ABSTRACT
Single-cell datasets often lack individual cell labels, making it challenging to identify cells associated with disease. To address this, we introduce Mixture Modeling for Multiple Instance Learning (MMIL), an expectation maximization method that enables the training and calibration of cell-level classifiers using patient-level labels. Our approach can be used to train e.g. lasso logistic regression models, gradient boosted trees, and neural networks. When applied to clinically-annotated, primary patient samples in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL), our method accurately identifies cancer cells, generalizes across tissues and treatment timepoints, and selects biologically relevant features. In addition, MMIL is capable of incorporating cell labels into model training when they are known, providing a powerful framework for leveraging both labeled and unlabeled data simultaneously. Mixture Modeling for MIL offers a novel approach for cell classification, with significant potential to advance disease understanding and management, especially in scenarios with unknown gold-standard labels and high dimensionality.
ABSTRACT
Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.
Subject(s)
Busulfan , Microglia , Progranulins , Animals , Microglia/metabolism , Microglia/drug effects , Progranulins/metabolism , Progranulins/genetics , Mice , Busulfan/pharmacology , Hematopoietic Stem Cell Transplantation , Aminopyridines/pharmacology , Brain/metabolism , Pyrroles/pharmacology , Mice, Inbred C57BL , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/cytology , Bone Marrow Transplantation , Male , Central Nervous System/metabolism , Mice, Knockout , Transplantation Conditioning/methods , Single-Cell Analysis , Cytokines/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitorsABSTRACT
The growth of omic data presents evolving challenges in data manipulation, analysis and integration. Addressing these challenges, Bioconductor provides an extensive community-driven biological data analysis platform. Meanwhile, tidy R programming offers a revolutionary data organization and manipulation standard. Here we present the tidyomics software ecosystem, bridging Bioconductor to the tidy R paradigm. This ecosystem aims to streamline omic analysis, ease learning and encourage cross-disciplinary collaborations. We demonstrate the effectiveness of tidyomics by analyzing 7.5 million peripheral blood mononuclear cells from the Human Cell Atlas, spanning six data frameworks and ten analysis tools.
Subject(s)
Software , Humans , Computational Biology/methods , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/cytology , Genomics/methods , Data AnalysisABSTRACT
The growth of omic data presents evolving challenges in data manipulation, analysis, and integration. Addressing these challenges, Bioconductor1 provides an extensive community-driven biological data analysis platform. Meanwhile, tidy R programming2 offers a revolutionary standard for data organisation and manipulation. Here, we present the tidyomics software ecosystem, bridging Bioconductor to the tidy R paradigm. This ecosystem aims to streamline omic analysis, ease learning, and encourage cross-disciplinary collaborations. We demonstrate the effectiveness of tidyomics by analysing 7.5 million peripheral blood mononuclear cells from the Human Cell Atlas3, spanning six data frameworks and ten analysis tools.
ABSTRACT
Chimeric antigen receptor (CAR) T cell therapy is a promising treatment for pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia (R/R B ALL). Cytokine release syndrome (CRS) is a common toxicity after CAR T cell therapy and fever is often the first symptom. Differentiating CRS from infection after CAR T cell therapy can be challenging. Plasma microbial cell free DNA (mcfDNA) is a novel diagnostic tool which allows for qualitative and quantitative assessment of over 1000 organisms. This pilot study sought to characterize mcfDNA results in pediatric patients with R/R B ALL in the first 2 months after CAR T cell therapy.
Subject(s)
Cell-Free Nucleic Acids , Immunotherapy, Adoptive , Humans , Child , Immunotherapy, Adoptive/adverse effects , Immunotherapy, Adoptive/methods , Male , Female , Cell-Free Nucleic Acids/blood , Child, Preschool , Adolescent , Receptors, Chimeric Antigen/immunology , Recurrence , Pilot Projects , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , Treatment Outcome , Infant , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/therapy , Cytokine Release Syndrome/blood , Cytokine Release Syndrome/diagnosisABSTRACT
Chimeric antigen receptor (CAR) T cells targeting CD22 (CD22-CAR) provide a therapeutic option for patients with CD22+ malignancies with progression after CD19-directed therapies. Using on-site, automated, closed-loop manufacturing, we conducted parallel Phase 1b clinical trials investigating a humanized CD22-CAR with 41BB costimulatory domain in children and adults with heavily treated, relapsed/refractory (r/r) B-ALL. Of 19 patients enrolled, 18 had successful CD22-CAR manufacturing, and 16 patients were infused. High grade (3-4) cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS) each occurred in only one patient; however, three patients experienced immune-effector-cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). Twelve of 16 patients (75%) achieved CR with an overall 56% MRD-negative CR rate. Duration of response was overall limited (median 77 days), and CD22 expression was downregulated in 4/12 (33%) available samples at relapse. In summary, we demonstrate that closed-loop manufacturing of CD22-CAR T cells is feasible and is associated with a favorable safety profile and high CR rates in pediatric and adult r/r B-ALL, a cohort with limited CD22-CAR reporting.
