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X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) is an extremely rare inborn error of immunity (IEI) caused by X-linked recessive inheritance and loss-of-function mutations in the MAGT1 gene, resulting in magnesium ion channel defects. This article reports 2 cases of systemic EBV-positive T-cell Lymphoma of childhood (SETLC) associated with XMEN, which have not been reported before. Whole exome sequencing (WES) in their family revealed previously unreported MAGT1 gene mutations (c.77T>C, p.I26T; c.956-957del: p.Ser319Tyrfs) inherited from their mothers. These mutations expand the spectrum of gene mutations in XMEN disease. The importance of genetic testing for MAGT1 mutations in the initial diagnosis of SETLC was emphasized. We also review the literature on this uncommon IEI., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Competing Interests: Declaration of interests R.S.M. is an employee of and has equity in Immusoft Corporation.

Base editing of hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematologic diseases. However, the feasibility of using adenine-base-edited HSPCs for treating X-linked severe combined immunodeficiency (SCID-X1), the influence of dose-response relationships on immune cell generation, and the potential risks have not been demonstrated in vivo. Here, a humanized SCID-X1 mouse model was established, and 86.67% ± 2.52% (n = 3) of mouse hematopoietic stem cell (HSC) pathogenic mutations were corrected, with no single-guide-RNA (sgRNA)-dependent off-target effects detected. Analysis of peripheral blood over 16 weeks post-transplantation in mice with different immunodeficiency backgrounds revealed efficient immune cell generation following transplantation of different amounts of modified HSCs. Therefore, a large-scale infusion of gene-corrected HSCs within a safe range can achieve rapid, stable, and durable immune cell regeneration. Tissue-section staining further demonstrated the restoration of immune organ tissue structures, with no tumor formation in multiple organs. Collectively, these data suggest that base-edited HSCs are a potential therapeutic approach for SCID-X1 and that a threshold infusion dose of gene-corrected cells is required for immune cell regeneration. This study lays a theoretical foundation for the clinical application of base-edited HSCs in treating SCID-X1., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.

Background: X-linked severe combined immunodeficiency is caused by IL2RG gene mutation. Several variations have been identified in the IL2RG gene, which potentially can prevent the production of nonfunctional proteins. Herein, a novel X-linked variant in the IL2RG gene is reported in twin brothers, associated with inflammatory bowel symptoms., Case Presentation: The patients were 26-month-old monozygotic twin middle-eastern males with failure to thrive and several inpatient admissions due to severe chronic nonbloody diarrhea that started at the age of 12 months. Pancolitis was revealed after performing upper and lower gastrointestinal endoscopies on the twin with more severe gastrointestinal symptoms. Flow cytometric evaluation of the peripheral blood cells showed low levels of CD4+ cells in both patients. Next generation sequencing-based gene panel test results of the two patients proved a novel heterozygous missense X-linked IL2RG mutation (70330011 A > G, p.Trp197Arg) in one of the patients, which was predicted to be deleterious (CADD score of 28), which soon after was confirmed by Sanger segregation in his twin brother. Both parents were wild types and had never experienced similar symptoms. The patients received an human leukocyte antigen (HLA)-matched cord blood transplant. The twin with more severe gastrointestinal symptoms died 1 month after transplantation. In his brother, watery diarrhea eventually subsided after transplantation., Conclusion: Intestinal involvement in X-linked severe combined immunodeficiency is a rare presentation that might be neglected. The increasing availability of genetic screening tests worldwide could be helpful for early detection of such lethal primary immunodeficiency diseases and in implementing effective interventions to handle the severe outcomes., (© 2023. The Author(s).)

