IL3 Has a Detrimental Effect on Hematopoietic Stem Cell Self-Renewal in Transplantation Settings.

The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34+ from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34+ cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols.

IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study.

BACKGROUND: Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment. METHODS AND FINDINGS: We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome. To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor's ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL. CONCLUSIONS: Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events.

Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression.

ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.

The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia.

The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycolysis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia.

Methods of Observing Variations in Physicians' Decisions: The Opportunities of Clinical Vignettes.

To support their efforts to promote high quality and efficient care, policymakers need to better understand the key factors associated with variations in physicians' decisions, and in particular, physician deviations from evidence-based care. Clinical vignette survey instruments hold potential for research in this area as an approach that both allows for practical, large-scale study and overcomes the data quality challenges posed by analysis of clinical data. These surveys present respondents with a narrative description of a hypothetical patient case and solicit responses to one or more questions regarding the care of the patient. In this review, we describe various methods for measuring variations in physicians' decisions and highlight a range of design features researchers should consider when developing a clinical vignette survey. We conclude by identifying areas for future research.

The Results Are Only as Good as the Sample: Assessing Three National Physician Sampling Frames.

BACKGROUND: Databases of practicing physicians are important for studies that require sampling physicians or counting the physician population in a given area. However, little is known about how the three main sampling frames differ from each other. OBJECTIVE: Our purpose was to compare the National Provider and Plan Enumeration System (NPPES), the American Medical Association Masterfile and the SK&A physician file. METHODS: We randomly sampled 3000 physicians from the NPPES (500 in six specialties). We conducted two- and three-way comparisons across three databases to determine the extent to which they matched on address and specialty. In addition, we randomly selected 1200 physicians (200 per specialty) for telephone verification. KEY RESULTS: One thousand, six hundred and fifty-five physicians (55 %) were found in all three data files. The SK&A data file had the highest rate of missing physicians when compared to the NPPES, and varied by specialty (50 % in radiology vs. 28 % in cardiology). NPPES and SK&A had the highest rates of matching mailing address information, while the AMA Masterfile had low rates compared with the NPPES. We were able to confirm 65 % of physicians' address information by phone. The NPPES and SK&A had similar rates of correct address information in phone verification (72-94 % and 79-92 %, respectively, across specialties), while the AMA Masterfile had significantly lower rates of correct address information across all specialties (32-54 % across specialties). CONCLUSIONS: None of the data files in this study were perfect; the fact that we were unable to reach one-third of our telephone verification sample is troubling. However, the study offers some encouragement for researchers conducting physician surveys. The NPPES and to a lesser extent, the SK&A file, appear to provide reasonably accurate, up-to-date address information for physicians billing public and provider insurers.

Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors.

Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients.

Utilizing Epigenetic Regulators to improve HSC-based lentiviral gene therapy.

Curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven for various diseases. However, the low number of true HSCs that can be collected from patients and subsequently in vitro maintenance and expansion of true HSCs for genetic correction remain challenging. Addressing this issue, we here focused on optimizing culture conditions to improve the ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explore the use of epigenetic regulators to enhance the effectiveness of HSC-based lentiviral (LV) gene therapy. The HDAC inhibitor Quisinostat and the bromodomain inhibitor CPI203 each promote ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single cell RNA-sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, while addition of Quisinostat or CPI203 improved expansion of HSCs in transduction protocols. Of note, we demonstrated that addition of Quisinostat improved LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effect of epigenetic regulators in hematopoietic stem cell biology and their clinical applications to advance HSC-based gene correction.

Calcineurin sets the bandwidth for discrimination of signals during thymocyte development.

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.

The reliability and validity of lung cancer and melanoma clinical quality survival measures.

