Defects in IL-2R Signaling Contribute to Diminished Maintenance of FOXP3 Expression in CD4+CD25+ Regulatory T-Cells of Type 1 Diabetic Subjects

OBJECTIVE In humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4+CD25+FOXP3+ regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2–dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects. RESEARCH DESIGN AND METHODS Persistence of Tregs was assessed by culturing sorted CD4+CD25hi natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4+CD25− T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis. RESULTS Maintenance of FOXP3 expression in CD4+CD25+ Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4+ T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling. CONCLUSIONS Aberrant IL-2R signaling in CD4+ T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity

The TYK2-P1104A Autoimmune Protective Variant Limits Coordinate Signals Required to Generate Specialized T Cell Subsets

TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within TYK2 encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant TYK2A1104 (TYK2P). To study this variant in vivo, we developed a knock-in murine model of this allele. Murine Tyk2P expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous Tyk2P T cells exhibited diminished in vitro Th1 skewing. Surprisingly, despite these signaling changes, in vivo formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that Tyk2P expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 in vitro. Consistent with these findings, homozygous, but not heterozygous, Tyk2P mice were fully protected in a murine model of MS. Homozygous Tyk2P mice had fewer infiltrating CD4+ T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17+/IFNγ+, double positive, pathogenic CD4+ T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the Tyk2P allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2P diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades

Successful Hematopoietic Stem Cell Transplantation in a Patient with a Novel DNA Ligase IV Mutation

Abstract DNA Ligase IV deficiency syndrome (LIG4 syndrome) is a rare autosomal recessive disorder caused by mutations in the DNA ligase IV gene (LIG4), which is essential for the repair of DNA double-strand breaks in mamalian cells by non-homologous end joining (O’Driscoll et al, 2001; Critchlow et al, 1998). LIG4-deficient patients are characterized by microcephaly, growth retardation, developmental delay, low birth weight, dysmorphic facial findings called bird-like face, immunodeficiency, pancytopenia, and pronounced clinical and cellular radiosensitivity (O’Driscoll et al, 2001). Herein, we report two siblings with a novel DNA ligase IV mutation, one of whom underwent hematopoietic stem cell transplantation (HSCT). Case 1, was a 10 year-old girl, whose pancytopenia started at 4 years of age. History revealed a birth weight of 2400 g. There was a first-degree consanguinity between parents. Body weight, height, and head circumference were below the third percentile. Low anterior hairline, prominent nasal bridge and bilateral epicanthus were present. Recurrent upper respiratory infection (URI) history was striking. Serum IgM was low for age. Pancytopenia deepened progressively and HSCT was performed from 6/6 HLA matched father after non-myeloablative conditioning; however autologous reconstruction developed and a 2nd HSCT was performed after busulfan, cyclophosphamide and ATG conditioning. Engraftment was achieved by +12 day. Case 2 was a 6 year-old boy, presented with widespread echymoses at three years of age. Microcephaly, body weight and height below the third percentile, inguinal hernia, prominent nasal bridge and bilateral epicanthi were positive findings. History revealed recurrent URI. Serum IgG and IgM were low for age. DEB and mitomycin-C induced chromosomal analyses were within normal limits, whereas spontaneous breakage was found to be increased. Bone marrow examination revealed hypercellularity in both siblings. Alpha-fetoprotein levels were within normal limits. Mutation analysis for Nijmegen breakage syndrome was negative, however sequencing revealed a novel mutation in the LIG4 gene. A homozygous sequence variant, 1762delAAG, which results in the amino acid deletion 588delK, was identified in case 2. Both parents were heterozygous for the alteration. This novel mutation is located in a highly conserved peptide in the ligase IV protein, adjacent to the ligase domain, with previously unknown function (Wei et al, 1995). Our results demonstrate that non-myeloablative conditioning may not be adequate for LIG4 syndrome patients with hypercellular bone marrow. However, successful treatment of LIG4 syndrome can be achieved by HSCT after more potent conditioning

PTPN22 R620W gene editing in T cells enhances low-avidity TCR responses

A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases and linked to altered TCR regulation and T cell activation. Here, we utilize Crispr/Cas9 gene editing with donor DNA repair templates in human cord blood-derived, naive T cells to generate PTPN22 risk edited (620W), non-risk edited (620R), or knockout T cells from the same donor. PTPN22 risk edited cells exhibited increased activation marker expression following non-specific TCR engagement, findings that mimicked PTPN22 KO cells. Next, using lentiviral delivery of T1D patient-derived TCRs against the pancreatic autoantigen, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), we demonstrate that loss of PTPN22 function led to enhanced signaling in T cells expressing a lower avidity self-reactive TCR, but not a high-avidity TCR. In this setting, loss of PTPN22 mediated enhanced proliferation and Th1 skewing. Importantly, expression of the risk variant in association with a lower avidity TCR also increased proliferation relative to PTPN22 non-risk T cells. Together, these findings suggest that, in primary human T cells, PTPN22 rs2476601 contributes to autoimmunity risk by permitting increased TCR signaling and activation in mildly self-reactive T cells, thereby potentially expanding the self-reactive T cell pool and skewing this population toward an inflammatory phenotype