Maternal engraftment is frequently present in X‑linked severe combined immunodeficiency (X‑SCID) patients caused by pathogenic mutations in IL2GR . However, the functional status of the engrafted cells remains unclear because of the difficulty in separately evaluating the function of the maternal and autologous cells. The present study reported an X‑SCID patient with a de novo c.677C>T (p.R226H) variant in exon 5 of IL2RG , exhibiting recurrent and persistent infections from 3‑months‑old. After the male patient suffering recurrent pneumonia and acute hematogenous disseminated tuberculosis when 13‑months‑old, single‑cell RNA sequencing was applied to characterize the transcriptome landscape of his bone marrow mononuclear cells (BMMNCs). A novel bioinformatic analysis strategy was designed to discriminate maternal and autologous cells at single‑cell resolution. The maternal engrafted cells consisted primarily of T, NKT and NK cells and the patient presented with the coexistence of autologous cells of these cell types. When compared respectively with normal counterparts, both maternal and autologous T and NKT cells increased the transcription of some important cytokines ( GZMB , PRF1 and NKG7 ) against infections, but decreased the expression of a number of key transcription factors ( FOS , JUN , TCF7 and LEF1 ) related to lymphocyte activation, proliferation and differentiation. Notably, the expression of multiple inhibitory factors ( LAG3 , CTLA4 and HAVCR2 ) were substantially enhanced in the T and NKT cells of both origins. In conclusion, both maternal and autologous T and NKT cells exhibited exhaustion‑like dysfunction in this X‑SCID patient suffering recurrent and persistent infections.

Abnormally high γδ T cell numbers among individuals with atypical SCID have been reported but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large γδ T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRvγ sequencing, and target cell killing. In contrast to his ⍺β T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in γδ T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells., (© 2022. The Author(s).)

Introduction: Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs., Methods: We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)- IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)- IL2RG donor following double-strand DNA break at the endogenous IL2RG locus., Results and Discussion: In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI- IL2RG HSPCs with efficient T cell development following TI- IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before ( in vitro ) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients., Competing Interests: BK is an inventor on patents and patent applications filed by Mass General Brigham that describe genome engineering technologies. BK is a consultant for Avectas Inc., EcoR1 capital, and ElevateBio, and is an advisor to Acrigen Biosciences and Life Edit Therapeutics. RM and GD are employees of CELLSCRIPT, LLC Madison, WI. Authors SL and XW were employed by Leidos Biomedical Research Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brault, Liu, Liu, Lawson, Choi, Kozhushko, Bzhilyanskaya, Pavel-Dinu, Meis, Eckhaus, Burkett, Bosticardo, Kleinstiver, Notarangelo, Lazzarotto, Tsai, Wu, Dahl, Porteus, Malech and De Ravin.)

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.

To address inborn errors of immunity (IEI) which were underdiagnosed in resource-limited regions, our centre developed and offered free genetic testing for the most common IEI by Sanger sequencing (SS) since 2001. With the establishment of The Asian Primary Immunodeficiency (APID) Network in 2009, the awareness and definitive diagnosis of IEI were further improved with collaboration among centres caring for IEI patients from East and Southeast Asia. We also started to use whole exome sequencing (WES) for undiagnosed cases and further extended our collaboration with centres from South Asia and Africa. With the increased use of Next Generation Sequencing (NGS), we have shifted our diagnostic practice from SS to WES. However, SS was still one of the key diagnostic tools for IEI for the past two decades. Our centre has performed 2,024 IEI SS genetic tests, with in-house protocol designed specifically for 84 genes, in 1,376 patients with 744 identified to have disease-causing mutations (54.1%). The high diagnostic rate after just one round of targeted gene SS for each of the 5 common IEI (X-linked agammaglobulinemia (XLA) 77.4%, Wiskott-Aldrich syndrome (WAS) 69.2%, X-linked chronic granulomatous disease (XCGD) 59.5%, X-linked severe combined immunodeficiency (XSCID) 51.1%, and X-linked hyper-IgM syndrome (HIGM1) 58.1%) demonstrated targeted gene SS should remain the first-tier genetic test for the 5 common X-linked IEI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chan, Wong, Leung, Yang, Fok, Mak, Yao, Ma, Mao, Zhao, Liang, Singh, Barbouche, He, Jiang, Liew, Le, Muktiarti, Santos-Ocampo, Djidjik, Belaid, Ismail, Abdul Latiff, Lee, Chen, Liu, Jin, Wang, Chien, Yu, Raj, Raj, Vaughan, Urban, Berg, Eley, Lee, Isa, Ang, Lee, Yeoh, Shek, Quynh Le, Nguyen, Phan Nguyen Lien, Capulong, Mallillin, Villanueva, Camonayan, Vera, Casis-Hao, Lobo, Foronda, Binas, Boushaki, Kechout, Phongsamart, Wongwaree, Jiratchaya, Lao-Araya, Trakultivakorn, Suratannon, Jirapongsananuruk, Chantveerawong, Kamchaisatian, Chan, Koh, Wong, Fong, Thong, Latiff, Noh, Silva, Jouhadi, Al-Saad, Vignesh, Jindal, Rawat, Gupta, Suri, Yang, Au, Kwok, Chan, Hui, Chua, Duque, Cheong, Chong, Ho, Lee, Wong, Yang, Lee, Tu, Yang and Lau.)

