Marita Bosticardo

Molecular characterization of human monogenic inborn errors of T cell immunity provides both biological insights and medical progress. We report rare biallelic deleterious variants in GTF3A , encoding transcription factor IIIA (TFIIIA), a zinc-finger protein required for transcription and chaperoning of 5S ribosomal RNA (rRNA). These variants were identified in ten patients from eight unrelated families and eight countries presenting with either T ^- B ⁺ NK ⁺ severe combined immunodeficiency (SCID) or combined immune deficiency (CID), characterized by T cell lymphopenia and variable antibody deficiency. The GTF3A variants disrupt TFIIIA function through distinct mechanisms, including defective DNA binding, aberrant nuclear localization, and reduced protein stability compromising TFIIIA-mediated transcription and chaperoning of 5S rRNA. Using artificial thymic organoids derived from TFIIIA-deficient CD34 ⁺ progenitors, an early developmental arrest at the T cell commitment stage was documented in vitro. Zebrafish deficient for gtf3aa recapitulated the impaired thymocyte development in vivo . Together, these findings establish TFIIIA deficiency as a novel cause of (S)CID, expanding the genetic and mechanistic landscape of inborn errors of T cell immunity and uncovering an essential role for TFIIIA in human adaptive immunity.

Mutations in DNA helicase RECQL4 can cause immunodeficiency, including SCID. The T cell defects are intrinsic to the hematopoietic system and amenable to hematopoietic stem cell transplant. Radiosensitivity due to DNA repair defects limits conditioning regimen options.

The transcription factor IKAROS, encoded by IKZF1 , is crucial for lymphocyte development and differentiation. Germline heterozygous IKZF1 mutations cause B cell immunodeficiency, but also affect T cells. Patients with IKZF1 haploinsufficiency (HI) or dimerization-defective (DD) variants show reduced naive and increased memory T cells, while dominant-negative (DN) mutations result in the opposite phenotype. Gain-of-function patients display variable patterns. To investigate IKAROS’s role in shaping the human naive/memory T cell phenotype, we performed IKAROS immunomodulation and knockdown experiments and analyzed early T cell development in an artificial thymic organoid (ATO) system using CD34 ⁺ cells from patients with representative IKZF1 variants. IKAROS inhibition by lenalidomide or silencing by small hairpin RNA directly altered expression of HNRNPLL, the master regulator of CD45 isoform splicing that defines CD45RA ⁺ /naive and CD45RO ⁺ /memory phenotypes. In the ATO system, IKAROS-DN precursor cells were blocked at the CD4 ^– CD8 ^– /double-negative stage and retained a CD45RA ⁺ phenotype, whereas IKAROS-HI cells inefficiently reached the CD4 ⁺ CD8 ⁺ /double-positive stage and partially transitioned from CD45RA to CD45RO. Analysis of public gene expression data showed high HNRNPLL expression in double-positive thymic cells, beyond the stages affected by IKZF1 DN and HI mutations. Collectively, these findings indicate that IKAROS regulates early and late T cell development by mechanisms, including HNRNPLL modulation.

STING gain-of-function (GOF) mutations cause STING-Associated Vasculopathy with onset in Infancy (SAVI), a severe autoinflammatory disease. Mice carrying STING GOF V154M mutation develop profound T cell lymphopenia, partly due to impaired thymic development. To investigate the mechanisms of peripheral T cell dysfunctions, we analyzed transcriptomic and phenotypic profiles of splenic T cells from these mice. We found a terminally exhausted T cell phenotype, established early in life upon entry into the periphery, independent of type I interferons and intrinsic STING activation in T cells or stromal cells. Mechanistically, naive T cells in the lymphopenic periphery experienced heightened stimulation of the IL-7 receptor and TCR, including NFAT pathway, a key factor in T cell exhaustion. Transplantation of STING GOF hematopoietic stem cells with wild-type bone marrow prevented exhaustion in this non-lymphopenic context, placing lymphopenia as a key driver. T cell exhaustion was also observed in lymphopenic mice carrying Rag1 hypomorphic mutations. In conclusion, our results highlight T cell exhaustion induced by lymphopenia and could have important implications for the management of patients with severe immune deficiencies.

