in the PT-CY Haplo group (relapse: HR, 0.83; CI, 0.69-0.99; P = .038). Unrelated 10/10 matched transplantation with ATG leads to lower GVHD rates and improved survival rates compared with PT-CY Haplo transplantation in Germany., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Steroid-refractory chronic graft-versus-host disease (cGvHD) is associated with significant morbidity and mortality, with ruxolitinib being the first drug approved for its treatment. We retrospectively analyzed the safety and efficacy of ruxolitinib for treatment of cGvHD at our center between 07/2015 and 12/2022 and identified 48 patients receiving ruxolitinib as second (18/48) or advanced (30/48) treatment line. Ruxolitinib was started on median day 340 (range 119-595) after cGvHD onset; median duration of administration was 176 (range, 79-294) days with 16/48 patients continuing treatment at last follow-up. National Institutes of Health organ grading and the intensity of immunosuppression were assessed at the start of ruxolitinib treatment and repeated after 1, 3, 6, and 12 months. Response assessment was terminated at the start of any additional new immunosuppressant treatment. The median time of follow-up was 582 (range, 104-1161) days. At the primary analysis after six months on ruxolitinib treatment, the overall response rate was 33%, and failure-free survival was 58%. Infectious adverse events ≥ CTCAE grade III were observed in 10/48 patients. The response rate was not associated with the severity of cGvHD, number of previous treatment lines, or number of additional agents combined with ruxolitinib applying a univariate regression model. At the time of the 12-month follow-up, four patients experienced recurrence of the underlying malignancy and two patients had experienced non-relapse-related mortality. Overall, ruxolitinib was relatively well-tolerated and showed outcomes comparable to the REACH3 trial in a heavily pretreated patient population., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR) T-cell therapy causes serious side effects including cytokine release syndrome (CRS). CRS-related coagulopathy is associated with hypofibrinogenemia that has up to now been considered the result of disseminated intravascular coagulation (DIC) and liver dysfunction. We investigated the incidence and risk factors for hypofibrinogenemia in 41 consecutive adult patients with hematologic malignancies (median age 69 years, range 38-83 years) receiving CAR T-cell therapy between January 2020 and May 2023 at the University Medical Center Regensburg. CRS occurred in 93% of patients and was accompanied by hypofibrinogenemia already from CRS grade 1. Yet DIC and liver dysfunction mainly occurred in severe CRS (≥ grade 3). After an initial increase during CRS, fibrinogen levels dropped after administration of tocilizumab in a dose-dependent manner (r = -0.44, P=0.004). In contrast, patients who did not receive tocilizumab had increased fibrinogen levels. Logistic regression analysis identified tocilizumab as an independent risk factor for hypofibrinogenemia (odds ratio = 486, P<0.001). We thus hypothesize that fibrinogen synthesis in CRS is up-regulated in an interleukin-6-dependent acute phase reaction compensating for CRS-induced consumption of coagulation factors. Tocilizumab inhibits fibrinogen upregulation resulting in prolonged hypofibrinogenemia. These observations provide novel insights into the pathophysiology of hypofibrinogenemia following CAR T-cell therapy, and emphasize the need for close fibrinogen monitoring after tocilizumab treatment of CRS.

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., (© 2024. The Author(s).)

