Chronic graft-versus-host disease (cGvHD) is a major cause of late morbidity after allogeneic hematopoietic stem-cell transplantation (alloHSCT), particularly in severe fibrosing forms. Although IFNγ-related cytokines have been implicated in cGvHD, their contributions to disease progression, survival, relapse, and response to immunosuppression remain unclear. In this retrospective study, 552 alloHSCT recipients with serum samples collected at cGvHD onset (or day 180 in controls) were analyzed for IFNγ-related cytokines. Among 14 cytokines, IL-12p40 and IL-18 emerged as divergent signatures. Both were elevated in patients who later developed severe fibrosing cGvHD, but their clinical associations differed markedly. IL-12p40 correlated with reduced relapse (cause-specific HR 0.88, 95% CI 0.84-0.93; p.

For most people living with Human immunodeficiency virus (HIV), virus control and sufficient immune function can be achieved with polypharmacotherapy. A minority is intolerant to thereto, and drug-resistant mutants are emerging. Additionally, people living with HIV (PLWH) have significantly higher risk of cardiovascular morbidity and malignant diseases. HIV is in principle immunogenic. If T-cell depletion could be avoided immunological HIV eradication should be achievable. The "Berlin patient" in whom allogeneic stem cell transplantation from a CCR5Δ32/Δ32 donor induced long-term remission without antiviral therapy raised the hope that stem cell gene therapy with CCR5-deleted autologous stem cells could provide a cure, but the strategy did not live up to expectations. We propose to test an alternative strategy, namely expression of a designer recombinase which specifically excises HIV provirus from the genome under a Tat-inducible promoter so that expression is restricted to infected cells. A cGMP-compliant manufacturing protocol and quality control strategy for this investigational medicinal product was developed, validated, and a manufacturing authorization obtained. A First-in-Human-clinical trial, testing engraftment, repopulation of peripheral specific immunity, and ability to control HIV infection without antiviral medication is in preparation. Protocols can be adapted to other stem cell gene therapies by transferring alternative lentviral cargo.

Codons function as translation units in open-reading-frames (ORF) of genes to encode for proteins. Transfer RNAs (tRNAs) mediate the connection of every codon to its cognate amino acid. Despite the cooperation between messenger and transfer RNA during translation, approaches to integrate codon usage and tRNA quantities remain to be established. Using matched mRNA- and tRNA-sequencing of peripheral blood cells, we apply a precision-biology approach quantitatively integrating transcriptomic codon- and corresponding tRNA-abundance. Thereby, we classify codons as highly or lowly supplied and compare optimality of synonymous codons. Additionally, we describe substantial differences regarding the conservation of a codon's tRNA-supply among healthy donors. A meta-ORF-analysis demonstrates depletion of lowly supplied codons at translation start sites. Discrepancy between codon- and tRNA-abundance, and codon-preference depending on the distance to the translation start site, seem to be non-random and could affect translational speed and thus provide a novel level of regulation of protein abundance.

