T-follicular helper cell expansion and chronic T-cell activation are characteristic immune anomalies in Evans syndrome.

Pediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor ß chain (TCR-ß) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

The role of the conditioning regimen for autologous and ex vivo genetically modified hematopoietic stem cell-based therapies: recommendations from the ISCT stem cell engineering committee.

BACKGROUND: The advent of autologous gene modified cell therapies to treat monogenic disorders has been a major step forward for the field of hematopoietic stem cell transplantation (HCT) and cellular therapies. The need for disease-specific conditioning to enable these products to provide a potential cure has required extrapolation from experience in myeloablative and non-myeloablative HCT for these disorders. METHODS: In this manuscript, we review the current datasets and clinical experience using different conditioning regimens for autologous gene therapies in hemoglobinopathies, metabolic and lysosomal disorders, inborn errors of immunity (IEI) and bone marrow failure (BMF) syndromes. RESULTS: The disease specific and unique conditioning requirements of each disorder are considered in order to achieve maximal benefit while minimizing associated toxicities. CONCLUSIONS: Standardized recommendations based on these data are made for each set of disorders to harmonize treatment. Future directions and the possibility of non-genotoxic conditioning regimens for autologous gene therapies are also discussed. Ethical Statement: The authors followed all relevant ethical considerations in writing this manuscript.

Strategic infection prevention after genetically modified hematopoietic stem cell therapies: recommendations from the International Society for Cell & Gene Therapy Stem Cell Engineering Committee.

There is lack of guidance for immune monitoring and infection prevention after administration of ex vivo genetically modified hematopoietic stem cell therapies (GMHSCT). We reviewed current infection prevention practices as reported by providers experienced with GMHSCTs across North America and Europe, and assessed potential immunologic compromise associated with the therapeutic process of GMHSCTs described to date. Based on these assessments, and with consensus from members of the International Society for Cell & Gene Therapy (ISCT) Stem Cell Engineering Committee, we propose risk-adapted recommendations for immune monitoring, infection surveillance and prophylaxis, and revaccination after receipt of GMHSCTs. Disease-specific and GMHSCT-specific considerations should guide decision making for each therapy.

Early Stage Professionals Committee proceedings from the International Society for Cell & Gene Therapy 2022 Annual Meeting.

In this Committee Proceedings, representatives from the Early Stage Professional (ESP) committee highlight the innovative discoveries and key take-aways from oral presentations at the 2022 International Society for Cell and Gene Therapy (ISCT) Annual Meeting that cover the following subject categories: Immunotherapy, Exosomes and Extracellular Vesicles, HSC/Progenitor Cells and Engineering, Mesenchymal Stromal Cells, and ISCT Late-Breaking Abstracts.

Transplant for non-malignant disorders: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee report on the role of alternative donors, stem cell sources and graft engineering.

Hematopoietic stem cell transplantation (HSCT) is curative for many non-malignant disorders. As HSCT and supportive care technologies improve, this life-saving treatment may be offered to more and more patients. With the development of new preparative regimens, expanded alternative donor availability, and graft manipulation techniques, there are many options when choosing the best regimen for patients. Herein the authors review transplant considerations, transplant goals, conditioning regimens, donor choice, and graft manipulation strategies for patients with non-malignant disorders undergoing HSCT.

International Society for Cell & Gene Therapy Stem Cell Engineering Committee: Cellular therapies for the treatment of graft-versus-host-disease after hematopoietic stem cell transplant.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplant is a curative approach for many malignant and non-malignant hematologic conditions. Despite advances in its prevention and treatment, the morbidity and mortality related to graft-versus-host disease (GVHD) remains. The mechanisms by which currently used pharmacologic agents impair the activation and proliferation of potentially alloreactive T cells reveal pathways essential for the detrimental activities of these cell populations. Importantly, these same pathways can be important in mediating the graft-versus-leukemia effect in recipients transplanted for malignant disease. This knowledge informs potential roles for cellular therapies such as mesenchymal stromal cells and regulatory T cells in preventing or treating GVHD. This article reviews the current state of adoptive cellular therapies focused on GVHD treatment. METHODS: We conducted a search for scientific literature in PubMed® and ongoing clinical trials in clinicaltrial.gov with the keywords "Graft-versus-Host Disease (GVHD)," "Cellular Therapies," "Regulatory T cells (Tregs)," "Mesenchymal Stromal (Stem) Cells (MSCs)," "Natural Killer (NK) Cells," "Myeloid-derived suppressor cells (MDSCs)," and "Regulatory B-Cells (B-regs)." All the published and available clinical studies were included. RESULTS: Although most of the existing clinical data focus on cellular therapies for GVHD prevention, there are observational and interventional clinical studies that explore the potential for cellular therapies to be safe modalities for GVHD treatment while maintaining the graft-versus-leukemia effect in the context of malignant diseases. However, there are multiple challenges that limit the broader use of these approaches in the clinical scenario. CONCLUSIONS: There are many ongoing clinical trials to date with the promise to expand our actual knowledge on the role of cellular therapies for GVHD treatment in an attempt to improve GVHD-related outcomes in the near future.