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

XMEN (X-linked immunodeficiency with magnesium defect) is caused by loss-of-function mutations in MAGT1 which is encoded on the X chromosome. The disorder is characterised by CD4 lymphopenia, severe chronic viral infections and defective T-lymphocyte activation. XMEN patients are susceptible to Epstein-Barr virus infections and persistently low levels of intracellular Mg 2+ . Here we describe a patient that presented with multiple recurrent infections and a subsequent diffuse B-cell lymphoma. Molecular genetic analysis by exome sequencing identified a novel hemizygous MAGT1 nonsense mutation c.1005T>A (NM_032121.5) p.(Cys335*), confirming a diagnosis of XMEN deficiency. Follow-up immunophenotyping was performed by antibody staining and flow cytometry; proliferation was determined by 3 H-thymidine uptake after activation by PHA and anti-CD3. Cytotoxic natural killer cell activity was assessed with K562 target cells using the NKTEST TM assay. While lymphocyte populations were superficially intact, B cells were largely naive with a reduced memory cell compartment. Translated NKG2D was absent on both NK and T cells in the proband, and normally expressed in the carrier mother. In vitro NK cell activity was intact in both the proband and his mother. This report adds to the growing number of identified XMEN cases, raising awareness of a, still rare, X-linked immunodeficiency., (© 2022. The Author(s).)

Loss-of-function mutations in magnesium transporter 1 (MAGT1) gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasm (X-MEN), a disease with quite diverse clinical and immunological consequences. The phenotypic characteristics of the initially described patients included CD4+ T cell lymphopenia, immune deficiency, EBV viremia, and EBV-related lymphoproliferative disease. To date, a total of 25 patients have been reported. The spectrum of the MAGT1 defect ranges from other viral infections (HSV, VZV, CMV, MCV) and sinopulmonary bacterial infections, autoimmune diseases, non-EBV driven lymphoproliferative disease, Castleman disease, HHV8+ Kaposi's sarcoma, vasculitis (Kawasaki) to glycosylation defects in new patients. Here, we report 2 patients from two different families with novel MAGT1 mutations and different clinical features. The first patient presented with B cell lymphoma and low IgM level without recurrent infections. The second patient presented with recurrent upper respiratory tract infections, Kawasaki-like disease, hypogammaglobulinemia, and T cell lymphopenia. X-MEN disease is the first phenotype identified due to MAGT1 mutation. The identification of new mutations and atypical presentations will clarify whether there is a relationship between the genotype and the phenotype and the characteristics of the disease.

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesin R171W ). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesin R171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesin R171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesin R171W . Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesin R171W , and will provide new insights into how the actin cortex regulates lymphocyte function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Avery, Robertson, Wu, Roy, Chauvin, Perkey, Vanderbeck, Maillard and Burkhardt.)

X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus (EBV) infection and N-linked glycosylation defect (XMEN) disease is an inborn error of immunity caused by loss-of-function mutations in the magnesium transporter 1 (MAGT1) gene. The original studies of XMEN patients focused on impaired magnesium regulation, leading to decreased EBV-cytotoxicity and the loss of surface expression of the activating receptor "natural killer group 2D" (NKG2D) on CD8 + T cells and NK cells. In vitro studies showed that supraphysiological supplementation of magnesium rescued these defects. Observational studies in 2 patients suggested oral magnesium supplementation could decrease EBV viremia. Hence, we performed a randomized, double-blind, placebo-controlled, crossover study in 2 parts. In part 1, patients received either oral magnesium L-threonate (MLT) or placebo for 12 weeks followed by 12 weeks of the other treatment. Part 2 began with 3 days of high-dose intravenous (IV) magnesium sulfate (MgSO 4 ) followed by open-label MLT for 24 weeks. One EBV-infected and 3 EBV-naïve patients completed part 1. One EBV-naïve patient was removed from part 2 of the study due to asymptomatic elevation of liver enzymes during IV MgSO 4 . No change in EBV or NKG2D status was observed. In vitro magnesium supplementation experiments in cells from 14 XMEN patients failed to significantly rescue NKG2D expression and the clinical trial was stopped. Although small, this study indicates magnesium supplementation is unlikely to be an effective therapeutic option in XMEN disease., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation., (© 2021 by The American Society of Hematology.)