OBJECTIVE: To develop a risk adjustment approach and test reliability and validity for oncology survival measures. DATA SOURCES AND STUDY SETTING: We used the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER)-Medicare data from 2010 to 2013, with mortality data through 2015. STUDY DESIGN: We developed 2-year risk-standardized survival rates (RSSR) for melanoma, non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). Patients were attributed to group practices based on the plurality of visits. We identified the risk-adjustment variables via bootstrap and calculated the RSSRs. Reliability was tested via three approaches: (1) signal-to-noise ratio (SNR) reliability, (2) split-half, and (3) test-retest using bootstrap. We tested known group validity by stage at diagnosis using Cohen's d. DATA COLLECTION/EXTRACTION METHODS: We selected all patients enrolled in Medicare and linked to SEER during the measurement period with an incident first primary diagnosis of stage I-IV melanoma, NSCLC, or SCLC. We excluded patients with missing data on month and/or stage of diagnosis. PRINCIPAL FINDINGS: Results are based on patients with melanoma (n = 4344); NSCLC (n = 16,080); and SCLC (n = 2807) diagnosed between 2012 and 2013. The median (interquartile range) for the RSSRs at the group practice-level were 0.89 (0.83-0.87) for melanoma, 0.37 (0.30-0.43) for NSCLC, and 0.19 (0.11-0.25) for SCLC. C-statistics for the models ranged from 0.725 to 0.825. The reliability varied by approach with median SNR 0.20, 0.25, and 0.13; median test-retest 0.59, 0.57, and 0.56; median split-half reliability 0.21, 0.29, and 0.29 for melanoma, NSCLC, and SCLC, respectively. Cohen's d for stage I-IIIa and IIIb+ was 1.27, 0.86, 0.60 for melanoma, NSCLC, and SCLC, respectively. CONCLUSIONS: Our results suggest that these cancer survival measures demonstrated adequate test-retest reliability and expected findings for the known-group validity analysis. If data limitations and feasibility challenges can be addressed, implementation of these quality measures may provide a survival metric used for oncology quality improvement efforts.

The quest for the holy grail: overcoming challenges in expanding human hematopoietic stem cells for clinical use.

Hematopoietic stem cell (HSC) transplantation has been the golden standard for many hematological disorders. However, the number of HSCs obtained from several sources, including umbilical cord blood (UCB), often is insufficient for transplantation. For decades, maintaining or even expanding HSCs for therapeutic purposes has been a "holy grail" in stem cell biology. Different methods have been proposed to improve the efficiency of cell expansion and enhance homing potential such as co-culture with stromal cells or treatment with specific agents. Recent progress has shown that this is starting to become feasible using serum-free and well-defined media. Some of these protocols to expand HSCs along with genetic modification have been successfully applied in clinical trials and some others are studied in preclinical and clinical studies. However, the main challenges regarding ex vivo expansion of HSCs such as limited growth potential and tendency to differentiate in culture still need improvements. Understanding the biology of blood stem cells, their niche and signaling pathways has provided possibilities to regulate cell fate decisions and manipulate cells to optimize expansion of HSCs in vitro. Here, we review the plethora of HSC expansion protocols that have been proposed and indicate the current state of the art for their clinical application.

Multi-omic analyses in immune cell development with lessons learned from T cell development.

Traditionally, flow cytometry has been the preferred method to characterize immune cells at the single-cell level. Flow cytometry is used in immunology mostly to measure the expression of identifying markers on the cell surface, but-with good antibodies-can also be used to assess the expression of intracellular proteins. The advent of single-cell RNA-sequencing has paved the road to study immune development at an unprecedented resolution. Single-cell RNA-sequencing studies have not only allowed us to efficiently chart the make-up of heterogeneous tissues, including their most rare cell populations, it also increasingly contributes to our understanding how different omics modalities interplay at a single cell resolution. Particularly for investigating the immune system, this means that these single-cell techniques can be integrated to combine and correlate RNA and protein data at the single-cell level. While RNA data usually reveals a large heterogeneity of a given population identified solely by a combination of surface protein markers, the integration of different omics modalities at a single cell resolution is expected to greatly contribute to our understanding of the immune system.

Real-World Adherence to Patient-Reported Outcome Monitoring as a Cancer Care Quality Metric.

