IKAROS regulates human T cell phenotype at a thymic and postthymic level.

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.