Mutations of the IL2RG gene, which encodes for the interleukin-2 receptor common gamma chain (γ C , CD132), can lead to X-linked severe combined immunodeficiency (X-SCID) associated with a T - B + NK - phenotype as a result of dysfunctional γ C -JAK3-STAT5 signaling. Lately, hypomorphic mutations of the IL2RG gene have been described causing atypical SCID with a milder phenotype. Here, we report three brothers with low-normal lymphocyte counts and susceptibility to recurrent respiratory infections and cutaneous warts. The clinical presentation combined with dysgammaglobulinemia suspected an inherited immunity disorder, which has been proven by Next Generation Sequencing as a novel c.458T > C; p.Ile153Thr IL2RG missense-mutation. Subsequent functional characterization revealed impaired T-cell proliferation, low TREC levels and a skewed TCR Vβ repertoire in all three patients. Interestingly, investigation of various subpopulations showed normal expression of CD132 but with partially impaired STAT5 phosphorylation compared to healthy controls. Additionally, we performed precise genetic analysis of subpopulations revealing spontaneous somatic reversion, predominately in lymphoid derived CD3 + , CD4 + and CD8 + T cells. Our data demonstrate that the atypical SCID phenotype noticed in these three brothers is due to the combination of hypomorphic IL-2RG function and somatic reversion.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA-corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1-/- and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival., (© 2021 by The American Society of Hematology.)

Cryptococcosis is an opportunistic disease caused by the fungus Cryptococcus neoformans and Cryptococcus gattii. It starts as a pulmonary infection that can spread to other organs, such as the brain, leading to the most serious occurrence of the disease, meningoencephalitis. The humoral response has already been described in limiting the progression of cryptococcosis where the B-1 cell seems to be responsible for producing natural IgM antibodies, crucial for combating fungal infections. The role of the B-1 cell in C. neoformans infection has been initially described, however the role of the humoral response of B-1 cells has not yet been evaluated during C. gattii infections. In the present study we tried to unravel this issue using XID mice, a murine model deficient in the Btk protein which compromises the development of B-1 lymphocytes. We use the XID mice compared to BALB/c mice that are sufficient for the B-1 population during C. gattii infection. Our model of chronic lung infection revealed that XID mice, unlike the sufficient group of B-1, had early mortality with significant weight loss, in addition to reduced levels of IgM and IgG specific to GXM isolated from the capsule of C. neoformans. In addition to this, we observed an increased fungal load in the blood and in the brain. We described an increase in the capsular size of C. gattii and the predominant presence of cytokines with a Th2 profile was also observed in these animals. Thus, the present study strongly points to a higher susceptibility of the XID mouse to C. gattii, which suggests that the presence of B-1 cells and anti-GXM antibodies is fundamental during the control of infection by C. gattii., (© 2021. The Author(s).)

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6-15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC., Competing Interests: The authors have declared that no competing interests exist.

Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990-2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription ("SIN" vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs., Competing Interests: LK is the recipient of an A. P. Giannini Foundation Postdoctoral Research Fellowship. DK is a member of the Scientific Advisory Board for Orchard Therapeutics that markets Strimvelis and has licensed a lentiviral vector for gene therapy of ADA SCID from the University of California, Los Angeles for which he is an inventor. DK is also the principal investigator for clinical trials discussed in this article on gene therapy for XCGD (California Institute for Regenerative Medicine CLIN2-08231 and Orchard Therapeutics) and LAD-I (California Institute for Regenerative Medicine CLIN2-11480 and Rocket Pharmaceuticals). He is the Chair of the Data Safety Monitoring Board for Leadiant Biosciences for Revcovi PEG-ADA ERT., (Copyright © 2021 Kohn and Kohn.)

Hematopoietic stem and progenitor cell (HSPC)-based ex vivo gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, ex vivo gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an in vivo gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3 + T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after in vivo gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with in vivo delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.

