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.

Over the last decade, the identification of hypomorphic variants in patients previously diagnosed with Common Variable Immunodeficiency (CVID) has led to the association of milder phenotypes with variants of the IL2RG gene that are usually related to severe combined immunodeficiency. Indeed, several revertant mosaicisms have been described in cases with hypomorphic variants in that gene. Our main objective herein was the functional characterization of p. (Pro58Thr) variant in the IL2RG gene in an adult patient with antibody deficiency and moderate CD4+ T cell lymphopenia. Evaluation of the patient included a clinical examination and a complete analysis of the peripheral blood phenotype. To further explore IL2RG functionality we selected downstream signaling readouts, namely STAT3 and STAT5 phosphorylation, NK degranulation and B- and T-cell proliferation capacity in vitro, which can be measured by flow cytometry, that reflect the strength of homeostatic signaling pathways in resting cells and after activation. The patient presented reduced CD132 expression and conserved T- and B-cell proliferation capacity in vitro. However, we found that intracellular signaling downstream of IL2γc is affected, with reduced STAT3 phosphorylation after IL-21 stimulation in B cells and CD4 T cells. In addition, CD4+ T cells showed a reduced STAT5 phosphorylation in response to IL-2, which was not so evident in CD8+ T cells. NK degranulation was impaired upon PHA and IL-2 as well as plasmablast differentiation in vitro. We conclude that p. (Pro58Thr) in the IL2RG gene is functionally a hypomorphic variant, as reported previously. Although the functionality of CD8+ is less impaired than the rest of the lymphocyte subsets, we did not detect a reversion of the variant in isolated CD8+, CD4+, CD19+ or NK cells.

Both innate and adaptive lymphocytes have critical roles in mucosal defense that contain commensal microbial communities and protect against pathogen invasion. Here we characterize mucosal immunity in patients with severe combined immunodeficiency (SCID) receiving hematopoietic stem cell transplantation (HSCT) with or without myeloablation. We confirmed that pretransplant conditioning had an impact on innate (natural killer and innate lymphoid cells) and adaptive (B and T cells) lymphocyte reconstitution in these patients with SCID and now show that this further extends to generation of T helper 2 and type 2 cytotoxic T cells. Using an integrated approach to assess nasopharyngeal immunity, we identified a local mucosal defect in type 2 cytokines, mucus production, and a selective local immunoglobulin A (IgA) deficiency in HSCT-treated SCID patients with genetic defects in IL2RG/GC or JAK3. These patients have a reduction in IgA-coated nasopharyngeal bacteria and exhibit microbial dysbiosis with increased pathobiont carriage. Interestingly, intravenous immunoglobulin replacement therapy can partially normalize nasopharyngeal immunoglobulin profiles and restore microbial communities in GC/JAK3 patients. Together, our results suggest a potential nonredundant role for type 2 immunity and/or of local IgA antibody production in the maintenance of nasopharyngeal microbial homeostasis and mucosal barrier function.

Accumulation of human CD21low B cells in peripheral blood is a hallmark of chronic activation of the adaptive immune system in certain infections and autoimmune disorders. The molecular pathways underpinning the development, function, and fate of these CD21low B cells remain incompletely characterized. Here, combined transcriptomic and chromatin accessibility analyses supported a prominent role for the transcription factor T-bet in the transcriptional regulation of these T-bethighCD21low B cells. Investigating essential signals for generating these cells in vitro established that B cell receptor (BCR)/interferon-γ receptor (IFNγR) costimulation induced the highest levels of T-bet expression and enabled their differentiation during cell cultures with Toll-like receptor (TLR) ligand or CD40L/interleukin-21 (IL-21) stimulation. Low proportions of CD21low B cells in peripheral blood from patients with defined inborn errors of immunity (IEI), because of mutations affecting canonical NF-κB, CD40, and IL-21 receptor or IL-12/IFNγ/IFNγ receptor/signal transducer and activator of transcription 1 (STAT1) signaling, substantiated the essential roles of BCR- and certain T cell–derived signals in the in vivo expansion of T-bethighCD21low B cells. Disturbed TLR signaling due to MyD88 or IRAK4 deficiency was not associated with reduced CD21low B cell proportions. The expansion of human T-bethighCD21low B cells correlated with an expansion of circulating T follicular helper 1 (cTfh1) and T peripheral helper (Tph) cells, identifying potential sources of CD40L, IL-21, and IFNγ signals. Thus, we identified important pathways to target autoreactive T-bethighCD21low B cells in human autoimmune conditions, where these cells are linked to pathogenesis and disease progression.