Background & Aims Recurrent glioblastoma (GB) still carries a dismal prognosis with few established treatment options. In the multicenter CAR2BRAIN phase I first-in-human clinical trial, we investigated repetitive local adoptive transfer of clonal chimeric antigen receptor (CAR)-NK cells (NK-92/5.28.z) targeting HER2 alone and in combination with the anti-PD-1 checkpoint inhibitor ezabenlimab in patients with recurrent HER2-positive GB. Methodology After establishing the dose of 1×108 irradiated CAR-NK cells as safe for intracerebral injection in the dose-escalation cohort in 9 patients, 6 patients were treated with repetitive doses of CAR-NK cells in the expansion cohort. In the subsequent CAR2BRAIN-Check cohort, 12 patients received a combination therapy with the anti-PD-1 checkpoint inhibitor ezabenlimab. CAR-NK cells were injected into the margins of the surgical cavity during relapse surgery, and repeatedly via an implanted reservoir into the resection cavity. Where feasible, we adhered to a biopsy-treat-resect-treat strategy, initiating study treatment before planned tumor resection. An in-depth analysis of tissue, cerebrospinal fluid (CSF) and blood samples before and after immunotherapy was performed to assess treatment-induced modulation of the tumor immune microenvironment. Results Repetitive intracranial injection of CAR-NK cells was feasible and safe and none of the patients developed a cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. Combination immunotherapy induced a local immune response with elevated pro-inflammatory cytokines and cell counts measured in CSF sampled from the resection cavity. We observed an increase of CD4+ and CD8+ T cells, and a decrease of regulatory CD4+FoxP3+ T cells. Median progression-free survival of the patients treated in the dose-escalation cohort was 7 weeks, compared to 10.5 and 14.5 weeks, respectively, for the patients of the expansion cohort and the CAR2BRAIN-Check chort. Median overall survival of the patients in the dose escalation cohort was 31 weeks and 44.5 weeks for both the expansion and CAR2BRAIN-Check cohort. Conclusion Immunotherapy with repetitive intracranial injection of HER2-targeted CAR-NK cells is feasible and safe both as monotherapy and in combination with the systemic checkpoint inhibitor ezabenlimab, and favorably modulates the intratumoral immune microenvironment. Given the highly promising results obtained so far, further trials are warranted to confirm the potential clinical benefit of this strategy.

Despite therapeutic advancements and improved patient outcomes, relapsed and refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) after anti-CD19 chimeric antigen receptor T-cell (CART19) therapy due to antigen loss remains a critical unmet clinical need. In this study, we identify that integrin α4 is consistently expressed on B-ALL cells before and after CART19. CRISPR/Cas9-mediated CD19 knockout in primary B-ALL cells did not alter integrin α4 expression, further suggesting stable integrin α4 expression independent of CD19 a stable target. Using the United States Food and Drug Administration (FDA)-approved anti-integrin α4 antibody natalizumab (NZM), we demonstrated effective disruption of leukemia cell adhesion to both VCAM-1, the primary integrin α4 ligand, and to stromal OP9 cells, thereby critically reducing interactions with the leukemia-supportive microenvironment. Most importantly, NZM treatment markedly extended survival in NSG mice engrafted with post-CART19-relapsed B-ALL compared with controls. Our work establishes integrin α4 as an ideal marker for identifying leukemia cells in patients receiving CART19.

Purpose Patients with high-risk (HR) leukemia remain at substantial risk of early relapse, treatment-related toxicity, and poor survival, underscoring the need for effective relapse prevention therapies. To our knowledge, this first-in-human, disease burden-guided study evaluated the feasibility, safety, and efficacy of donor-derived allogeneic interleukin-15-activated cytokine-induced killer cells (IL15-CIK) combining T-cell and natural killer cell properties for post-transplant disease control. Methods In a prospective, multicenter phase I/II trial (EudraCT 2013-005446-11) and an identically designed pilot study, 53 adult and pediatric patients with HR leukemia received 56 courses of IL15-CIK monotherapy after human leukocyte antigen (HLA)-matched or HLA-mismatched transplantation. Treatment intent was categorized as consolidation (13%), preemptive (61%), or salvage (27%) with 169 infusions administered as a single dose (29%) or according to adaptable dose-escalation regimens (71%). Results Acute graft-versus-host disease (GVHD) grades 1-2 and grade 3 occurred in 27% and 4% of cases, respectively; no extensive chronic GVHD or treatment-related mortality was observed. IL15-CIK-associated adverse events were predominantly mild. Disease clearance, assessed by the cumulative incidence of complete molecular remission, peaked at day 700, reaching 74% in the preemptive and 13% in the salvage setting. The five-year progression-free survival was 50% overall and highest (69%) in pediatric acute myeloid leukemia. The five-year overall survival (OS) was 71% in the consolidation, 61% in the preemptive, and 20% in the salvage setting. Multivariable analysis demonstrated significantly lower relapse rates with Campath compared with ATG, superior OS in myeloid malignancies, and reduced IL15-CIK efficacy in advanced disease. The median follow-up was 7.3 years. Conclusion IL15-CIK monotherapy is feasible and safe and demonstrates promising relapse-preventive activity after hematopoietic stem-cell transplantation. Clinical outcomes are strongly influenced by disease burden at treatment initiation and previous serotherapy, supporting optimized patient selection and timing in future post-transplant immunotherapeutic strategies.