Background: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency disorder (PID) characterized by microthrombocytopenia, bloody diarrhea, eczema, recurrent infections, and a high incidence of autoimmunity and malignancy., Objective: To investigate the mechanism of thrombocytopenia and infections in four boys of consanguineous parents from Lebanon., Methods: Patient gDNA was studied using Next Generation Sequencing and Sanger Sequencing. Protein expression was determined by immunoblotting, and mRNA expression by semi-quantitative RT-PCR. F-actin polymerization and cellular proliferation were assayed by flow cytometry., Results: We identified a threonine to a methionine change at position 45 (T45M) of the WAS protein (WASp) that abolished protein expression and disturbed F-actin polymerization and T cell proliferation, but not B cell proliferation. In addition, the levels of the WAS-interacting protein (WIP) were significantly decreased in the patients., Conclusion: The mutation identified severely destabilizes WASp and affects the downstream signaling events important for T cell function, but not B cell function. It was previously known that the stability of WASp depends on WIP. In this manuscript, we report that the stability of WIP also depends on WASp. Finally, it is important to suspect X-linked PIDs even in consanguineous families., Clinical Implications: The patients are above the optimal age for transplant in WAS, and it is difficult to identify one or more donors for four patients, therefore, they represent ideal candidates for gene therapy or interleukin-2 therapy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Backgound: The potential of a single progenitor cell to establish and maintain long-term protective T-cell immunity in humans is unknown. For genetic disorders disabling T-cell immunity, somatic reversion was shown to support limited T-cell development attenuating the clinical phenotype. However, the cases reported so far deteriorated over time leaving unanswered the important question of long-term activity of revertant precursors and the robustness of the resulting T-cell system., Methods: We applied TCRβ-CDR3 sequencing and mass cytometry on serial samples of a now 18 year-old SCIDX1 patient with somatic reversion to analyse the longitudinal diversification and stability of a T-cell system emerging from somatic gene rescue., Findings: We detected close to 10 5 individual CDR3β sequences in the patient. Blood samples of equal size contained about 10-fold fewer unique CDR3β sequences compared to healthy donors, indicating a surprisingly broad repertoire. Despite dramatic expansions and contractions of individual clonotypes representing up to 30% of the repertoire, stable diversity indices revealed that these transient clonal distortions did not cause long-term repertoire imbalance. Phenotypically, the T-cell system did not show evidence for progressive exhaustion. Combined with immunoglobulin substitution, the limited T-cell system in this patient supported an unremarkable clinical course over 18 years., Interpretation: Genetic correction in the appropriate cell type, in our patient most likely in a T-cell biased self-renewing hematopoietic progenitor, can yield a diverse T-cell system that provides long-term repertoire stability, does not show evidence for progressive exhaustion and is capable of providing protective and regulated T-cell immunity for at least two decades., Funding: DFG EH 145/9-1, DFG SCHW 432/4-1 and the German Research Foundation under Germany's Excellence Strategy-EXC-2189-Project ID: 390939984., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Model animals are indispensable for the study of human diseases, and in general, of complex biological processes. The Syrian hamster is an important model animal for infectious diseases, behavioral science and metabolic science, for which more experimental tools are becoming available. Here, we describe the generation and characterization of an interleukin-2 receptor subunit gamma ( Il2rg ) knockout (KO) Syrian hamster strain. In humans, mutations in IL2RG can result in a total failure of T and natural killer (NK) lymphocyte development and nonfunctional B lymphocytes (X-linked severe combined immunodeficiency; XSCID). Therefore, we sought to develop a non-murine model to study XSCID and the infectious diseases associated with IL2RG deficiency. We demonstrated that the I l2rg KO hamsters have a lymphoid compartment that is greatly reduced in size and diversity, and is impaired in function. As a result of the defective adaptive immune response, I l2rg KO hamsters developed a more severe human adenovirus infection and cleared virus less efficiently than immune competent wild-type hamsters. Because of this enhanced virus replication, I l2rg KO hamsters developed more severe adenovirus-induced liver pathology than wild-type hamsters. This novel hamster strain will provide researchers with a new tool to investigate human XSCID and its related infections., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

