Faculty Bibliography

Ca2+ signals regulate the function of many immune cells and promote immune responses to infection, cancer, and autoantigens. Ca2+ influx in immune cells is mediated by store-operated Ca2+ entry (SOCE) that results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels. The CRAC channel is formed by three plasma membrane proteins, ORAI1, ORAI2, and ORAI3. Of these, ORAI1 is the best studied and plays important roles in immune function. By contrast, the physiological role of ORAI3 in immune cells remains elusive. We show here that ORAI3 is expressed in many immune cells including macrophages, B cells, and T cells. To investigate ORAI3 function in immune cells, we generated Orai3-/- mice. The development of lymphoid and myeloid cells in the thymus and bone marrow was normal in Orai3-/- mice, as was the composition of immune cells in secondary lymphoid organs. Deletion of Orai3 did not affect SOCE in B cells and T cells but moderately enhanced SOCE in macrophages. Orai3-deficient macrophages, B cells, and T cells had normal effector functions in vitro. Immune responses in vivo, including humoral immunity (T cell dependent or independent) and antitumor immunity, were normal in Orai3-/- mice. Moreover, Orai3-/- mice showed no differences in susceptibility to septic shock, experimental autoimmune encephalomyelitis, or collagen-induced arthritis. We conclude that despite its expression in myeloid and lymphoid cells, ORAI3 appears to be dispensable or redundant for physiological and pathological immune responses mediated by these cells.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N⁶-methyladenosine (m⁶A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m⁶A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m⁶A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt⁺ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m⁶A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m⁶A modification impacts on TCR signal transduction and determines activation and survival of T cells.

TCR stimulation triggers Ca²⁺ signals that are critical for T cell function and immunity. Several pore-forming α and auxiliary β subunits of voltage-gated Ca²⁺ channels (VGCC) were reported in T cells, but their mechanism of activation remains elusive and their contribution to Ca²⁺ signaling in T cells is controversial. We here identify Ca_Vβ1, encoded by Cacnb1, as a regulator of T cell function. Cacnb1 deletion enhances apoptosis and impairs the clonal expansion of T cells after lymphocytic choriomeningitis virus (LCMV) infection. By contrast, Cacnb1 is dispensable for T cell proliferation, cytokine production and Ca²⁺ signaling. Using patch clamp electrophysiology and Ca²⁺ recordings, we are unable to detect voltage-gated Ca²⁺ currents or Ca²⁺ influx in human and mouse T cells upon depolarization with or without prior TCR stimulation. mRNAs of several VGCC α1 subunits are detectable in human (Ca_V3.3, Ca_V3.2) and mouse (Ca_V2.1) T cells, but they lack transcription of many 5' exons, likely resulting in N-terminally truncated and non-functional proteins. Our findings demonstrate that although Ca_Vβ1 regulates T cell function, these effects are independent of VGCC channel activity.

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.

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.

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.