HES6 knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.

Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models. Using bulk and scRNA-seq data, we show that HES6 is expressed during erythroid/megakaryocyte and pDC development, as well as in multipotent precursors and at specific stages of T- and B-cell development following preBCR and preTCR signalling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, pDCs, Band T-cells. In addition, HES6 knockdown HSPCs displayed reduced colony forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression impacts cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that impact these perturbations. Thus, our study uncovers new insights for a role of HES6 in human hematopoiesis.

Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum.

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively, these data support the feasibility, safety, and immunogenicity of this treatment in resected NSCLC.

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated, site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study, we demonstrate the ability to target the integration of WAS2-12-containing constructs into intron 1 of the endogenous WAS gene of primary CD34+ hematopoietic stem and progenitor cells (HSPCs), as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore, TI restored WASp-dependent function to WAS patient T cells. Edited CD34+ HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient's CD34+ cells.

The Wiskott-Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.

Single-cell profiling identifies a novel human polyclonal unconventional T cell lineage.

In the human thymus, a CD10+ PD-1+ TCRαß+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8ß expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny.

Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles.

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage.

Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF.

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

T-BET drives the conversion of human type 3 innate lymphoid cells into functional NK cells.

Type 3 innate lymphoid cells (ILC3s) are characterized by RORγt expression and they produce IL-22 upon activation. ILC3s play a role in maintenance of barrier integrity in the intestine. Under inflammatory conditions, the ILC composition of the mucosal tissues is altered due to a high degree of plasticity. It has been extensively demonstrated that both murine and human ILC3s convert into ILC1s to mediate appropriate immune responses. However, plasticity between human ILC3s and NK cells is less well documented. As T-BET and EOMES are key transcription factors in NK cell differentiation, we investigated whether ectopic T-BET or EOMES expression converts human ILC3s into NK cells. ILC3s with ectopic T-BET and EOMES expression downregulate RORγt expression, while T-BET-overexpressing ILC3s additionally upregulate EOMES expression. High E ctopic T-BET expression in ILC3s results in transdifferentiation towards CD94+ NK cells, whereas ectopic EOMES overexpression results in dedifferentiation of ILC3s into CD94-CD117-/low cells but is ineffective in NK cell generation. Dedifferentiating ILC3s from both T-BET and EOMES overexpression cultures upregulate NK cell receptors, perforin and granzyme B. Finally, IL-22 secretion is completely blocked in transdifferentiating ILC3s with both T-BET and EOMES ectopic expression, whereas only T-BET overexpression increases IFN-γ secretion and cytotoxicity. Altogether, these findings demonstrate that human ILC3s can convert into functional NK cells, wherein T-BET, and not EOMES, is the main driver.

TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality.

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

Immunopeptidomics-based design of mRNA vaccine formulations against Listeria monocytogenes.

Listeria monocytogenes is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. Attenuated Listeria strains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen for Listeria peptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68 Listeria immunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8+ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine against Listeria and aid to improve attenuated Listeria vaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

Identification of distinct functional thymic programming of fetal and pediatric human γδ thymocytes via single-cell analysis.

Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human.

Transcriptional dynamics and epigenetic regulation of E and ID protein encoding genes during human T cell development.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards αß and γδ T cells. Although many of the mechanisms that control αß- and γδ-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of γδ T cells. In contrast, in αß T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a cross-species and cross-developmental level.

The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency.

Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rß expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.

Small-scale manufacturing of neoantigen-encoding messenger RNA for early-phase clinical trials.

Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA. The authors have developed MIDRIXNEO, a personalized mRNA-loaded dendritic cell vaccine targeting tumor neoantigens, which is currently being evaluated in a phase 1 clinical study in lung cancer patients. To facilitate this study, the authors set up a Good Manufacturing Practice (GMP)-compliant production process for the manufacture of small batches of personalized neoantigen-encoding mRNA. In this article, the authors describe the complete mRNA production process and the extensive quality assessment to which the mRNA is subjected. Validation runs have shown that the process delivers mRNA of reproducible, high quality. This process is now successfully applied for the production of neoantigen-encoding mRNA for the clinical evaluation of MIDRIXNEO. To the authors' knowledge, this is the first time that a GMP-based production process of patient-tailored neoantigen mRNA has been described.

Photothermal nanofibres enable safe engineering of therapeutic cells.

Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR-Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.

T-BET and EOMES Accelerate and Enhance Functional Differentiation of Human Natural Killer Cells.

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies.

Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

In vitro OP9-DL1 co-culture and subsequent maturation in the presence of IL-21 generates tumor antigen-specific T cells with a favorable less-differentiated phenotype and enhanced functionality.

T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms' tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable TSCM-like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML.

A Novel Non-Coding Variant in DCLRE1C Results in Deregulated Splicing and Induces SCID Through the Generation of a Truncated ARTEMIS Protein That Fails to Support V(D)J Recombination and DNA Damage Repair.

Severe Combined Immune Deficiency (SCID) is a primary deficiency of the immune system in which opportunistic and recurring infections are often fatal during neonatal or infant life. SCID is caused by an increasing number of genetic defects that induce an abrogation of T lymphocyte development or function in which B and NK cells might be affected as well. Because of the increased availability and usage of next-generation sequencing (NGS), many novel variants in SCID genes are being identified and cause a heterogeneous disease spectrum. However, the molecular and functional implications of these new variants, of which some are non-coding, are often not characterized in detail. Using targeted NGS, we identified a novel homozygous c.465-1G>C splice acceptor site variant in the DCLRE1C gene in a T-B-NK+ SCID patient and fully characterized the molecular and functional impact. By performing a minigene splicing reporter assay, we revealed deregulated splicing of the DCLRE1C transcript since a cryptic splice acceptor in exon 7 was employed. This induced a frameshift and the generation of a p.Arg155Serfs*15 premature termination codon (PTC) within all DCLRE1C splice variants, resulting in the absence of full-length ARTEMIS protein. Consistently, a V(D)J recombination assay and a G0 micronucleus assay demonstrated the inability of the predicted mutant ARTEMIS protein to perform V(D)J recombination and DNA damage repair, respectively. Together, these experiments molecularly and functionally clarify how a newly identified c.465-1G>C variant in the DCLRE1C gene is responsible for inducing SCID. In a clinical context, this demonstrates how the experimental validation of new gene variants, that are identified by NGS, can facilitate the diagnosis of SCID which can be vital for implementing appropriate therapies.