The interleukin-2 receptor γ (IL-2Rγ, or γc) is a crucial component of several cytokine receptor complexes. Deficiencies in γc lead to X-linked severe combined immunodeficiency (X-SCID), characterized by recurrent infections due to the absence or dysfunction of T and NK cells, and nonfunctional B cells. Missense variants in the γc extracellular region are linked to atypical X-SCID with normal counts of T, B, and NK cells and less severe symptoms, yet the underlying cellular and molecular mechanisms are not well understood. This study describes a case of atypical X-SCID with a missense variant (c.420 A > T, p.R140S) in the γc extracellular domain, associated with recurrent bacterial, fungal, and viral infections. We found that the R140S variant leads to reduced surface expression and variably affects cytokine receptor signaling. Specifically, STAT5 phosphorylation and proliferation in CD4+ T and CD8+ T cells are impaired in response to IL-7, a cytokine essential for T cell survival, proliferation and function. Notably, γcR140S predominantly localizes to the endoplasmic reticulum, in contrast to WT γc, which is found in acidic compartments. Despite this mislocalization, γcR140S does not trigger unfolded protein responses, and its protein stability and degradation pathways remain unaffected. Nevertheless, cells expressing high levels of γcR140S exhibited a competitive disadvantage in culture compared to those expressing WT γc, resulting in the enrichment of cells expressing lower levels of γcR140S. These findings extend our understanding of how mutations in the extracellular domain of γc can lead to reduced protein expression and influence the pathophysiology of atypical X-SCID.

X-linked severe combined immunodeficiency (X-SCID) caused by full mutation of the IL2RG gene leads to T- B+ NK- phenotype and is usually associated with severe opportunistic infections, diarrhea, and failure to thrive. When IL2RG hypomorphic mutation occurs, diagnosis could be delayed and challenging since only moderate reduction of T and NK cells may be present. Here, we explored phenotypic insights and the impact of the p.R222C hypomorphic mutation (IL2RGR222C ) in distinct cell subsets in an 8-month-old patient with atypical X-SCID. We found reduced CD4+ T cell counts, a decreased frequency of naïve CD4+ and CD8+ T cells, and an expansion of B cells. Ex vivo STAT5 phosphorylation was impaired in CD4+ CD45RO+ T cells, yet compensated by supraphysiological doses of IL-2. Sanger sequencing on purified cell subsets showed a partial reversion of the mutation in total CD3+ cells, specifically in recent thymic emigrants (RTE), effector memory (EM), and CD45RA+ terminally differentiated EM (EMRA) CD4+ T cells. Of note, patient's NK cells had a normal frequency compared to age-matched healthy subjects, but displayed an expansion of CD56bright cells with higher perforin content and cytotoxic potential, associated with accumulation of NK-cell stimulatory cytokines (IL-2, IL-7, IL-15). Overall, this report highlights an alteration in the NK-cell compartment that, together with the high disease-phenotype variability, should be considered in the suspicion of X-SCID with hypomorphic IL2RG mutation.

Inborn errors in interleukin 2 receptor, gamma (IL2RG) perturb signaling of the common gamma chain family cytokines and cause severe combined immunodeficiency (SCID). Here, we report two brothers suffering from chronic cryptosporidiosis, severe diarrhea, and cholangitis. Pan T, B, and NK cell numbers were normal, but immunophenotyping revealed defective B cell differentiation. Using whole exome sequencing, we identified a base pair deletion in the first exon of IL2RG predicted to cause a frameshift and premature stop. However, flow cytometry revealed normal surface expression of the IL-2Rγ chain. While IL-2, IL-7, and IL-15 signaling showed only mild defects of STAT5 phosphorylation in response to the respective cytokines, IL-4- and IL-21-induced phosphorylation of STAT3 and STAT6 was markedly reduced. Examination of RNA isoforms detected alternative splicing downstream of IL2RG exon 1 in both patients resulting in resolution of the predicted frameshift and 16 mutated amino acids. In silico modeling suggested that the IL-2Rγ mutation reduces the stabilization of IL-4 and IL-21 cytokine binding by affecting the N-terminal domain of the IL-2Rγ. Thus, our study shows that IL2RG deficiency can be associated with differential signaling defects. Confounding effects of alternative splicing may partially rescue genetic defects and should be considered in patients with inborn errors of immunity.

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor γ chain gene (IL2RG), and it is characterized by profound defects in T, B, and natural killer (NK) cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early murine leukemia retrovirus (MLV)-derived gammaretroviral vectors showed restoration of T cell immunity in patients, but it resulted in vector-induced insertional oncogenesis. We developed a self-inactivating (SIN) lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1α short promoter (EFS-IL2RG), and we tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin- HSPCs in an Il2rg-/-/Rag2-/- mouse model. The study showed restoration of T, B, and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile, and it showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase I/II clinical trial aimed at restoring both T and B cell immunity in SCID-X1 children upon non-myeloablative conditioning.

JAK3 is a tyrosine kinase essential for signaling downstream of the common gamma chain subunit shared by multiple cytokine receptors. JAK3 deficiency results in T-B+NK- severe combined immune deficiency (SCID). We report a patient with SCID due to a novel mutation in the JAK3 JH4 domain. The function of the JH4 domain remains unknown. This is the first report of a missense mutation in the JAK3 JH4 domain, thereby demonstrating the importance of the JH4 domain of JAK3 in host immunity.