ThPOK is a transcription factor that acts as a master regulator of CD4+ T cell lineage commitment. We report the first human disease caused by a genetic alteration in ThPOK, specifically, a damaging heterozygous de novo variant in ThPOK (NM_001256455.2:c.1080A>C, p.K360N). This patient exhibited the unusual constellation of persistent CD4+ T cell deficiency, allergy, interstitial lung disease, corneal vascularization and scarring, developmental delay, and growth failure. The ThPOKK360N variant displayed abnormal multimorphic activity, interfering with ThPOKWT (antimorph), failing to bind wild-type ThPOK consensus sequences (amorph), and showing novel DNA-binding specificity (neomorph). Single-cell RNA sequencing revealed defects in CD4+ and CD8+ T cell maturation and activation (hypomorph). Recapitulated in lentivirally transduced healthy control T cells and fibroblasts, the transcriptomic analysis showed ThPOKK360N-transduced T cells had impaired TCR activation and ThPOKK360N-transduced fibroblasts with increased profibrotic gene expression. This novel human disease confirms ThPOK’s role in CD4+ T cell development but also uncovers novel roles in TCR activation and regulation of fibrotic pathways in fibroblasts.

Partial RAG deficiency (pRD) can manifest with systemic and tissue-specific immune dysregulation, with inflammatory bowel disease (IBD) in 15% of the patients. We aimed at identifying the immunopathological and microbial signatures associated with IBD in patients with pRD and in a mouse model of pRD (Rag1w/w) with spontaneous development of colitis. pRD patients with IBD and Rag1w/w mice showed a systemic and colonic Th1/Th17 inflammatory signature. Restriction of fecal microbial diversity, abundance of pathogenic bacteria, and depletion of microbial species producing short-chain fatty acid were observed, which were associated with impaired induction of lamina propria peripheral Treg cells in Rag1w/w mice. The use of vedolizumab in Rag1w/w mice and of ustekinumab in a pRD patient were ineffective. Antibiotics ameliorated gut inflammation in Rag1w/w mice, but only bone marrow transplantation (BMT) rescued the immunopathological and microbial signatures. Our findings shed new light in the pathophysiology of gut inflammation in pRD and establish a curative role for BMT to resolve the disease phenotype.

Human recombination-activating gene (RAG) deficiency can manifest with distinct clinical and immunological phenotypes. By applying a multiomics approach to a large group of RAG -mutated patients, we aimed at characterizing the immunopathology associated with each phenotype. Although defective T and B cell development is common to all phenotypes, patients with hypomorphic RAG variants can generate T and B cells with signatures of immune dysregulation and produce autoantibodies to a broad range of self-antigens, including type I interferons. T helper 2 (T _H 2) cell skewing and a prominent inflammatory signature characterize Omenn syndrome, whereas more hypomorphic forms of RAG deficiency are associated with a type 1 immune profile both in blood and tissues. We used cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis to define the cell lineage–specific contribution to the immunopathology of the distinct RAG phenotypes. These insights may help improve the diagnosis and clinical management of the various forms of the disease.

Recent technological advances have transformed our understanding of the human thymus. Innovations such as high-resolution imaging, single-cell omics, and organoid cultures, including thymic epithelial cell (TEC) differentiation and culture, and improvements in biomaterials, have further elucidated the thymus architecture, cellular dynamics, and molecular mechanisms underlying T cell development, and have unraveled previously unrecognized levels of stromal cell heterogeneity. These advancements offer unprecedented insights into thymic biology and hold promise for the development of novel therapeutic strategies for immune-related disorders.

ThPOK is best known as a regulator of CD4+ T cell lineage commitment, although it was initially cloned as a suppressor of collagen expression in the skin. The role of ThPOK has not been formally established in humans since individuals with damaging variants in ThPOK have not yet been identified. Here, we report the first case of a human with a damaging heterozygous de novo variant in ThPOK causing a syndrome encompassing CD4+ T cell deficiency, allergy, fibroinflammatory interstitial lung disease, developmental delay, and growth failure. The patient variant, ThPOK K360N, exhibited abnormal multimorphic activity, including interfering with ThPOK WT in regulating gene regulation (antimorph). Protein-DNA interaction assays showed inability of ThPOK K360N to bind to wild-type consensus sequences (amorph) and revealed a novel DNA-binding specificity (neomorph). Single-cell RNA sequencing of peripheral blood revealed potential developmental defects in maturation and activation of CD4+ and CD8+ T cells (hypomorph). To establish causality, we recapitulated the observed cellular defects in lentivirally transduced healthy control T cells and pulmonary fibroblasts. Transcriptomic analysis showed that T cells transduced with ThPOK K360N lacked the upregulation of activation, proliferation, and functional pathways observed in ThPOKWT-transduced cells. When overexpressed in healthy control fibroblasts, ThPOK K360N significantly increased the expression of pro-fibrotic genes implicated in pulmonary fibrosis, indicating a defect in regulating collagen expression. This novel human disease caused by a multimorphic variant in ThPOK confirms its role in CD4+ T cell development in an intact human context, while also revealing an unanticipated role for ThPOK in T cell function and the regulation of fibrotic pathways in fibroblasts.