Faculty Bibliography

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.

Calcium (Ca²⁺) signals play fundamental roles in immune cell function. The main sources of Ca²⁺ influx in mammalian lymphocytes following antigen receptor stimulation are Ca²⁺ release-activated Ca²⁺ (CRAC) channels. These are formed by ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the endoplasmic reticulum (ER). Human loss-of-function (LOF) mutations in ORAI1 and STIM1 that abolish Ca²⁺ influx cause a unique disease syndrome called CRAC channelopathy that is characterized by immunodeficiency autoimmunity and non-immunological symptoms. Studies in mice lacking Stim and Orai genes have illuminated many cellular and molecular mechanisms by which these molecules control lymphocyte function. CRAC channels are required for the differentiation and function of several T lymphocyte subsets that provide immunity to infection, mediate inflammation and prevent autoimmunity. This review examines new insights into how CRAC channels control T cell-mediated immunity.

T cells are an essential component of the immune system that provide antigen-specific acute and long lasting immune responses to infections and tumors, ascertain the maintenance of immunological tolerance and, on the flipside, mediate autoimmunity in a variety of diseases. The activation of T cells through antigen recognition by the T cell receptor (TCR) results in transient and sustained Ca²⁺ signals that are shaped by the opening of Ca²⁺ channels in the plasma membrane and cellular organelles. The dynamic regulation of intracellular Ca²⁺ concentrations controls a variety of T cell functions on the timescale of seconds to days after signal initiation. Among the more recently identified roles of Ca²⁺ signaling in T cells is the regulation of metabolic pathways that control the function of many T cell subsets. In this review, we discuss how Ca²⁺ regulates several metabolic programs in T cells such as the activation of AMPK and the PI3K-AKT-mTORC1 pathway, aerobic glycolysis, mitochondrial metabolism including tricarboxylic acid (TCA) cycle function and oxidative phosphorylation (OXPHOS), as well as lipid metabolism.

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca²⁺ signals generated by Orai1 activate Ca²⁺-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca²⁺ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca²⁺ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca²⁺]_i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP₂-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca²⁺ depletion by binding to Orai1 and caused local and global [Ca²⁺]_i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP₂-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca²⁺ influx to NFAT1 activation.

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca²⁺ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca²⁺ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca²⁺ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell-specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases.

B lymphocytes are an important component of the adaptive and innate immune system because of their ability to secrete antibodies and to present antigens to T cells, which is critical for immune responses to many pathogens. Abnormal B cell function is the cause of diseases including autoimmune, paraneoplastic, and immunodeficiency disorders. The development, survival, and function of B cells depend on signaling through the B cell receptor (BCR) and costimulatory receptors. One of the signaling pathways induced by antigen binding to the BCR is store-operated Ca²⁺ entry (SOCE), which depends on the Ca²⁺ channel ORAI1 and its activators stromal interaction molecule (STIM) 1 and 2. A recent study by Berry et al. [1] reports that B cells lacking STIM1 and STIM2 fail to survive and proliferate because abolished SOCE results in impaired expression of two key anti-apoptotic genes and blunted activation of mTORC1 and c-Myc signaling. The associated Ca²⁺ regulated checkpoints of B cell survival and proliferation can be bypassed, at least partially, by costimulation through CD40 or TLR9. This study provides important new insights on how SOCE controls B cell function.

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca²⁺ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca²⁺ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca²⁺ entry (ROCE). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca²⁺ influx and cell function in TRPC1/6-deficient (TRPC1/6^-/-) and STIM1/2- deficient (STIM1/2^ΔpmLF) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2- deficient pmLF and ROCE was decreased in TRPC1/6^-/- pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6^-/- pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLF.