Thymic stromal cells are essential for T-cell progenitor proliferation and differentiation. While thymic epithelial cells are well studied, non-epithelial stromal populations–particularly neural crest—derived mesenchymal cells–are increasingly recognized for their roles in thymus formation and function. Mesenchymal defects contribute to thymic abnormalities in congenital syndromes such as DiGeorge syndrome (DGS), CHARGE syndrome, and Trisomy 21 (T21), yet human studies are limited by the rarity of these conditions and restricted access to thymic tissue.
We generated iPSCs from individuals with TBX1, CHD7, HOXA3, and PAX1 mutations, as well as DGS and T21 patients. Control and patient iPSCs, derived from PBMCs or skin biopsies, were differentiated into mesenchymal stem cells (MSCs). Primary thymic mesenchymal cells (ThyMCs) were also isolated from human thymi. All cell populations were analyzed by flow cytometry and bulk RNA-seq. Tri-lineage differentiation assays (adipogenic, chondrogenic, osteogenic) were performed to assess functional pathway defects.
Flow cytometry confirmed robust expression of MSC markers (CD73, CD146, CD105) in all MSC and ThyMC samples, absent in undifferentiated iPSCs. Principal component analysis revealed clear segregation of iPSCs, MSCs, and ThyMCs. Transcriptomic profiling showed that all patient-derived MSCs acquired mesenchymal identity, but each disease group displayed distinct transcriptional changes. MSCs with TBX1, HOXA3, or PAX1 mutations had the highest number of differentially expressed genes, affecting pathways such as extracellular matrix and cartilage development.DGS-derived MSCs and ThyMCs showed marked upregulation of ECM and collagen genes (FBLN5, PCOLCE, EMILIN1, COL3A1, COL1A2).
Our findings reveal disease-specific mesenchymal defects underlying thymic abnormalities in congenital syndromes.This platform enables mechanistic studies of thymic stromal dysfunction and advances understanding of immune deficits in these disorders.
This work was supported by the Division of Intramural Research, NIAID, NIH.
Hematopoiesis and Immune System Development (HEM)
Giuseppe Sangiorgio, Francesca Pala, Kayla Amini et al.· Journal of Immunology· 0 citations
Heterozygous single-nucleotide variants cause many inborn errors of immunity (IEI), yet most functional genomics tools generate homozygous edits, limiting insight into dominant-negative (DN) and dosage-dependent variant effects. Expression imbalance of the mutant and wild-type alleles can further complicate genotype—phenotype relationships, and has been nearly impossible to interrogate experimentally. To overcome this, we built a scalable platform to engineer heterozygous variants in primary immune cells and directly link allelic expression bias to phenotype.
We harnessed an innovative CRISPR-based editing system, helicase-assisted continuous editing (HACE), in primary human T cells to engineer heterozygous variants across CARD11, a key regulator of immune signaling, in which heterozygous variants cause diverse IEI with variable penetrance. In parallel, we developed single-cell Allele-Integrated Multi-omics sequencing (sc-AIMseq), which integrates transcriptomics, surface proteomics, and quantification of mutant and wild-type allele expression at single cell resolution.
HACE screens recovered known pathogenic DN variants in CARD11 and revealed novel DN and haploinsufficient variants not evident in homozygous models. sc-AIMseq of HACE-edited T cells uncovered marked cell-to-cell variability in CARD11 allelic expression bias, which dramatically influenced T cell phenotype. We also performed sc-AIMseq on PBMCs from multiple patients with DN CARD11 mutations, which revealed that CARD11 allelic expression bias is cell-type dependent, providing a mechanistic basis for variable penetrance.
This work establishes a scalable strategy to model heterozygous genetic disorders in primary and patient-derived immune cells, and dissect their variable penetrance. By directly coupling heterozygous variant engineering, allelic expression bias, and phenotype at single-cell resolution, our approach enables unprecedented functional variant interpretation and advances precision immunology.
Z.H.W. has received research funding from NCI F30CA298572 and the Melanoma Research Foundation.
Technological Innovations in Immunology (TECH)
Zachary H. Walsh, C. Frangieh, Shudipto Wahed et al.· Journal of Immunology· 0 citations