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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 Ca(2+) signals that are shaped by the opening of Ca(2+) channels in the plasma membrane and cellular organelles. The dynamic regulation of intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

ObjectiveInflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses and constitutes a major clinical challenge in need of new treatment modalities to improve patient care. Store-operated Ca2+ entry (SOCE) is the predominant Ca2+ influx pathway in T cells and other immune cells, regulating many of their functional properties. It is currently unknown whether the pharmacologic blockade of SOCE represents a suitable drug-target for IBD treatment.DesignUsing mass and flow cytometry the effects of SOCE inhibition on lamina propria (LP) immune cells of patients with ulcerative colitis (UC) and Crohn’s disease (CD) were investigated. Primary organoid cultures served to study the impact of SOCE inhibition on the function, differentiation and survival of intestinal epithelial cells (IEC). T cell transfer models of colitis were applied to examine how the genetic or pharmacologic ablation of SOCE affects the clinical course of IBD in mice.ResultsWe observed that the LP of IBD patients is characterized by an enrichment of innate lymphoid cells (ILC), CD4+ and CD8+ effector- as well as T regulatory cells producing IL-17 and TNFα. The pharmacologic inhibition of SOCE attenuated the production of pathogenic cytokines including IL-2, IL-4, IL-6, IL-17, TNFα and IFNγ by human colonic T cells and ILC, reduced the production of IL-6 by B cells and the production of IFNγ by myeloid cells, without affecting the viability, differentiation and function of primary IEC. T cell-specific genetic deletion of the SOCE signaling components Orai1, Stim1 or Stim2 revealed that the magnitude of SOCE correlates with the function of T cells and intestinal inflammation in mice. Moreover, the pharmacologic inhibition of SOCE alleviated the clinical course of colitic mice.ConclusionOur data suggest that SOCE inhibition may serve as a new pharmacologic strategy for treating IBD.

T follicular helper (Tfh) cells are essential for germinal center B cell responses. The molecular mechanism underlying the initial Tfh cell differentiation, however, is still incompletely understood. In this study, we show that in vivo, despite enhanced non–Tfh cell effector functions, the deletion of transcription factor Bach2 results in preferential Tfh cell differentiation. Mechanistically, the deletion of Bach2 leads to the induction of CXCR5 expression even before the upregulation of Ascl2. Subsequently, we have identified a novel regulatory element in the murine CXCR5 locus that negatively regulates CXCR5 promoter activities in a Bach2-dependent manner. Bach2 deficiency eventually results in a collapsed CD4+ T cell response with severely impaired CD4+ T cell memory, including Tfh cell memory. Our results demonstrate that Bach2 critically regulates Tfh cell differentiation and CD4+ T cell memory.

Liver-resident NK cells are distinct from conventional NK cells and play an important role in the maintenance of liver homeostasis. How liver-resident NK cells participate in autoimmune cholangitis remains unclear. Here, we extensively investigated the impact of NK cells in the pathogenesis of autoimmune cholangitis utilizing the well-established dnTGFβRII cholangitis model, NK cell-deficient (Nfil3(−/−)) mice, adoptive transfer and in vivo antibody-mediated NK cell depletion. Our data demonstrated that disease progression was associated with a significantly reduced frequency of hepatic NK cells. Depletion of NK cells resulted in exacerbated autoimmune cholangitis in dnTGFβRII mice. We further confirmed that the DX5(−)CD11c(hi) liver-resident NK cell subset colocalized with CD4(+) T cells and inhibited CD4(+) T cell proliferation. Gene expression microarray analysis demonstrated that liver-resident NK cells had a distinct gene expression pattern consisting of the increased expression of genes involved in negative regulatory functions in the context of the inflammatory microenvironment.

T follicular helper (Tfh) cells play an essential role in the formation of germinal centers (GC) and generation of high-affinity Abs. The homing of activated CD4+ T cells into B cell follicles and the involvement of key costimulatory and coinhibitory molecules are critical in controlling both the initiation and the magnitude of GC responses. Meanwhile, studies have shown that a high number of single clone B cells leads to intraclonal competition, which inhibits the generation of high-affinity Abs. Our previous work has shown that transcription factor Foxp1 is a critical negative regulator of Tfh cell differentiation. In this study, we report that the deletion of Foxp1 leads to a high proportion of activated CD4+ T cells homing into B cell follicles with faster kinetics, resulting in earlier GC formation. In addition, we show that Foxp1-deficient Tfh cells restore the generation of high-affinity Abs when cotransferred with high numbers of single clone B cells. We find that Foxp1 regulates the expression levels of cytotoxic T lymphocyte–associated Ag-4 (CTLA-4) in activated CD4+ T cells and that Ctla4 is a direct Foxp1 target. Finally, we demonstrate that CTLA-4 expression on conventional CD4+ T cells plays a cell-intrinsic role in Tfh cell differentiation in vivo, and CTLA-4 blockade helps abolish the intraclonal competition of B cells in generating high-affinity Abs.

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4(-/-)Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4(-/-)Tg mice with established disease at 8-9 weeks of age with C57BL/6 control mice. Such parabiotic "twins" had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4(-/-)Tg and CD8(-/-) mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8(+) T effector cells in the presence of wild type CD4 cells was noted. In conclusion, "correcting" the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.

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.