"X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia" (XMEN) disease is an inborn error of glycosylation and immunity caused by loss of function mutations in the magnesium transporter 1 (MAGT1) gene. It is a multisystem disease that strongly affects certain immune cells. MAGT1 is now confirmed as a non-catalytic subunit of the oligosaccharyltransferase complex and facilitates Asparagine (N)-linked glycosylation of specific substrates, making XMEN a congenital disorder of glycosylation manifesting as a combined immune deficiency. The clinical disease has variable expressivity, and impaired glycosylation of key MAGT1-dependent glycoproteins in addition to Mg 2+ abnormalities can explain some of the immune manifestations. NKG2D, an activating receptor critical for cytotoxic function against EBV, is poorly glycosylated and invariably decreased on CD8 + T cells and natural killer (NK) cells from XMEN patients. It is the best biomarker of the disease. The characterization of EBV-naïve XMEN patients has clarified features of the genetic disease that were previously attributed to EBV infection. Extra-immune manifestations, including hepatic and neurological abnormalities, have recently been reported. EBV-associated lymphomas remain the main cause of severe morbidity. Unfortunately, treatment options to address the underlying mechanism of disease remain limited and Mg 2+ supplementation has not proven successful. Here, we review the expanding clinical phenotype and recent advances in glycobiology that have increased our understanding of XMEN disease. We also propose updating XMEN to "X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect" in light of these novel findings.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

In addition to their detection in typical X-linked severe combined immunodeficiency, hypomorphic mutations in the interleukin (IL)-2 receptor common gamma chain gene (IL2RG) have been described in patients with atypical clinical and immunological phenotypes. In this leaky clinical phenotype the diagnosis is often delayed, limiting prompt therapy in these patients. Here, we report the biochemical and functional characterization of a nonsense mutation in exon 8 (p.R328X) of IL2RG in two siblings: a 4-year-old boy with lethal Epstein-Barr virus-related lymphoma and his asymptomatic 8-month-old brother with a T low B + natural killer (NK) + immunophenotype, dysgammaglobulinemia, abnormal lymphocyte proliferation and reduced levels of T cell receptor excision circles. After confirming normal IL-2RG expression (CD132) on T lymphocytes, signal transducer and activator of transcription-1 (STAT-5) phosphorylation was examined to evaluate the functionality of the common gamma chain (γ c ), which showed partially preserved function. Co-immunoprecipitation experiments were performed to assess the interaction capacity of the R328X mutant with Janus kinase (JAK)3, concluding that R328X impairs JAK3 binding to γ c . Here, we describe how the R328X mutation in IL-2RG may allow partial phosphorylation of STAT-5 through a JAK3-independent pathway. We identified a region of three amino acids in the γ c intracellular domain that may be critical for receptor stabilization and allow this alternative signaling. Identification of the functional consequences of pathogenic IL2RG variants at the cellular level is important to enable clearer understanding of partial defects leading to leaky phenotypes., (© 2019 British Society for Immunology.)

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient's clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.

Variants in MAGT1 have been identified as the cause of an immune deficiency termed X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection and neoplasia (XMEN) disease. Here, we describe 2 cases of XMEN disease due to novel mutations in MAGT1, one of whom presented with classical features of XMEN disease and another who presented with a novel phenotype including probable CNS vasculitis, HHV-8 negative multicentric Castelman disease and severe molluscum contagiosum, thus highlighting the clinical diversity that may be seen in this condition. Peripheral blood immunophenotyping of these 2 patients, together with an additional 4 XMEN patients, revealed reduced NKG2D expression, impaired CD28 expression on CD8 + T cells, CD4 + T cell lymphopenia, an inverted CD4:CD8 ratio and decreased memory B cells. In addition, we showed for the first time alterations to the CD8 + T cell memory compartment, reduced CD56 hi NK cells, MAIT and iNKT cells, as well as compromised differentiation of naïve CD4 + T cells into IL-21-producing Tfh-type cells in vitro. Both patients were treated with supplemental magnesium with limited benefit. However, one patient has undergone allogeneic haematopoietic stem cell transplant, with full donor chimerism and immune reconstitution. These results expand our understanding of the clinical and immunological phenotype in XMEN disease, adding to the current literature, which we further discuss here.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαβ+ T cells (αβDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Background: Mutations in the IL2RG gene are known to cause X linked severe combined immunodeficiency (XSCID). More than 250 unique variants of the IL2RG gene have been reported to be identified in SCID patients so far, while many of them are of unknown significance which complicate the interpretation of mutation analysis results; furthermore, there are still many novel variants seen in clinical practice., Methods: In this study we reviewed the testing results in three unrelated SCID families. All three probands had very severe immunodeficiency phenotypes and died in infancy. Next generation sequencing methods based on either SCID gene panel or exome sequencing were applied in causal variant screening for three probands. Sanger sequencing was used for verification of the variants of interest and carrier status study for the family members. STR analysis using the GoldeneyeTM DNA ID system (PeopleSpot Inc., China) was applied to verify the kinship of the family members when a de novo mutation was identified., Results: Causal mutations were identified in all three SCID male probands, among which one was novel (c.557dupT), one was reported to be identified in a common variable immunodeficiency patient in literature (Leu87Pro), and one was a "hot-spot-mutation" (Arg226Cys). The patient with the missense mutation Leu87Pro in this study had much more severe infection phenotypes compared with the reported case in literature., Conclusions: Combining our findings and the published evidence together, Leu87Pro can be classified as a pathogenic variant following the ACMG guidelines. Correct and undoubted classification of the variants is of great importance for clinical gene testing.

