Faculty Bibliography

B cells play critical roles in humoral immunity to infection, vaccination, and autoimmunity. The differentiation of B cells into antibody-producing plasma cells (PCs) has been extensively studied, but the role of metabolic transporters that mediate nutrient uptake during PC differentiation is not well-understood. Here, we characterized the dependence of B cells and PC differentiation on the neutral amino acid transporter SLC7A5. We demonstrate that SLC7A5 promotes B cell functions including proliferation and PC differentiation in vitro and in vivo after immunization with T dependent and independent antigens. Deletion of SLC7A5 in B cells suppressed the function of mTORC1 and enforced mTORC1 activity rescued PC differentiation. The role of SLC7A5 in B cells appears to be unrelated to leucine uptake because B cells were insensitive to extracellular leucine depletion. Defects in SLC7A5-deficient B cells could, however, be rescued by extracellular methionine supplementation, suggesting a role for methionine in SLC7A5-dependent B cell function and PC differentiation. Our study provides evidence for a leucine-independent role of SLC7A5 in B cell function and PC differentiation.

Chaperone-mediated autophagy (CMA) contributes to proteostasis maintenance by selectively degrading a subset of proteins in lysosomes. CMA declines with age in most tissues, including skeletal muscle. However, the role of CMA in skeletal muscle and the consequences of its decline remain poorly understood. Here we demonstrate that CMA regulates skeletal muscle function. We show that CMA is upregulated in skeletal muscle in response to starvation, exercise and tissue repair, but declines in ageing and obesity. Using a muscle-specific CMA-deficient mouse model, we show that CMA loss leads to progressive myopathy, including reduced muscle force and degenerative myofibre features. Comparative proteomic analyses reveal CMA-dependent changes in the mitochondrial proteome and identify the sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase (SERCA) as a CMA substrate. Impaired SERCA turnover in CMA-deficient skeletal muscle is associated with defective calcium (Ca²⁺) storage and dysregulated Ca²⁺ dynamics. We confirm that CMA is also downregulated with age in human skeletal muscle. Remarkably, genetic upregulation of CMA activity in old mice partially ameliorates skeletal muscle ageing phenotypes. Together, our work highlights the contribution of CMA to skeletal muscle homoeostasis and myofibre integrity.

Loss of function mutations of ORAI1 suppress store-operated Ca²⁺ entry (SOCE) and cause an immunodeficiency disorder called Ca²⁺ release-activated Ca²⁺ (CRAC) channelopathy. Here we report an infant patient who is compound heterozygous for p.His134Pro and p.Leu194Pro mutations in ORAI1 and whose T cells have strongly reduced SOCE. Whereas the p.Leu194Pro mutant ORAI1 protein is not expressed at the plasma membrane, the p.His134Pro mutation results in a constitutively open channel that is unresponsive to activation by stromal interaction molecule 1 (STIM1). The patient suffered from a severe form of combined immunodeficiency (CID), hemophagocytic lymphohistiocytosis (HLH) and fatal chronic cytomegalovirus infection. His immunodeficiency was characterized by an altered composition of T and NK cell compartments, impaired stimulation-induced cytokine production and signs of CD4⁺ T cell and NK cell activation but attenuated CD8⁺ T effector memory cell function. Our findings demonstrate that small constitutive SOCE through a mutant ORAI1 channel is not sufficient to provide immunity to viral infection.

Pathogenic CD4 T cells drive autoimmunity in diseases such as multiple sclerosis (MS) and inflammatory bowel disease (IBD). Through a forward genetic screen, we identified chloride nucleotide-sensitive channel 1A (CLNS1A) as a key regulator of inflammation in the experimental autoimmune encephalomyelitis (EAE) model of MS. CLNS1A is expressed in several subsets of CD4 T cells, including pathogenic T helper 17 (pT_H17) cells. Deletion of Clns1a in T cells resulted in DNA damage, cell cycle arrest, impaired T cell proliferation, and effector function, thereby protecting mice from both EAE and IBD. We found that CLNS1A interacts with protein arginine methyl transferase 5 (PRMT5). Moreover, CLNS1A regulates symmetric histone dimethylation and the expression of genes involved in DNA repair, replication, and cell cycle progression. Thus, CLNS1A plays an important role in CD4 T cells by promoting genome stability and cell cycle progression.