Curative therapy for hemoglobinopathies: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee review comparing outcomes, accessibility and cost of ex vivo stem cell gene therapy versus allogeneic hematopoietic stem cell transplantation.

Thalassemia and sickle cell disease (SCD) are the most common monogenic diseases in the world and represent a growing global health burden. Management is limited by a paucity of disease-modifying therapies; however, allogeneic hematopoietic stem cell transplantation (HSCT) and autologous HSCT after genetic modification offer patients a curative option. Allogeneic HSCT is limited by donor selection, morbidity and mortality from transplant conditioning, graft-versus-host disease and graft rejection, whereas significant concerns regarding long-term safety, efficacy and cost limit the broad applicability of gene therapy. Here the authors review current outcomes in allogeneic and autologous HSCT for transfusion-dependent thalassemia and SCD and provide our perspective on issues surrounding accessibility and costs as barriers to offering curative therapy to patients with hereditary hemoglobinopathies.

Viral infection in hematopoietic stem cell transplantation: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee review on the role of cellular therapy in prevention and treatment.

Despite recent advances in the field of HSCT, viral infections remain a frequent causeof morbidity and mortality among HSCT recipients. Adoptive transfer of viral specific T cells has been successfully used both as prophylaxis and treatment of viral infections in immunocompromised HSCT recipients. Increasingly, precise risk stratification of HSCT recipients with infectious complications should incorporate not only pretransplant clinical criteria, but milestones of immune reconstitution as well. These factors can better identify those at highest risk of morbidity and mortality and identify a population of HSCT recipients in whom adoptive therapy with viral specific T cells should be considered for either prophylaxis or second line treatment early after inadequate response to first line antiviral therapy. Broadening these approaches to improve outcomes for transplant recipients in countries with limited resources is a major challenge. While the principles of risk stratification can be applied, early detection of viral reactivation as well as treatment is challenging in regions where commercial PCR assays and antiviral agents are not readily available.

Characterization and cost-benefit analysis of automated bioreactor-expanded mesenchymal stem cells for clinical applications.

BACKGROUND: Expanding quantities of mesenchymal stem cells (MSCs) sufficient to treat large numbers of patients in cellular therapy and regenerative medicine clinical trials is an ongoing challenge for cell manufacturing facilities. STUDY DESIGN AND METHODS: We evaluated options for scaling up large quantities of bone marrow-derived MSCs (BM-MSCs) using methods that can be performed in compliance with Good Manufacturing Practices (GMP). We expanded BM-MSCs from fresh marrow aspirate in αMEM supplemented with 5% human platelet lysate using both an automated cell expansion system (Quantum, Terumo BCT) and a manual flask-based method using multilayer flasks. We compared MSCs expanded using both methods and assessed their differentiation to adipogenic and osteogenic tissue, capacity to suppress T-cell proliferation, cytokines, and growth factor secretion profile and cost-effectiveness of manufacturing enough BM-MSCs to administer a single dose of 100 × 106 cells per subject in a clinical trial of 100 subjects. RESULTS: We have established that large quantities of clinical-grade BM-MSCs manufactured with an automated hollow-fiber bioreactor were phenotypically (CD73, CD90, CD105) and functionally (adipogenic and osteogenic differentiation and cytokine and growth factor secretion) similar to manually expanded BM-MSCs. In addition, MSC manufacturing costs significantly less and required less time and effort when using the Quantum automated cell expansion system over the manual multilayer flasks method. CONCLUSION: MSCs manufactured by an automated bioreactor are physically and functionally equivalent to the MSCs manufactured by the manual flask method and have met the standards required for clinical application.

Modifiers of mesenchymal stem cell quantity and quality.

BACKGROUND: Clinical trials involving mesenchymal stem cell (MSC) therapy have variable outcomes. We hypothesize this is largely attributed to donor-to-donor variability and tissue of origin. STUDY DESIGN AND METHODS: We examined proliferation rates, cytokine secretion profiles, and differentiation capability of seven bone marrow-derived MSCs (BM-MSCs) and 16 adipose tissue-derived MSCs (AD-MSCs) from 23 donors. RESULTS: AD-MSCs had the capacity to undergo more than 40 population doublings, while the BM-MSC proliferation rate was found to be considerably slower. We observed more donor-to-donor variability in proliferation rates of BM-MSCs than with AD-MSCs. Cytokine analysis revealed that secretion of eight cytokines was significantly increased by AD-MSCs at Passage (P)3 compared with P1, while for BM-MSCs at P3 relative to P1, only interleukin-8 and RANTES secretion was significantly increased. By P5, secretion of all cytokines by AD-MSCs was either decreased or unchanged relative to P1. In contrast, cytokine secretion by BM-MSCs at P5 was mostly unchanged, although secretion of six cytokines was significantly increased relative to P1. When we compared cytokine secretion between AD-MSCs and BM-MSCs at P3, AD-MSCs significantly secreted higher concentrations of cytokines than BM-MSCs while the opposite was observed at P5. This suggests that BM-MSCs are relatively more potent at P5 while AD-MSCs are relatively more potent at P3. AD-MSCs and BM-MSCs exhibited the capacity for chondrogenic differentiation. AD-MSCs and BM-MSCs appeared to display a more enhanced inclination toward adipogenic and osteogenic differentiation, respectively. CONCLUSION: MSC physiology is significantly influenced by donor variability and tissue of origin and this should be considered when designing clinical trials.