PURPOSE: Routine collection of patient-reported outcomes (PROs) for patients with advanced solid malignancies is an evidence-based practice and critical component of high-quality cancer care, but real-world adherence is poorly characterized. We sought to describe real-world adherence to PRO monitoring and its potential predictors. METHODS: We conducted a retrospective cross-sectional study using deidentified electronic health record data from a National Cancer Institute Cancer Center, encompassing one academic and two community sites. Participants included individuals with lung cancer receiving systemic therapy from January 1 to December 31, 2019. The primary outcome was patient-level adherence, defined as the proportion of treatment visits during which a PRO questionnaire (spanning symptoms, functional status, and global quality-of-life domains) was completed within 30 days. Practice-level performance was calculated as unadjusted mean patient-level adherence. We modeled patient-level adherence using multivariable ordinary least squares regression and identified covariates associated with adherence using a significance threshold of P < .05. RESULTS: In 2019, there were 18,604 encounters for 1,105 patients with lung cancer (mean [standard deviation] age 65.8 [10.2] years; 621 [56.2%] female; 216 [19.6%] Black) receiving systemic therapy. The mean patient-level PRO adherence ranged from 27.2% to 70.0% across sites and was 49.4% overall. Advanced age (≥ 65 years) and Black or African American race were negatively associated with PRO adherence (P < .01). CONCLUSION: Across this real-world cohort of patients undergoing treatment for lung cancer, adherence to PRO monitoring lagged that achieved in seminal clinical trials, with potential age- and race-based disparities, demonstrating an implementation gap that could be addressed with standardized reporting of an adherence-based quality metric.

Climate change, fire return intervals and the growing risk of permanent forest loss in boreal Eurasia.

Climate change has driven an increase in the frequency and severity of fires in Eurasian boreal forests. A growing number of field studies have linked the change in fire regime to post-fire recruitment failure and permanent forest loss. In this study we used four burned area and two forest loss datasets to calculate the landscape-scale fire return interval (FRI) and associated risk of permanent forest loss. We then used machine learning to predict how the FRI will change under a high emissions scenario (SSP3-7.0) by the end of the century. We found that there are currently 133,000 km2 forest at high, or extreme, risk of fire-induced forest loss, with a further 3 M km2 at risk by the end of the century. This has the potential to degrade or destroy some of the largest remaining intact forests in the world, negatively impact the health and economic wellbeing of people living in the region, as well as accelerate global climate change.

MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

Rearrangements that drive ectopic MEF2C expression have recurrently been found in patients with human early thymocyte progenitor acute lymphoblastic leukemia (ETP-ALL). Here, we show high levels of MEF2C expression in patients with ETP-ALL. Using both in vivo and in vitro models of ETP-ALL, we demonstrate that elevated MEF2C expression blocks NOTCH-induced T cell differentiation while promoting a B-lineage program. MEF2C activates a B cell transcriptional program in addition to RUNX1, GATA3, and LMO2; upregulates the IL-7R; and boosts cell survival by upregulation of BCL2. MEF2C and the Notch pathway, therefore, demarcate opposite regulators of B- or T-lineage choices, respectively. Enforced MEF2C expression in mouse or human progenitor cells effectively blocks early T cell differentiation and promotes the development of biphenotypic lymphoid tumors that coexpress CD3 and CD19, resembling human mixed phenotype acute leukemia. Salt-inducible kinase (SIK) inhibitors impair MEF2C activity and alleviate the T cell developmental block. Importantly, this sensitizes cells to prednisolone treatment. Therefore, SIK-inhibiting compounds such as dasatinib are potentially valuable additions to standard chemotherapy for human ETP-ALL.

Single-cell immune profiling reveals thymus-seeding populations, T cell commitment, and multilineage development in the human thymus.

T cell development in the mouse thymus has been studied extensively, but less is known regarding T cell development in the human thymus. We used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of prelineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step toward monitoring and guiding regenerative therapies in patients after hematopoietic stem cell transplantation.

Stem Cell-Based Disease Models for Inborn Errors of Immunity.

The intrinsic capacity of human hematopoietic stem cells (hHSCs) to reconstitute myeloid and lymphoid lineages combined with their self-renewal capacity hold enormous promises for gene therapy as a viable treatment option for a number of immune-mediated diseases, most prominently for inborn errors of immunity (IEI). The current development of such therapies relies on disease models, both in vitro and in vivo, which allow the study of human pathophysiology in great detail. Here, we discuss the current challenges with regards to developmental origin, heterogeneity and the subsequent implications for disease modeling. We review models based on induced pluripotent stem cell technology and those relaying on use of adult hHSCs. We critically review the advantages and limitations of current models for IEI both in vitro and in vivo. We conclude that existing and future stem cell-based models are necessary tools for developing next generation therapies for IEI.