Subject(s)
Immunotherapy, Adoptive , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Chimeric Antigen , Sialic Acid Binding Ig-like Lectin 2 , Humans , Sialic Acid Binding Ig-like Lectin 2/immunology , Child , Adult , Female , Male , Adolescent , Immunotherapy, Adoptive/methods , Immunotherapy, Adoptive/adverse effects , Young Adult , Receptors, Chimeric Antigen/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Child, Preschool , Middle Aged , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.
Subject(s)
Inosine , T-Lymphocytes , Humans , T-Lymphocytes/metabolismABSTRACT
A t(4;11) leukemia model established from CRISPR-engineered chromosomal translocations between the KMT2A and AFF1 genes recapitulate proteomic, epigenomic, and transcriptomic features of primary patient leukemias.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Translocation, Genetic , Humans , Gene Editing , Proteomics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , DNA-Binding Proteins/genetics , Transcriptional Elongation Factors/geneticsABSTRACT
Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies, with multiple CAR T cell products approved for numerous indications by regulatory agencies worldwide. However, significant work remains to be done to enhance these treatments. In March 2023, a group of experts in CAR T cell therapy assembled at the National Institutes of Health in Bethesda, Maryland at the Insights in Pediatric CAR T Cell Immunotherapy: Recent Advances and Future Directions (INSPIRED) Symposium to identify key areas for research for the coming years. In session 4B, correlative studies to be incorporated into future clinical trials and real-world settings were discussed. Active areas of research identified included (1) optimizing CAR T cell product manufacturing; (2) ensuring adequate lymphodepletion prior to CAR T cell administration; (3) overcoming immunoregulatory cells and tumor stroma present in the tumor microenvironment, particularly in solid tumors; (4) understanding tumor intrinsic properties that lead to CAR T cell immunotherapy resistance; and (5) uncovering biomarkers predictive of treatment resistance, treatment durability, or immune-related adverse events. Here we review the results of previously published clinical trials and real-world studies to summarize what is currently known about each of these topics. We then outline priorities for future research that we believe will be important for improving our understanding of CAR T cell therapy and ultimately leading to better outcomes for patients.
Subject(s)
Neoplasms , Receptors, Chimeric Antigen , United States , Humans , Child , Receptors, Chimeric Antigen/genetics , T-Lymphocytes , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/therapeutic use , Neoplasms/therapy , Immunotherapy, Adoptive/adverse effects , Tumor MicroenvironmentABSTRACT
Hematopoietic stem cell transplantation (HSCT) represents a consolidated therapeutic strategy for high-risk pediatric acute lymphoblastic leukemia (ALL), offering the potential for curative treatment. This manuscript delves into the debate around the more universal application of HSCT for pediatric ALL in the modern era, considering the ubiquitous availability of suitable donors. In fact, despite significant advancements in chemotherapy, targeted therapy, and immunotherapy, a subset of pediatric patients with ALL with high-risk features or relapse continue to encounter poor prognostic outcomes. For this subgroup of patients, HSCT often remains the only potentially curative measure, leveraging the graft-versus- leukemia effect for long-term disease control. Nevertheless, the procedure's complexity and associated risks have traditionally curtailed its widespread use. However, the scenario is shifting with improvements in HLA matching, availability of alternative donor sources, less toxic conditioning regimens, and improved supportive care protocols. Concurrently, emerging therapies like CD19+ CAR T cells present new considerations for definitive therapy selection in relapsed/ refractory ALL. This article reviews critical current evidence and debates the potential of HSCT as a more universal treatment for ALL, reevaluating traditional treatment stratification in light of the constant availability of stem cell donors.
Subject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Child , Hematopoietic Stem Cell Transplantation/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Tissue Donors , Prognosis , Transplantation Conditioning/methods , Graft vs Host Disease/etiologyABSTRACT
Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn-/- mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.