Patients with acute graft-versus-host disease (aGvHD) not responding to steroids or further-line treatment, including ruxolitinib, face a poor prognosis. We conducted a multi-center, retrospective analysis of real-world data from 140 treatment episodes in 139 pediatric patients with steroid- or treatment-refractory aGvHD receiving the random-donor mesenchymal stromal cell (MSC) preparation tomostrocel. Most patients received 4–6 infusions of 1–2 million MSCs/kg body weight after a median of 3.5 prior therapies, including ruxolitinib in 76 patients. At baseline, over two-thirds had grade III/IV aGvHD, mostly with lower gastrointestinal involvement. At day 28, overall response (OR) rate was 62.9% (74.1%, 75.0% and 53.2% for grade II, III and IV aGvHD, respectively). OR was similar with versus without ruxolitinib pre-treatment. Organ stage with skin, liver, lower gastrointestinal and upper gastrointestinal involvement improved at day 28 in 69.4%, 57.5%, 58.6% and 39.5% of patients, respectively. Most responses were sustained. Six-month overall survival was 64.1%. Favorable survival factors were younger age, lower grade aGvHD and OR at day 28. The favorable safety profile of tomostrocel was consistent with prior MSC reports. Treatment with tomostrocel resulted in good responses with a favorable safety profile in heavily pre-treated pediatric patients, including those pre-treated with ruxolitinib.

Introduction: Acute graft-versus-host disease (aGvHD) is a potentially life-threatening complication that can occur following allogeneic haematopoietic stem cell transplantation. Although corticosteroids remain the standard first-line therapy, a considerable number of patients fail to respond adequately, leading to significant morbidity. Methods: Mesenchymal stromal cells (MSCs) are being investigated as a therapeutic option in this setting due to their immunomodulating and tissue-regenerative properties. Two ongoing clinical trials - IDUNN (NCT04629833) and BALDER (NCT06075706) - are assessing the role of the innovative product MSC-FFM/MC0518 in managing steroid-refractory aGvHD (SR-aGvHD). In parallel, a dedicated surveillance programme is collecting long-term efficacy and safety data to inform clinical use beyond the controlled trial environment. Conclusion: The IDUNN and BALDER trials will deliver definitive evidence on the efficacy and safety of MC0518 in SR-aGvHD and may shape future therapeutic strategies.

Abstract How transfer RNA (tRNA) expression changes as hematopoietic stem cells are specified into human blood cell types remains unknown. tRNAs translate the 64-codon genetic code into the 21-amino acid protein code, and their relative abundance can influence proteomic output. Here, we introduce simultaneous single-cell tRNA and mRNA sequencing (sc-STM-seq), a scalable, high-throughput approach for profiling tRNAs alongside mRNA transcriptomes in individual cells. Applying sc-STM-seq to human bone marrow, we map tRNA expression and splicing across hematopoietic differentiation trajectories and identify tRNAs associated with stemness, differentiation, and specific cell lineages. Our study provides an atlas of the hematopoietic tRNA landscape and establishes tRNA expression as a previously underappreciated layer of cellular heterogeneity during human blood cell development.