Background & Aims: Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID., Methods: We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID., Results: We demonstrated that X-SCID pigs completely lacked Peyer's patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG., Conclusions: These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Foamy viruses (FVs) are nonpathogenic retroviruses that infect various animals including bovines, felines, nonhuman primates (NHPs), and can be transmitted to humans through zoonotic infection. Due to their non-pathogenic nature, broad tissue tropism and relatively safe integration profile, FVs have been engineered as novel vectors (foamy virus vector, FVV) for stable gene transfer into different cells and tissues. FVVs have emerged as an alternative platform to contemporary viral vectors (e.g., adeno associated and lentiviral vectors) for experimental and therapeutic gene therapy of a variety of monogenetic diseases. Some of the important features of FVVs include the ability to efficiently transduce hematopoietic stem and progenitor cells (HSPCs) from humans, NHPs, canines and rodents. We have successfully used FVV for proof of concept studies to demonstrate safety and efficacy following in-vivo delivery in large animal models. In this review, we will comprehensively discuss FVV based in-vivo gene therapy approaches established in the X-linked severe combined immunodeficiency (SCID-X1) canine model.

Here, we have investigated the possible effect of B-1 cells on the activity of peritoneal macrophages in E. cuniculi infection. In the presence of B-1 cells, peritoneal macrophages had an M1 profile with showed increased phagocytic capacity and index, associated with the intense microbicidal activity and a higher percentage of apoptotic death. The absence of B-1 cells was associated with a predominance of the M2 macrophages, reduced phagocytic capacity and index and microbicidal activity, increased pro-inflammatory and anti-inflammatory cytokines production, and higher percentual of necrosis death. In addition, in the M2 macrophages, spore of phagocytic E. cuniculi with polar tubular extrusion was observed, which is an important mechanism of evasion of the immune response. The results showed the importance of B-1 cells in the modulation of macrophage function against E. cuniculi infection, increasing microbicidal activity, and reducing the fungal mechanisms involved in the evasion of the immune response., Competing Interests: The authors have declared that no competing interests exist.

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.

X-linked severe combined immunodeficiency disease (SCID) is caused by mutations in the interleukin (IL)-2 receptor γ (IL2RG) gene and patients usually present with a TBNK SCID phenotype. Nevertheless, a minority of these patients present with a TBNK phenotype, similar to the IL-7R-deficient patients. We report a patient with a novel missense p.Glu297Gly mutation in the IL2RG gene presenting with a leaky TBNK SCID with delayed onset, moderate susceptibility to infections, and nodular regenerative hyperplasia. He presents with preserved STAT5 tyrosine phosphorylation in response to IL-15 stimulation but not in response to IL-2 and IL-7, resulting in the NK phenotype.

Background: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia., Methods: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1., Results: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven., Conclusions: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.)., (Copyright © 2019 Massachusetts Medical Society.)

The common cytokine receptor γ chain, γ c , is a component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21. Mutation of the gene encoding γ c results in X-linked severe combined immunodeficiency in humans, and γ c family cytokines collectively regulate development, proliferation, survival, and differentiation of immune cells. Here, we review the basic biology of these cytokines, highlighting mechanisms of signaling and gene regulation that have provided insights for immunodeficiency, autoimmunity, allergic diseases, and cancer. Moreover, we discuss how studies of this family stimulated the development of JAK3 inhibitors and present an overview of current strategies targeting these pathways in the clinic, including novel antibodies, antagonists, and partial agonists. The diverse roles of these cytokines on a range of immune cells have important therapeutic implications., (Copyright © 2019. Published by Elsevier Inc.)