International Society for Cell & Gene Therapy Stem Cell Engineering Committee report on the current state of hematopoietic stem and progenitor cell-based genomic therapies and the challenges faced.

Genetic manipulation of hematopoietic stem cells (HSCs) is being developed as a therapeutic strategy for several inherited disorders. This field is rapidly evolving with several novel tools and techniques being employed to achieve desired genetic changes. While commercial products are now available for sickle cell disease, transfusion-dependent ß-thalassemia, metachromatic leukodystrophy and adrenoleukodystrophy, several challenges remain in patient selection, HSC mobilization and collection, genetic manipulation of stem cells, conditioning, hematologic recovery and post-transplant complications, financial issues, equity of access and institutional and global preparedness. In this report, we explore the current state of development of these therapies and provide a comprehensive assessment of the challenges these therapies face as well as potential solutions.

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.

Advancements in γδT cell engineering: paving the way for enhanced cancer immunotherapy.

Comprising only 1-10% of the circulating T cell population, γδT cells play a pivotal role in cancer immunotherapy due to their unique amalgamation of innate and adaptive immune features. These cells can secrete cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and can directly eliminate tumor cells through mechanisms like Fas/FasL and antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike conventional αßT cells, γδT cells can target a wide variety of cancer cells independently of major histocompatibility complex (MHC) presentation and function as antigen-presenting cells (APCs). Their ability of recognizing antigens in a non-MHC restricted manner makes them an ideal candidate for allogeneic immunotherapy. Additionally, γδT cells exhibit specific tissue tropism, and rapid responsiveness upon reaching cellular targets, indicating a high level of cellular precision and adaptability. Despite these capabilities, the therapeutic potential of γδT cells has been hindered by some limitations, including their restricted abundance, unsatisfactory expansion, limited persistence, and complex biology and plasticity. To address these issues, gene-engineering strategies like the use of chimeric antigen receptor (CAR) T therapy, T cell receptor (TCR) gene transfer, and the combination with γδT cell engagers are being explored. This review will outline the progress in various engineering strategies, discuss their implications and challenges that lie ahead, and the future directions for engineered γδT cells in both monotherapy and combination immunotherapy.

Vaccine Associated Measles Complicated by Suspected Measles Inclusion Body Encephalitis in a Pediatric Leukemia Patient and Stem Cell Transplant Recipient: A Focus on Clinical Evolution and Management.

BACKGROUND: Immunocompromised individuals are at increased risk for severe disease and complications from viral infections, highlighting the importance of vaccination. However, in extremely rare situations, vaccine associated viral infections can be associated with disseminated disease and complications in immunocompromised hosts. CASE: Herein, we present a case of a 1-year-old child diagnosed with acute myeloid leukemia less than 2 weeks after receiving live viral vaccines who developed acute vaccine-strain measles virus disease, later complicated by central nervous system involvement following hematopoietic stem cell transplantation. A brain biopsy specimen was positive for vaccine-strain measles virus detected by reverse transcriptase polymerase chain reaction. MANAGEMENT AND OUTCOME: She was treated with intravenous ribavirin, inosine pranobex, intrathecal interferon-alpha and donor lymphocyte infusion following measles-mumps-rubella vaccine boost. Despite these measures, the patient suffered neurologic decline and dysautonomia, expiring after compassionate extubation. Management and ideal risk mitigation strategies are discussed within the context of existing literature for this rare complication.

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.

Extracellular release of damaged mitochondria induced by prehematopoietic stem cell transplant conditioning exacerbates GVHD.

ABSTRACT: Despite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.

Effect of Rabbit ATG PK on Outcomes after TCR-αß/CD19-depleted Pediatric Haploidentical HCT for Hematologic Malignancy.

We hypothesized that inferior disease-free survival (DFS) seen in older patients undergoing αß/CD19-T-cell depleted (AB-TCD) haploidentical hematopoietic cell transplantation (HCT) for patients with hematologic malignancies was due to excessive exposure to rabbit antithymocyte globulin (rATG; Thymoglobulin®). Between 2015-2023, 163 patients with a median age of 13 years (range, 0.4-27.4) underwent AB-TCD haploidentical HCT for treatment of ALL (n=98), AML/MDS (n=49), or other malignancies (n=16) at nine centers on two prospective trials. Exposures of rATG pre- and post-HCT were predicted with a validated pharmacokinetic (PK) model. ROC curves were used to identify optimal target windows of rATG exposure related to outcomes. We identified four quadrants of rATG exposure - quadrant 1 (n=52): high pre-HCT AUC (≥50 AU*day/mL) and low post-HCT (<12 AU*day/L); quadrant 2 (n=47): both low pre-HCT and post-HCT AUCs, quadrant 3 (n=13): low pre-HCT AUC and high post-HCT, and quadrant 4 (n=51): both high pre- and post-HCT AUCs. Quadrant 1 had a 3-year DFS of 86.5% (95% CI, 76.3-96.7%), compared to quadrant 2 (64.6%; 95% CI, 49.1-80.1%), quadrant 3 (32.9%; 95% CI, 0.1-80.5%) or quadrant 4 (48.2%; 95% CI, 22.1-63.3%) (p<0.001). Adjusted regression analysis demonstrated additional factors associated with increased hazard for worse DFS: MRD-positivity (HR=2.45; 1.36-4.41; p=0.003) and CMV R+/D- serostatus (HR=3.33; 1.8-6.16; p<0.001). Non-optimal rATG exposure exhibited the strongest effect in unadjusted (HR=4.24; 1.79-10.03; p=0.001) and adjusted (MRD status or CMV serostatus) analyses (HR=3.84, 1.63-9.05; p=0.002). High exposure to rATG post-HCT is associated with inferior DFS following AB-TCD haploidentical HCT for pediatric patients with hematologic malignancies. Model-based dosing of rATG to achieve optimal exposure may improve DFS. Clinical trials: NCT02646839 & NCT04337515.

Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.