This multicenter real-world study identifies critical determinants of outcome for tisagenlecleucel (tisa-cel) in treating post-HSCT relapse in 220 children/young adults with B-ALL from 31 European centers. Median follow-up was 30.0 months, with a 43.6% 2-year event-free survival (EFS), 67.2% overall-survival (OS), and 57.1% incidence of CAR-T failure. CAR-T for relapse after transplant from a matched sibling donor (MSD) compared to alternative donors was associated with lower 2-year-OS (MSD 59.1%, mismatched donor MMD 80.2%, matched family/unrelated donor MFD/MUD 68.3%, p = 0.046). Two-year incidence of CAR-T failure was highest for MSD (MSD 73.8%, MFD/MUD 49.7%, MMD 52.2%, p = 0.006). Patients who had relapsed early (< 6 months post HSCT) showed inferior 2-year-EFS (23.7%) and OS (47.2%) compared to patients with late relapse, ≥ 6 months after HSCT (EFS 49.8%, p = 0.001; OS 73.9%, p < 0.001). Early relapse was associated with a higher incidence of CAR-T failure and relapse after tisa-cel, particularly CD19+ relapses. Outcomes correlated with disease burden at lymphodepletion: 2-year-OS was 81.7% for MRD-, 69.2% for MRD+, and 55.2% for patients in non-remission (p = 0.003), with incidence of CAR-T failure highest in non-remission. Prior transplant from an MSD, early post-HSCT relapse, and disease burden at lymphodepletion identify patients at increased risk of CAR-T failure after HSCT.

The BCL-2 inhibitor venetoclax has transformed the treatment of acute myeloid leukemia (AML), but relapse due to resistance of leukemic stem cells (LSCs) remains a major challenge. By molecular and functional profiling of LSCs from >150 patients, we identify four LSC subtypes. These mirror distinct hematopoietic lineage stages, which determine the expression ratio between the venetoclax target BCL-2 and resistance-inducing proteins MCL-1 and BCL-xL (MAC-score). Longitudinal analyses reveal that venetoclax resistance mostly arises in LSCs through plasticity toward a megakaryocytic/erythroid-progenitor (MEP)-LSC state that switches survival dependency from BCL-2 to BCL-xL. In rare cases, mature monocytic/dendritic (MoDe)-LSCs, found within LAMP5+ monocytic AMLs, drive venetoclax resistance. LSC subtyping improves genetic risk stratification and provides subtype-specific therapies: venetoclax-resistant MEP-LSCs respond to BCL-xL inhibitors, whereas MoDe-LSCs are sensitive to MEK1/2 inhibition. Our findings reveal four distinct LSC types with unique vulnerabilities and propose biomarker-guided treatment strategies that complement genetic profiling to overcome venetoclax resistance.

Bioreactors enable scalable cell cultivation by providing controlled environments for temperature, oxygen, and nutrient regulation, maintaining viability and enhancing expansion efficiency. Automated systems improve reproducibility and minimize contamination risks, making them ideal for high-density cultures. While fed-batch bioreactors dominate biologics production, continuous systems like perfusion cultures offer superior resource efficiency and productivity. The Quantum hollow-fiber perfusion bioreactor supports cell expansion via semi-permeable capillary membranes and a closed modular design, allowing continuous media exchange while retaining key molecules. We developed a multiple-harvest protocol for suspension cells in the Quantum system, yielding 2.5 × 1010 MEL-745A cells within 29 days, with peak densities of 4 × 107 cells/mL—a 15-fold increase over static cultures. Viability averaged 91.3%, with biweekly harvests yielding 3.1 × 109 viable cells per harvest. Continuous media exchange required more basal media to maintain glucose and lactate levels but meaningfully less growth supplement than the 2D culture. Stable transgene expression suggested phenotypic stability. Automated processing reduced hands-on time by one-third, achieving target cell numbers 12 days earlier than 2D culture. Despite higher media use, total costs for the automated were lower compared to the manual process. Quantum enables high-density suspension cell expansion with cost advantages over conventional methods.