Background: Prevention of toilet-to-patient transmission of multidrug-resistant Pseudomonas aeruginosa (MDR PA) poses management-related challenges at many bone marrow transplant units (BMTUs)., Aim: To conduct a longitudinal retrospective analysis of the toilet-to-patient transmission rate for MDR PA under existing infection control (IC) measures at a BMTU with persistent MDR PA toilet colonization., Methods: The local IC bundle comprised: (1) patient education regarding IC; (2) routine patient screening; (3) toilet flushing volume of 9 L; (4) bromination of toilet water tanks, and (5) toilet decontamination using hydrogen peroxide. Toilet water was sampled periodically between 2016 and 2021 (minimum every three months: 26 intervals). Upon MDR PA detection, disinfection and re-sampling were repeated until ≤3 cfu/100 mL was reached. Whole-genome sequencing (WGS) was performed retrospectively on all available MDR PA isolates (90 out of 117 positive environmental samples, 10 out of 14 patients, including nine nosocomial)., Findings: WGS of patient isolates identified six sequence types (STs), with ST235/CT1352/FIM-1 and ST309/CT3049/no-carbapenemase being predominant (three isolates each). Environmental sampling consistently identified MDR PA ST235 (65.5% ST235/CT1352/FIM-1), showing low genetic diversity (difference of ≤29 alleles by core-genome multi-locus sequence typing (cgMLST)). This indicates that direct toilet-to-patient transmission was infrequent although MDR PA was widespread (detection on 79 occasions, detection in every toilet). Only three MDR PA patient isolates can be attributed to the ST235/CT1352/FIM-1 toilet MRD PA population over six years., Conclusion: Stringent targeted toilet disinfection can reduce the potential risk for MDR PA acquisition by patients., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Induction of donor-specific tolerance is a promising approach to achieve long-term graft patency in transplantation with little to no maintenance immunosuppression. Changes to the recipient's T cell receptor (TCR) repertoire are understood to play a pivotal role in the establishment of a robust state of tolerance in chimerism-based transplantation protocols., Methods: We investigated changes to the TCR repertoires of patients participating in an ongoing prospective, controlled, phase I/IIa trial designed to evaluate the safety and efficacy of combination cell therapy in living donor kidney transplantation. Using high-throughput sequencing, we characterized the repertoires of six kidney recipients who also received bone marrow from the same donor (CKBMT), together with an infusion of polyclonal autologous Treg cells instead of myelosuppression., Findings: Patients undergoing combination cell therapy exhibited partial clonal deletion of donor-reactive CD4 + T cells at one, three, and six months post-transplant, compared to control patients receiving the same immunosuppression regimen but no cell therapy (p = 0.024). The clonality, R20 and turnover rates of the CD4 + and CD8 + TCR repertoires were comparable in both groups, showing our protocol caused no excessive repertoire shift or loss of diversity. Treg clonality was lower in the case group than in control (p = 0.033), suggesting combination cell therapy helps to preserve Treg diversity., Interpretation: Overall, our data indicate that combining Treg cell therapy with CKBMT dampens the alloimmune response to transplanted kidneys in humans in the absence of myelosuppression., Funding: This study was funded by the Vienna Science and Technology Fund (WWTF)., Competing Interests: Declaration of interests The authors have declared that no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In clinical situations, peripheral blood accessible CD3 + CD4 + CXCR5 + T-follicular helper (T FH ) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of T FH cells in peripheral blood versus tonsils, CD3 + CD4 + CD45RA - CXCR5 + cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3 + CD4 + CXCR5 + T-cell subpopulations also appear in tonsils, there with some supplementary T FH characteristics, while peripheral blood-derived T FH cells display markers of proliferation and migration. Three further subsets of T FH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4 + CXCR5 + subpopulation presents pronounced cytotoxic properties. Those 'killer T FH (T FK ) cells' can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other T FH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4 + CXCR5 + T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts., (© 2024. The Author(s).)