There have been major advances in defining the immunological events associated with fibrosis in various chronic liver diseases. We have taken advantage of this data to focus on the mechanisms of action of a unique multi-kinase inhibitor, coined sorafenib, on CCl4-induced murine liver fibrosis, including the effects of this agent in models of both acute and chronic CCl4-mediated pathology. Importantly, sorafenib significantly attenuated chronic liver injury and fibrosis, including reduction in liver inflammation and histopathology as well as decreased expression of liver fibrosis-related genes, including α-smooth muscle actin, collagen, matrix metalloproteinases and the tissue inhibitor of metalloproteinase-1. Furthermore, sorafenib treatment resulted in translocation of cytoplasmic STAT3 to the nucleus in its active form. Based on this observation, we used hepatocyte-specific STAT3 knockout (STAT3(Hep-/-)) mice to demonstrate that hepatic STAT3 was critical for sorafenib-mediated protection against liver fibrosis, and that the upregulation of STAT3 phosphorylation was dependent on Kupffer cell-derived IL-6. In conclusion, these data reflect the clinical potential of the multi-kinase inhibitor sorafenib for the prevention of fibrosis as well as the treatment of established liver fibrosis and illustrate the immunological mechanisms that underlie the protective effects of sorafenib.

// Yan-Qing Yang 1 , Wei Yang 1 , Yuan Yao 1 , Hong-Di Ma 1 , Yin-Hu Wang 1 , Liang Li 1 , Qingfa Wu 2 , M. Eric Gershwin 3 and Zhe-Xiong Lian 1,4 1 Liver Immunology Laboratory, Institute of Immunology and The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei, China 2 The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China, Hefei, China 3 Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA, United States of America 4 Innovation Center for Cell Signaling Network, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China Correspondence to: Zhe-Xiong Lian, email: // Hong-Di Ma, email: // Keywords : B1a cell, autoimmune cholangitis, dysregulation, Breg, peritoneal cavity, Immunology and Microbiology Section, Immune response, Immunity Received : March 15, 2016 Accepted : April 13, 2016 Published : April 20, 2016 Abstract Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease with progressive cholestasis and liver fibrosis. Similar to human patients with PBC, p40 -/- IL-2Rα -/- mice spontaneously develop severe autoimmune cholangitis. Although there has been considerable work on immune regulation and autoimmunity, there is a relative paucity of work directed at the functional implications of the key peritoneal cavity (PC) B cell subset, coined B1a cells in PBC. We used flow cytometry and high-resolution microarrays to study the qualitative and quantitative characteristics of B cells, particularly B1a cells, in the PC of p40 -/- IL-2Rα -/- mice compared to controls. Importantly, B1a cell proliferation was markedly lower as the expression of Ki67 decreased. Meanwhile, the apoptosis level was much higher. These lead to a reduction of B1a cells in the PC of p40 -/- IL-2Rα -/- mice compared to controls. In contrast, there was a dramatic increase of CD4 + and CD8 + T cells accompanied by elevated production of IFN-γ. In addition, we found a negative correlation between the frequency of B1a cells and the presence of autoreactive CD8 + T cells in both liver and PC of p40 -/- IL-2Rα -/- mice. From a functional perspective, B cells from p40 -/- IL-2Rα -/- mice downregulated IL-10 production and CTLA-4 expression, leading to loss of B cell regulatory function. We suggest that the dysfunction of B1a cells in the PC in this murine model of autoimmune cholangitis results in defective regulatory function. This highlights a new potential therapeutic target in PBC.

Previously we have shown that transcription factor Foxp1 plays an essential role in maintaining naive T cell quiescence; in the absence of Foxp1, mature naive CD8+ T cells proliferate in direct response to homeostatic cytokine IL-7. In this study, we report that the deletion of Foxp1 in naive CD8+ T cells leads to enhanced activation of the PI3K/Akt/mammalian target of rapamycin signaling pathway and its downstream cell growth and metabolism targets in response to IL-7. We found that Foxp1 directly regulates PI3K interacting protein 1, a negative regulator of PI3K. Additionally, we found that deletion of Foxp1 in naive CD8+ T cells results in increased expression levels of E2fs, the critical components for cell cycle progression and proliferation, in a manner that is not associated with increased phosphorylation of retinoblastoma protein. Taken together, our studies suggest that Foxp1 enforces naive CD8+ T cell quiescence by simultaneously repressing key pathways in both cellular metabolism and cell cycle progression.

Thymic CD4 + FoxP3 + regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell–cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3 + Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFβ. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells.

Understanding the mechanisms that lead to autoimmunity is critical for defining potential therapeutic pathways. In this regard there have been considerable efforts in investigating the interacting roles of TGF-β and IL-2 on the function regulatory T cells. We have taken advantage of dnTGF-βRII Il2ra−/− (abbreviated as Il2ra−/−Tg) mouse model, which allows a direct mechanistic approach to define the relative roles of TGF-β and IL-2 on Treg development. Il2ra−/−Tg mice spontaneously developed multi-organ autoimmune diseases with expansion of pathogenic T cells and enhanced germinal center response at 3–4 weeks of age. Importantly, peripheral Treg cells from Il2ra−/−Tg mice demonstrated an activated Th1-like stable phenotype and normal in vitro suppressive function, while thymus Treg increased but manifested decreased suppressive function. Interestingly, neither thymus nor peripheral Treg cells of Il2ra−/−Tg mice contained Neuropilin-1+ or PD-1hi phenotype, resulting in defective follicular regulatory T (Tfr) cell development. Such defective Tfr development led to elevated follicular T helper cells, enhanced germinal center responses and increased plasma cell infiltration. These data demonstrate an important synergetic role of TGF-β and IL-2 in the generation, activation and stability of Treg cells, as well as their subsequent development into Tfr cells.