Sjogren's Disease (SjD) is an autoimmune disorder characterized by salivary and lacrimal gland dysfunction and immune cell infiltration leading to gland inflammation and destruction. Although SjD is a common disease, its pathogenesis is not fully understood. In this study, we conducted a single-cell transcriptome analysis of peripheral blood mononuclear cells (PBMC) from patients with SjD and symptomatic non-SjD controls to identify cell types and functional changes involved in SjD pathogenesis. All PBMCs populations showed marked differences in gene expression between SjD patients and controls, particularly an increase in interferon (IFN) signaling gene signatures. T and B cells of SjD patients displayed a depletion of ribosomal gene expression and pathways linked to protein translation. SjD patients had increased frequencies of naive B cells, which featured a unique gene expression profile (GEP) distinct from controls and had hallmarks of B cell hyperactivation. Non-negative matrix factorization (NMF) also identified several non-overlapping GEPs in CD4⁺ and CD8⁺ T cells with differential usage in SjD patients and controls. Of these, only the Th1 activation GEP was enriched in T cells of SjD patients whereas the other two GEPs were depleted in T cells, emphasizing the important role of Th1 cells in SjD. Our study provides evidence for aberrant and unique gene expression patterns in both B and T lymphocytes of SjD patients that point to their altered activation states and may provide new insights into the pathogenesis of SjD.

Stromal interaction molecule 1 (STIM1) is critical for store-operated Ca²⁺ entry (SOCE) and T cell activation. T helper 1 (T_H1) cells, which express T-bet (encoded by TBX21), mediate immunity to intracellular pathogens. Although SOCE is known to regulate other T_H lineages, its role in Th1 differentiation remains unclear. Here, we report a patient with an intronic loss-of-function mutation in STIM1, which abolishes SOCE and causes immunodeficiency. We demonstrate that SOCE promotes nuclear factor of activated T cells (NFAT) binding to conserved noncoding sequence (CNS)-12 in the TBX21 enhancer and enables NFAT to synergize with STAT1 to mediate TBX21 expression. While SOCE-deficient CD4⁺ T cells have reduced expression of TBX21 in the absence of interleukin-12 (IL-12), their expression of IL-12 receptors β1 and β2 is increased, sensitizing them to IL-12 signaling and allowing IL-12 to rescue T-bet expression. Our study reveals that the STIM1-SOCE-NFAT signaling axis is essential for the differentiation of Th1 cells depending on the cytokine milieu.

Ca2+ signaling via the store operated Ca2+ entry (SOCE) mediated by STIM1 and STIM2 proteins and the ORAI1 Ca2+ channel is important in saliva fluid secretion and has been associated with Sjogren's disease (SjD). However, there are no studies addressing STIM1/2 dysfunction in salivary glands or SjD in animal models. We report that mice lacking Stim1 and Stim2 (Stim1/2K14Cre(+)) in salivary glands exhibited reduced Ca2+ levels and hyposalivate. SOCE was functionally required for the activation of the Ca2+ activated Cl- channel ANO1. Ageing Stim1/2K14Cre(+) mice showed no evidence of lymphocytic infiltration or increased levels of autoantibodies characteristic of SjD, possibly associated with a downregulation of toll-like receptor 8 (Tlr8) expression. Salivary gland biopsies of SjD patients showed increased expression of STIM1 and TLR7/8. Our study shows that SOCE activates ANO1 function and fluid secretion in salivary glands and highlights a potential link between SOCE and TLR signaling in SjD.

Sjögren's disease (SjD) is an autoimmune disorder characterized by progressive salivary and lacrimal gland dysfunction, inflammation, and destruction, as well as extraglandular manifestations. SjD is associated with autoreactive B and T cells, but its pathophysiology remains incompletely understood. Abnormalities in regulatory T (T_reg) cells occur in several autoimmune diseases, but their role in SjD is ambiguous. We had previously shown that the function and development of T_reg cells depend on store-operated Ca²⁺ entry (SOCE), which is mediated by ORAI1 Ca²⁺ channels and stromal interaction protein 1 (STIM1) and STIM2. Here, we show that mice with a Foxp3⁺ T_reg cell-specific deletion of Stim1 and Stim2 develop a phenotype that fulfills all classification criteria of human SjD. Mutant mice have salivary and lacrimal gland inflammation characterized by strong lymphocyte infiltration and transcriptional signatures dominated by T helper 1 (T_H1) and interferon (IFN) signaling. CD4⁺ T cells from mutant mice are sufficient to induce SjD-like disease in an IFN-γ-dependent manner. Inhibition of IFN signaling with the JAK1/2 inhibitor baricitinib alleviated CD4⁺ T cell-induced SjD in mice. These findings are consistent with the transcriptional profiles of CD4⁺ T cells from patients with SjD, which indicate enhanced T_H1 but reduced memory T_reg cell function. Together, our study provides evidence for a critical role of dysfunctional T_reg cells and IFN-γ-producing T_H1 cells in the pathogenesis of SjD.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca²⁺ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.