Abstract: Immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is slow and patients carry a high and prolonged risk of opportunistic infections. We hypothesized that the adoptive transfer of donor B cells can foster after HSCT immuno-reconstitution. Here, we report, to our knowledge, the results of a first-in-human phase 1/2a study aimed to evaluate the feasibility and safety of adoptively transferred donor B cells and to test their activity upon recall vaccination. Good manufactoring practice (GMP) B-cell products were generated from donor apheresis products using 2-step magnetic cell separation. Fifteen patients who had undergone allo-HSCT were enrolled and treated after taper of immunosuppression (median, day +148; range, 130-160). Patients received 4 different doses of B cells (0.5 × 106 to 4.0 × 106 B cells per kg body weight). To test the activity of infused donor memory B cells in vivo, patients were vaccinated with a pentavalent vaccine 7 days after B-cell transfer. We observed the mobilization of plasmablasts and an increase in serum titers against vaccine antigens, with a stronger response in patients receiving higher B-cell numbers. Analysis of immunoglobulin VH-sequences by next-generation sequencing revealed that plasmablasts responding to vaccination originated from memory B-cell clones from the donor. Donor B-cell transfer was safe, as no Epstein-Barr virus (EBV) reactivation was observed, and only low-grade graft-versus-host disease (GVHD) occurred in 4 out of 15 patients. This pilot trial may pave the way for further studies exploring the adoptive transfer of memory B cells to reduce the frequency of infections after allo-HSCT. This trial was registered at ClinicalTrial.gov as #NCT02007811., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The adoptive transfer of regulatory T cells is a promising strategy to prevent graft-versus-host disease after allogeneic bone marrow transplantation. Here, we use a major histocompatibility complex-mismatched mouse model to follow the fate of in vitro expanded donor regulatory T cells upon migration to target organs. Employing comprehensive gene expression and repertoire profiling, we show that they retain their suppressive function and plasticity after transfer. Upon entering non-lymphoid tissues, donor regulatory T cells acquire organ-specific gene expression profiles resembling tissue-resident cells and activate hallmark suppressive and cytotoxic pathways, most evidently in the colon, when co-transplanted with graft-versus-host disease-inducing conventional T cells. Dominant T cell receptor clonotypes overlap between organs and across recipients and their relative abundance correlates with protection efficacy. Thus, this study reveals donor regulatory T cell selection and adaptation mechanisms in target organs and highlights protective features of Treg to guide the development of improved graft-versus-host disease prevention strategies., (© 2024. The Author(s).)

Introduction: Chronic graft-versus-host disease (cGvHD) is a serious late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT)., Methods: This multicenter analysis determined the cumulative incidence (CI) of cGvHD and late acute GvHD (laGvHD) and its impact on transplantation-related mortality (TRM), relapse (R), and overall survival (OS) in 317 patients [296 adults, 21 pediatrics (<12 years of age)] who underwent their first allo-HSCT in 2017., Results: The CI of laGvHD was 10.5% in adults and 4.8% in pediatrics, and the CI of cGvHD was 43.0% in all adult transplant patients and 50.2% in the adult at-risk cohort at the study end. The onset of cGvHD was de novo in 42.0% of patients, quiescent in 52.1%, and progressive in 5.9%. In adults, prophylactic use of antithymocyte globulin or posttransplant cyclophosphamide was associated with a significantly lower incidence of cGvHD (28.7%) vs. standard prophylaxis with calcineurin inhibitors (30.6%) and methotrexate/mycophenolate mofetil (58.4%) (all p  < 0.01). TRM was significantly higher in patients with aGvHD (31.8%) vs. cGvHD (12.6%) and no GvHD (6.3%) (all p  = 0.0001). OS in the adult at-risk cohort was significantly higher in patients with cGvHD (78.9%) vs. without (66.2%; p  = 0.0022; HR 0.48) due to a significantly lower relapse rate (cGvHD: 14.5%; without cGvHD: 27.2%; p  = 0.00016, HR 0.41). OS was also significantly higher in patients with mild (80.0%) and moderate (79.2%) cGvHD vs. without cGvHD (66.2%), excluding severe cGvHD (72.7%) (all p  = 0.0214)., Discussion: The negative impact of severe cGvHD on OS suggests a focus on prevention of severe forms is warranted to improve survival and quality of life., Competing Interests: DW received research funds from Novartis and honoraria from Amgen, Neovii, Novartis, Mallinckrodt, Behring, and Takeda. RV received honoraria from Novartis, Pfizer, Abbvie, and MSD. AL received honoraria from Novartis. JM received honoraria for participation in an advisory board from Novartis. HG received honoraria for participation in advisory boards and speakers' bureau from Gilead, Novartis, Neovii, Sanofi, Takeda, and Therakos. SG is an employee of Novartis Pharma AG. AP received honoraria from Alexion, BMS/Celgene, Pfizer, Novartis and Mallinckrodt. DP received honoraria from Novartis and Takeda. LD received honoraria from Novartis and Takeda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Langer, Lelas, Rittenschober, Piekarska, Sadowska-Klasa, Sabol, Desnica, Greinix, Dickinson, Inngjerdingen, Lawitschka, Vrhovac, Pulanic, Güneş, Klein, Moritz Middeke, Grube, Edinger, Herr and Wolff.)

The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)