Characterization of the cellular and molecular bases of inborn errors of immunity

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

The main results of the study performed in FY24 have been the following: 1) Identification and characterization of the molecular and cellular bases of novel forms of IEI. In a series of collaborative studies with colleagues from various institutes, we have identified the molecular and cellular bases of new forms of IEI. In particular, we have established that autosomal recessive deficiency of the human lymphocyte receptor LY9 underlines genetic susceptibility to tuberculosis due to selective impairment of interferon-gamma (IFN-gamma) production by Th1* cells (1). These data establish a critical role for LY9 to induce optimal Th1* cell- and IFN-gamma-dependent protective immunity to Mycobacterium tuberculosis in humans. In another manuscript, we have reported the first example of human disease due to a heterozygous de novo variant in the THPOK gene, encoding for a master regulator of human CD4+ T cell development (2). In addition to CD4+ cell deficiency, the patient also manifested allergy, interstitial lung disease, corneal vascularization and scarring, developmental delay, and growth failure. In vitro studies have shown that the THPOK variant showed novel DNA-binding specificity, and therefore behaved as a neomorphic variant. Moreover, the variant conferred profibrotic property to fibroblasts, accounting for the scarring and interstitial lung disease. Finally, we have unravelled unexpected consequences of balletic loss of function hypomorphic variants in the IL7 gene, encoding for interleukin-7 (IL-7) (3). This cytokines plays a critical role in human T-cell development by interacting with the IL-7 receptor (IL-7R). However, while IL-7R deficiency causes severe T-cell lymphopenia and high susceptibility to life-threatening infections since infancy, the immunological and clinical phenotype of IL-7 deficiency is milder, allowing survival into adulthood, with milder susceptibility to infections (with high frequency of warts and increased risk of squamous cell carcinoma) and partially preserved capacity to produce TCR gamma/delta T cells, as well as Th1, T follicular helper (Tfh( and regulatory T (Treg) cells. We have hypothesized that the difference in clinical and immunological phenotype between IL-7 and IL-7R deficiency may be accounted for by a role of thymic stromal lymphopoietin (which also signals through IL-7R) in supporting some T-cell development in patients with IL-7 deficiency. 2) Characterization of the molecular and cellular bases of immunopathology, and definition of the clinical spectrum of known forms of IEI In an original study entirely conducted by our group, we have performed the largest multi-omics characterization of human RAG deficiency, encompassing the entire phenotypic spectrum of the disease, in 157 patients (4). Although defective T and B cell development was common to all phenotypes, patients with hypomorphic mutations can generate residual number of T and B cells with signatures of immune dysregulation. A prominent type 2 signature was present in Omenn syndrome, with more hypomorphic forms of RAG deficiency showing a type 1 immune profile. In a collaborative study with Dr. Safavi's group at the NIH, we have established that a novel heterozygous loss-of-expression variant in the PIK3R1 gene (encoding for a regulatory subunit of PI3 kinase) causes slowly progressive amyotrophic lateral sclerosis (ALS) (5). In vitro differentiation of patient-derived induced pluripotent stem cells to neurons revealed disruption of electrical activity and increased apoptosis. Finally, in we have reported on the molecular and cellular bases of IEI due to thymus-intrinsic defects (6). 3) To investigate the bases underlying immune dysregulation associated with IEI We had previously shown that human hypomorphic (partial) RAG deficiency is frequently associated with immune dysregulation, with approximately 13% of the patients manifesting inflammatory bowel disease. In an original study conducted by our group by combining studies in patients and in animal model of partial RAG deficiency, we have shown that this condition is associated with a prominent TH1 and Th17 signature in the colon, with infiltration by oligoclonal Th1 and Th17 cells (7). Restriction of fecal microbial diversity, abundance of pathogenic bacteria and depletion of microbial species producing short-chain fatty acids (SCFA) were observed, along with markedly reduced representation of IL-10-producing Treg cells in the lamina propria. Treatment of Rag-mutated mice with vedolizumab (to prevent infiltration of gut-homing lymphocytes) was ineffective. Antibiotics