ABSTRACT
The advent of high-dimensional single-cell technologies has enabled detection of cellular heterogeneity and functional diversity of immune cells during health and disease conditions. Because of its multiplexing capabilities and limited compensation requirements, mass cytometry or cytometry by time of flight (CyTOF) has played a superior role in immune monitoring compared with flow cytometry. Further, it has higher throughput and lower cost compared with other single-cell techniques. Several published articles have utilized CyTOF to identify cellular phenotypes and features associated with disease outcomes. This article introduces CyTOF-based assays to profile immune cell-types, cell-states, and their applications in clinical research.
Subject(s)
Flow Cytometry , Flow Cytometry/methodsABSTRACT
PURPOSE: This study evaluated whether early brace treatment of curves < 25° decreased the prevalence of curve progression and surgery. METHODS: In a retrospective review, patients with idiopathic scoliosis Risser stages 0 to 2 braced at < 25° were followed until brace discontinuation, skeletal maturity, or surgery. Patients with predominantly primary thoracolumbar/lumbar curves were prescribed nighttime braces (NTB) and thoracic curves were prescribed fulltime braces (FTB). Comparisons were made for TLSO type (NTB vs. FTB) and triradiate cartilage (TRC) status (open vs. closed) at brace prescription. RESULTS: 283 patients were included, 81% who were Risser stage 0 with curves averaging 21.8° ± 2.1° at brace prescription. The average curve change was 2.4° ± 11.2°. Curves improved ≥ 6° in 23% of patients. Patients who were not skeletally mature at brace discontinuation (n = 39) had lower Cobb angles (16.7° vs. 23.9°, p < 0.001), better curve improvement (- 4.7° vs. 2.1°, p < 0.001), and were braced for a shorter period of time (1.8 years vs. 2.3 years, p = 0.011) than those who were skeletally mature at brace discontinuation (n = 239). Only 7% of patients in NTB and 8% of patients in FTB with open TRC required surgery. The numbers needed to treat to prevent surgery in patients in FTB with open TRC was calculated to be 4. CONCLUSION: Early brace treatment (Cobb < 25° and open TRC) may not only reduce curve progression and the need for surgical treatment but may also result in curve improvement, thus challenging the paradigm that the goal of bracing is merely to stop curve progression. LEVEL OF EVIDENCE: 3-retrospective cohort study.
ABSTRACT
Summary: While many algorithms for analyzing high-dimensional cytometry data have now been developed, the software implementations of these algorithms remain highly customized-this means that exploring a dataset requires users to learn unique, often poorly interoperable package syntaxes for each step of data processing. To solve this problem, we developed {tidytof}, an open-source R package for analyzing high-dimensional cytometry data using the increasingly popular 'tidy data' interface. Availability and implementation: {tidytof} is available at https://github.com/keyes-timothy/tidytof and is released under the MIT license. It is supported on Linux, MS Windows and MacOS. Additional documentation is available at the package website (https://keyes-timothy.github.io/tidytof/). Supplementary information: Supplementary data are available at Bioinformatics Advances online.
ABSTRACT
Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing.
ABSTRACT
Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.
Subject(s)
Burkitt Lymphoma , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Humans , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Dasatinib/pharmacology , Dasatinib/therapeutic use , Receptors, Glucocorticoid/genetics , Apoptosis , Proteomics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Recurrence , Drug Resistance, Neoplasm/genetics , Cell Line, TumorABSTRACT
Cancer immunotherapies have unique toxicities. Establishment of grading scales and standardized grade-based treatment algorithms for toxicity syndromes can improve the safety of these treatments, as observed for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) in patients with B cell malignancies treated with chimeric antigen receptor (CAR) T cell therapy. We have observed a toxicity syndrome, distinct from CRS and ICANS, in patients treated with cell therapies for tumors in the central nervous system (CNS), which we term tumor inflammation-associated neurotoxicity (TIAN). Encompassing the concept of 'pseudoprogression,' but broader than inflammation-induced edema alone, TIAN is relevant not only to cellular therapies, but also to other immunotherapies for CNS tumors. To facilitate the safe administration of cell therapies for patients with CNS tumors, we define TIAN, propose a toxicity grading scale for TIAN syndrome and discuss the potential management of this entity, with the goal of standardizing both reporting and management.