Uncovering early gene network changes of human hematopoietic stem cells (HSCs) leading to differentiation induction is of utmost importance for therapeutic manipulation. We employed single cell proteo-transcriptomic sequencing to FACS-enriched bone marrow hematopoietic stem and progenitor cells (HSPCs) from 15 healthy donors. Pseudotime analysis reveals four major differentiation trajectories, which remain consistent upon aging, with an early branching point into megakaryocyte-erythroid progenitors. However, young donors suggest a more productive differentiation from HSPCs to committed progenitors of all lineages. tradeSeq analysis depicts continuous changes in gene expression of HSPC-related genes ( DLK1, ADGRG6 ), and provides a roadmap of gene expression at the earliest branching points. We identify CD273/PD-L2 to be highly expressed in a subfraction of immature multipotent HSPCs with enhanced quiescence. Functional experiments confirm the immune-modulatory function of CD273/PD-L2 on HSPCs in regulating T-cell activation and cytokine release. Here, we present a molecular map of early HSPC differentiation across human life.

Background: Rhabdomyosarcoma (RMS) is the most common type of soft-tissue sarcoma in children, and it remains a challenging cancer with poor outcomes in high-risk and metastatic patients. This study reports the use of epidermal growth factor receptor (EGFR)-targeted chimeric antigen receptor (CAR) natural killer (NK) cells in combination with radiotherapy as a novel immunotherapeutic approach for RMS treatment.Methods: Primary human NK cells from healthy donors were engineered using lentiviral transduction to express a cetuximab-based EGFR-specific CAR. The ability of the engineered NK cells to lyse RMS cells was then assessed in vitro in RMS monolayers and spheroids, as well as against chemotherapy-resistant and primary patient-derived RMS cells. Migratory properties of NK cells were observed in a subcutaneous RMS xenograft model using in vivo imaging, and the efficacy of EGFR-CAR NK cells in combination with localized fractionated radiotherapy was analyzed.Results: Primary human EGFR-CAR NK cells demonstrated enhanced cytotoxicity against multiple RMS cell lines in both two-dimensional culture and three-dimensional spheroid models. Furthermore, EGFR-CAR NK cells were highly efficient against chemotherapy-resistant RMS cells and patient-derived samples. Importantly, EGFR-CAR NK cells also exhibited improved tumor homing compared with non-transduced NK cells in an in vivo RMS xenograft model. Notably, the combination of EGFR-CAR NK cell therapy with fractionated radiotherapy further enhanced NK cell infiltration into the tumor and reduced tumor growth.Conclusion: This study provides a proof-of-concept for EGFR-CAR NK cells as a promising immunotherapy for RMS, particularly when combined with radiotherapy to overcome barriers of solid tumors. This combinatorial approach may hold potential to improve outcomes for patients with RMS and other EGFR-expressing malignancies.

IntroductionCAR-T cell therapy, though successful in hematologic malignancies, faces challenges in solid tumors due to limitations of autologous T cells. Cytokine-induced killer (CIK) cells can be given safely across allogeneic barriers and constitute alternative effector cells generated from healthy donors. CIK cells are a heterogenous population of predominantly T cells with a mixed natural killer (NK) phenotype and combine non-MHC-restricted cytotoxicity with potent anti-tumor capacity of the adaptive immune system. Here, we characterize and compare efficacy, phenotypic subpopulations and modes of action of CAR-CIK cells and conventional CAR-T cells from same-donor samples in ErbB2+ rhabdomyosarcoma (RMS).MethodsTo benchmark CAR-CIK against conventional CAR-T cells, effector cells were generated from same-donor samples and lentivirally transduced with a second generation CD28-CD3ζ CAR. Effector subpopulations and their dynamics upon target cell exposure were phenotypically characterized by flow cytometry. Efficacy was assessed in human ErbB2+ RMS cancer cell lines and primary patient samples in vitro and ex vivo using cytotoxicity and spheroid co-incubation assays. Modes of action were assessed by comparing cytokine secretion profiles using bead-based multiplexed flow cytometry and by liquid chromatography mass spectrometry whole cell proteomics. Finally, we used an in vivo model of RMS mimicking minimal metastatic residual disease to compare anti-tumor potency of CAR-CIK vs. CAR-T cells and to assess their target organ infiltration.ResultsIn vitro assays demonstrated superior cytotoxicity of CAR-CIK cells against RMS cell lines and primary tumor samples. Long-term co-incubation with tumor spheroids led to expansion of CAR-CIK cells and enrichment of CD3+CD56+ TNK cells. CAR-CIK cell cytokine signature showed significantly increased secretion of effector molecules like interferon-γ, perforin and granulysin, and lower secretion of Th2 cytokines IL-2, IL-4 and IL-10. Whole cell proteomics showed corresponding upregulation of chemokine signaling and NK-cytotoxicity pathways in CAR-CIK cells. In NSG mice xenografted with ErbB2+ RMS, a single injection of either CAR-effector cells strongly impeded metastatic tumor development and significantly improved survival.ConclusionOur results demonstrate that CAR-CIK cells are at least equipotent to CAR-T cells. Combined with their favorable safety profile and allogeneic applicability, these findings position CAR-CIK cells as promising immune effectors for solid tumors.