helped reduce the inflammation, but did not alter the skewing to Th1/Th17 cells. Bone marrow transplantation was the only treatment that normalized both the immunological phenotype and the microbiome composition and diversity. In another collaborative manuscript with colleagues from Seattle, we have investigated the variability of clinical and immunological phenotype associated with hypomorphic RAG deficiency (8). Accumulation of unstitched dysreactive CD21low B cells was associated with features of an autoreactive antibody repertoire and expansion of polyclonal marginal zone-like B cells utilizing the unmutated autoreactive VH4-34 gene. Finally, D. Delmonte from our group has contributed to a collaborative study showing that STAT3 gain-of-function (GOF) mutations alter purinergic signaling in CD8+ T cells, increasing the capacity of these cells to produce IFN-gamma and tumor necrosis factor alpha (9). 4) To develop novel tools to model human IEI in vitro and in vivo Our group had previously demonstrated that artificial thymic organoids (ATO) represent powerful tool to investigate the cellular bases of severe congenital T-cell lymphopenia, distinguishing between hematopoietic cell autonomous and thymus-intrinsic defects of T-cell development. Using the ATO platform, we have demonstrated that severe congenital T-cell lymphopenia in an infant with a heterozygous TP63 variant was thymus-intrinsic (10). Following thymus implantation, the infant showed emergence of CD4+ recent thymic emigrants suggestive of restored thymopoiesis. In another manuscript, we investigated the cellular basis of SCID and Omenn syndrome in an infant with a heterozygous de novo pathogenic variant in PSMB10, encoding for a subunit of the immunoproteasome (11). In vitro differentiation of patient-derived CD34+ cells in the ATO platform showed normal generation of early T-cell progenitors, but slightly impaired generation of more mature CD3+ TCRalpha/beta+ cells. Single cell RNA sequencing of normal human thymus revealed that PSMB10 is expressed in various subsets of cortical and medullary thymic epithelial cells. These data indicate that PSMB10 plays critical roles in both hematopoietic and extrahematopoietic immune system development and function. Finally, the experience with use of in vitro studies of human CD34+ cell differentiation to T cells in two centers in North America (including ours at the NIH) has been reviewed in another manuscript (12). 5) To gain novel insights into the mechanisms that govern human immune system development The thymus plays a critical role in the generation of T lymphocytes and in shaping the peripheral T cell repertoire. In recent years, major advances have been made in the characterization of the cellular composition of the human thymus. In a collaborative study with the groups of Sarah Teichmann in Cambridge (UK), Tom Taghon in Ghent (Belgium) and Ron Germain at the NIH, our group has mapped the micro anatomical underpinnings of the process of T-cell development. By combining single-cell RNA sequencing, spatial transcriptomics and high-resolution multiplex imaging data, we have generated an atlas of the human thymus, established trajectories of canonical thymocyte development, defined niches occupied by distinct subsets of thymic stromal cells, and revealed temporal dynamics that regulate composition and organization of the human thymus at early age (13). 6) To participate at multicenter studies aimed at defining the natural history of IEI in humans, and at evaluating outcome of cellular and molecular therapy for these disorders In a collaborative multicenter study, we have reviewed clinical manifestations, treatment modalities and outcome of pre than 30,000 patients with IEI reported in the Registry of the European Society for Immune Deficiencies (14). A natural history study of 117 patients with Wiskott-Aldrich syndrome revealed important phenotypic variability of this condition, with patients with severe disease manifesting invasive infections and being at higher risk for severe autoimmune manifestations. Among 71 patients who received hematopoietic cell transplantation, 25-year overall survival was 75%. Gene therapy was performed in 10 patients, all of which have survived after 5 years of follow-up (15). Finally, in a multi center study of the Primary Immune Deficiency Treatment Consortium (PIDTC), we have demonstrated that carriers of X-linked chronic granulomatous disease (XL-CGD) have a high frequency of inflammatory and autoimmune manifestations, and a minority of them also showed susceptibility to CGD-associated infections. Nearly half of the carriers reported overall good health, but a quarter had work-related restrictions to health-related symptoms (16).