Subject(s)
Neoplasms , Neurotoxicity Syndromes , Humans , Neoplasms/therapy , Immunotherapy, Adoptive/adverse effects , Immunotherapy , Inflammation , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/therapy , Neurotoxicity Syndromes/etiologyABSTRACT
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse. MAIN BODY: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD <0.01%). Positron emission tomography with 18F-fluorodeoxyglucose revealed diffuse leukemia with innumerable bone and lymph node lesions, interestingly sparing her sacrum, the site of her bone marrow aspirate sampling. CONCLUSIONS: We highlight this case as both peripheral blood MRD and 18F-fluorodeoxyglucose positron emission tomography imaging were more sensitive than standard bone marrow aspirate testing in detecting this patient's post-CAR relapse. Clinical/Biologic Insight: In the multiply relapsed B-ALL setting, where relapse patterns may include patchy medullary and/or EM disease, peripheral blood MRD and/or whole body imaging, may carry increased sensitivity at detecting relapse in patient subsets, as compared with standard bone marrow sampling.
Subject(s)
Burkitt Lymphoma , Leukemia , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Humans , Child , Female , Fluorodeoxyglucose F18 , Bone Marrow/diagnostic imaging , Neoplasm, Residual , Positron-Emission Tomography , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnostic imaging , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapyABSTRACT
Childhood cancer is the second leading cause of death in children aged 1 to 14. Although survival rates have vastly improved over the past 40 years, cancer resistance and relapse remain a significant challenge. Advances in single-cell technologies enable dissection of tumors to unprecedented resolution. This facilitates unraveling the heterogeneity of childhood cancers to identify cell subtypes that are prone to treatment resistance. The rapid accumulation of single-cell data from different modalities necessitates the development of novel computational approaches for processing, visualizing, and analyzing single-cell data. Here, we review single-cell approaches utilized or under development in the context of childhood cancers. We review computational methods for analyzing single-cell data and discuss best practices for their application. Finally, we review the impact of several studies of childhood tumors analyzed with these approaches and future directions to implement single-cell studies into translational cancer research in pediatric oncology.
Subject(s)
Neoplasms , Child , Humans , Neoplasms/etiology , Neoplasms/therapy , Medical Oncology/methods , ProteomicsABSTRACT
BACKGROUND: Immunocompromised patients are at increased risk of SARS-CoV-2 infections. Patients undergoing chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory B-cell malignancies are uniquely immunosuppressed due to CAR T-mediated B-cell aplasia (BCA). While SARS-CoV-2 mortality rates of 33%-40% are reported in adult CAR T-cell recipients, outcomes in pediatric and young adult CAR T-cell recipients are limited. METHODS: We created an international retrospective registry of CAR T recipients aged 0-30 years infected with SARS-CoV-2 within 2 months prior to or any time after CAR T infusion. SARS-CoV-2-associated illness was graded as asymptomatic, mild, moderate, or severe COVID-19, or multisystem inflammatory syndrome in children (MIS-C). To assess for risk factors associated with significant SARS-CoV-2 infections (infections requiring hospital admission for respiratory distress or supplemental oxygen), univariate and multivariable regression analyses were performed. RESULTS: Nine centers contributed 78 infections in 75 patients. Of 70 SARS-CoV-2 infections occurring after CAR T infusion, 13 (18.6%) were classified as asymptomatic, 37 (52.9%) mild, 11 (15.7%) moderate, and 6 (8.6%) severe COVID-19. Three (4.3%) were classified as MIS-C. BCA was not significantly associated with infection severity. Prior to the emergence of the Omicron variant, of 47 infections, 19 (40.4%) resulted in hospital admission and 7 (14.9%) required intensive care, while after the emergence of the Omicron variant, of 23 infections, only 1 (4.3%) required admission and the remaining 22 (95.7%) had asymptomatic or mild COVID-19. Death occurred in 3 of 70 (4.3%); each death involved coinfection or life-threatening condition. In a multivariable model, factors associated with significant SARS-CoV-2 infection included having two or more comorbidities (OR 7.73, CI 1.05 to 74.8, p=0.048) and age ≥18 years (OR 9.51, CI 1.90 to 82.2, p=0.014). In the eight patients infected with SARS-CoV-2 before CAR T, half of these patients had their CAR T infusion delayed by 15-30 days. CONCLUSIONS: In a large international cohort of pediatric and young adult CAR-T recipients, SARS-CoV-2 infections resulted in frequent hospital and intensive care unit admissions and were associated with mortality in 4.3%. Patients with two or more comorbidities or aged ≥18 years were more likely to experience significant illness. Suspected Omicron infections were associated with milder disease.