Dual roles of autologous CD8+ T cells in hematopoietic progenitor cell mobilization and engraftment.

BACKGROUND: Poor marrow cellularity alone cannot explain poor hematopoietic progenitor cell (HPC) mobilization. This study assessed the role of CD8+ T cells in HPC cell mobilization and engraftment. STUDY DESIGN AND METHODS: Mobilization and engraftment were assessed in 192 autologous HPC donors. CD34+, CD4+, and CD8+ T-cell contents in apheresis products were evaluated. Using a chemotaxis assay, we assessed the effect of purified autologous CD8+ T cells from low and high mobilizers on HPC migration from high to low stromal cell-derived factor (SDF-1α) concentration gradients. We also assessed CD8+ T-cell content association with days to neutrophil engraftment. RESULTS: The median number of CD34+ cells/kg was 4.7 × 10(6) . Patients were categorized according to their total CD34+ cell collection quartile distribution into low, moderate, and high mobilizers. We found that HPC products from low mobilizers contained more CD8+ T cells than HPC products from moderate and high mobilizers. Chemotaxis assays showed depletion of CD8+ T cells enhances HPC mobilization independent of SDF-1α concentration. Neutrophil engraftment analysis showed that the higher the CD8+ T-cell content per unit CD34+ cell, the faster the rate of engraftment. CONCLUSION: Our findings suggest CD8+ T cells inhibit HPC mobilization and facilitate homing and engraftment.

Non-genotoxic conditioning facilitates hematopoietic stem cell gene therapy for hemophilia A using bioengineered factor VIII.

Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.

Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application.

Growing stem cells on Earth is very challenging and limited to a few population doublings. The standard two-dimensional (2D) culture environment is an unnatural condition for cell growth. Therefore, culturing stem cells aboard the International Space Station (ISS) under a microgravity environment may provide a more natural three-dimensional environment for stem cell expansion and organ development. In this study, human-derived mesenchymal stem cells (MSCs) grown in space were evaluated to determine their potential use for future clinical applications on Earth and during long-term spaceflight. MSCs were flown in Plate Habitats for transportation to the ISS. The MSCs were imaged every 24-48 h and harvested at 7 and 14 days. Conditioned media samples were frozen at -80 °C and cells were either cryopreserved in 5% dimethyl sulfoxide, RNAprotect, or paraformaldehyde. After return to Earth, MSCs were characterized to establish their identity and cell cycle status. In addition, cell proliferation, differentiation, cytokines, and growth factors' secretion were assessed. To evaluate the risk of malignant transformation, the space-grown MSCs were subjected to chromosomal, DNA damage, and tumorigenicity assays. We found that microgravity had significant impact on the MSC capacity to secrete cytokines and growth factors. They appeared to be more potent in terms of immunosuppressive capacity compared to their identical ground control. Chromosomal, DNA damage, and tumorigenicity assays showed no evidence of malignant transformation. Therefore, it is feasible and potentially safe to grow MSCs aboard the ISS for potential future clinical applications.

Effect of Comedications and Endotoxins on Mesenchymal Stem Cell Secretomes, Migratory and Immunomodulatory Capacity.

Mesenchymal stem cells (MSCs) are becoming an increasingly popular therapeutic option among patients with a broad range of ailments to modulate immunity and induce regeneration. The majority of patients receiving these MSC therapies are on concurrent medication or have ongoing infection. In the present study, we examined the effect of immunosuppressive drugs and lipopolysaccharides (LPS)/endotoxins on the secretory profile, migration towards site of injury, and suppression of lymphocyte proliferation of bone marrow-derived MSCs (BMSCs). Generally, LPS coculture augmented the secretory capacity of BMSCs while exposure to immunosuppressive drugs resulted primarily in no change or attenuated secretion, with some cases of increased secretion, dependent on the cytokine assayed. Among the immunosuppressants evaluated, Hydrocortisone had the most widespread inhibitory effect, while LPS from E. coli O111:B4 had the most potent stimulatory effect. In addition, we also showed that Hydrocortisone or LPS from E. coli O111:B4 affected the migratory and immunosuppressive capacity of BMSCs. Following simulation with Hydrocortisone, BMSC migration was attenuated, and immunosuppressive capacity against T cell proliferation was enhanced, however, the opposite effects were seen with LPS from E. coli O111:B4. Our data suggests that the clinical outcomes of MSC-based therapy are affected by the use of immunosuppressive medication or the presence of endotoxemia in patients.