Hematopoietic stem and progenitor cells (HSPC) from mobilized blood are the preferred graft source for allogeneic and autologous stem cell transplantation. The efficiency of CD34+ cell mobilization with granulocyte colony-stimulating factor (G-CSF) varies significantly between individuals, but is reproducible across mobilization cycles within an individual, suggesting a genetic component, a hypothesis that has been previously investigated by testing for candidate single-nucleotide polymorphisms (SNP) associations. As the genetic determinants of HSPC mobilization have not been analyzed on the genomic scale so far, we performed a genome-wide association study (GWAS) in a German population of 564 healthy G-CSF mobilized allogeneic stem cell donors. None of the association between about 5 million variants and the primary outcome investigated (CD34+ cell frequency in peripheral blood) reached genome-wide significance. Focused analysis of 11 variants previously shown to be associated with basal CD34+ cell levels confirmed an association of CXCR4-rs11688530 (A>G) and ARHGAP45-rs36084354 (A>G) with higher CD34+ frequency in G-CSF mobilized healthy donors showing an explained variance of 1.07% (p=0.004) and 0.86% (p=0.01), respectively. Demographic analysis revealed an association of peripheral blood CD34+ cell frequency with sex (Varex = 8.1%) and BMI (Varex = 7.2%) that exceeded the contribution of single variants. The current study is the first GWAS in mobilized stem cell donors and had a statistical power of 80% to detect SNPs with explained variance of ≥6.7% at genome-wide significance. The study results exclude a monogenetic cause of population G-CSF responsiveness and support the view that polygenetic risk scores are required as predictors.

The existing heterogeneity of the human hematopoietic stem cell (HSC) compartment imposes significant challenges in understanding their physiology and molecular constitution. The hematopoietic system is hierarchically organized, with HSCs at the apex, responsible for maintaining homeostasis by ensuring a life-long supply of blood cells. HSCs are highly potent but rare, making their pure isolation challenging. To address this, flow-cytometry-based methods are commonly used to isolate HSCs, bridging the gap between surface marker expression and understanding their functional and molecular properties. However, detailed methodology papers providing practical guidance for the prospective isolation of distinct human hematopoietic stem and progenitor cell (HSPC) populations are rare, hindering reproducible applications across different research groups. Here, we present a comprehensive protocol for isolating multipotent long-term repopulating HSCs (LT-HSCs) and define multipotent progenitor populations (MPPs) from human mobilized peripheral blood (mPB) after leukapheresis using fluorescence-activated cell sorting (FACS). By highlighting the workflow, outlining critical considerations and emphasizing recent advancements in the field, we provide an extensive overview of FACS-based human HSC isolation. This facilitates the enrichment of these rare cells for downstream analysis and enables researchers to improve our understanding of the heterogeneity within the HSC compartment.