Circulating follicular helper T cells exhibit reduced ICOS expression and impaired function in narcolepsy type 1 patients.

Despite genetic and epidemiological evidence strongly supporting an autoimmune basis for narcolepsy type 1, the mechanisms involved have remained largely unknown. Here, we aimed to investigate whether the frequency and function of circulating follicular helper and follicular regulatory T cells are altered in narcolepsy type 1. Peripheral blood mononuclear cells were collected from 32 patients with narcolepsy type 1, including 11 who developed disease after Pandemrix® vaccination, and 32 age-, sex-, and HLA-DQB1*06:02-matched healthy individuals. The frequency and phenotype of the different circulating B cell and follicular T cell subsets were examined by flow cytometry. The function of follicular helper T cells was evaluated by assessing the differentiation of naïve and memory B cells in a co-culture assay. We revealed a notable increase in the frequency of circulating B cells and CD4+CXCR5+ follicular T cells in narcolepsy patients compared to age-, sex- and HLA-matched healthy controls. However, the inducible T-cell costimulator molecule, ICOS, was selectively down-regulated on follicular T cells from patients. Reduced frequency of activated ICOS+ and PD1high blood follicular T cells was also observed in the narcolepsy group. Importantly, follicular T cells isolated from patients with narcolepsy type 1 had a reduced capacity to drive the proliferation/survival and differentiation of memory B cells. Our results provide novel insights into the potential involvement of T cell-dependent B cell responses in the pathogenesis of narcolepsy type 1 in which down-regulation of ICOS expression on follicular helper T cells correlates with their reduced function. We hypothesize that these changes contribute to regulation of the deleterious autoimmune process after disease onset.

Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions.

BACKGROUND: In addition to calcium release-activated calcium channel/ORAI calcium channels, the role of voltage-gated calcium (Cav1) channels in T-cell calcium signaling is emerging. Cav1 channels are formed by α1 (CaV1.1 to CaV1.4) and auxiliary subunits. We previously demonstrated that mouse TH2 cells selectively overexpressed CaV1.2 and CaV1.3 channels. Knocking down these channels with Cav1 antisense (AS) oligonucleotides inhibited TH2 functions and experimental asthma. OBJECTIVE: We investigated the expression profile and role of Cav1 channels in human T-cell subsets, with a focus on TH2 cells. METHODS: We compared the profile of CaV1 channel subunit expression in T-cell subsets isolated ex vivo from the blood of healthy donors, as well as in vitro-polarized T-cell subsets, and tested the effect of the Cav1 inhibitors nicardipine and Cav1.2AS on their functions. RESULTS: CaV1.4 expression was detectable in CD4(+) T cells, ex vivo TH1 cells, and TH17 cells, whereas Cav1.2 channels predominated in TH2 cells only. T-cell activation resulted in Cav1.4 downregulation, whereas Cav1.2 expression was selectively maintained in polarized TH2 cells and absent in TH1 or TH9 cells. Nicardipine and CaV1.2AS decreased Ca(2+) and cytokine responses in TH2, but not TH1, cells. Protein kinase C (PKC) α/ß inhibition decreased Ca(2+) and cytokine responses, whereas both calcium and cytokine responses induced by PKC activation were inhibited by nicardipine or Cav1.2AS in TH2 cells. CONCLUSION: This study highlights the selective expression of Cav1.2 channels in human TH2 cells and the role of PKC-dependent Cav1.2 channel activation in TH2 cell function. Blocking PKC or Cav1.2 channel activation in TH2 cells might represent new strategies to treat allergic diseases in human subjects.

Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis.

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17ß-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Impaired NFAT transcriptional activity in antigen-stimulated CD8 T cells linked to defective phosphorylation of NFAT transactivation domain.

NFAT transcription factors play critical roles in CD4 T cell activation and differentiation. Their function in CD8 T cell is, however, unknown. We show in this study that, in contrast to CD4 T cells, Ag-stimulated CD8 T cells do not demonstrate NFAT transcriptional activity despite normal regulation of NFAT nuclear shuttling. Further analysis of the signaling defect shows that phosphorylation of the (53)SSPS(56) motif of the NFAT transactivation domain is essential for NFAT-mediated transcription in primary T cells. Although Ag stimulation induces in CD4 T cells extensive phosphorylation of this motif, it does so only minimally in CD8 T cells. Although Ag stimulation triggers only modest activation of the p38 MAPK in CD8 T cells as opposed to CD4 T cells, p38 MAPK is not the upstream kinase that directly or indirectly phosphorylates the NFAT (53)SSPS(56) motif. These findings reveal an unsuspected difference between CD4 and CD8 T cells in the TCR downstream signaling pathway. Therefore, whereas in CD4 T cells TCR/CD28 engagement activates a yet unknown kinase that can phosphorylate the NFAT (53)SSPS(56) motif, this pathway is only minimally triggered in CD8 T cells, thus limiting NFAT transcriptional activity.

Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma.

RATIONALE: Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions. OBJECTIVES: Because dihydropyridines bind to and modulate voltage-dependent calcium (Ca(v)1) channel in excitable cells, we aimed at showing that Th2 cells selectively express functional Ca(v)1-related channels, the inhibition of which may prevent asthma. METHODS: We looked for Ca(v)1 channel expression in Th2 and Th1 cells by real-time polymerase chain reaction and Western blotting. We sequenced the isoforms expressed by Th2 cells and tested whether Ca(v)1 antisense oligodeoxynucleotides (Ca(v)1AS) affected Ca(2+) signaling and cytokine production. Finally, we tested the effect of Ca(v)1AS in the passive asthma model by injection of ovalbumin-specific Th2 cells transfected with Ca(v)1AS into BALB/c mice challenged with intranasal ovalbumin and in the active model of asthma by intranasal delivery of Ca(v)1AS together with soluble ovalbumin in BALB/c mice previously immunized with ovalbumin in alum. MEASUREMENTS AND MAIN RESULTS: We show that mouse Th2 but not Th1 cells expressed Ca(v)1.2 and Ca(v)1.3 channels. Th2 cells transfected with Ca(v)1AS had impaired Ca(2+) signaling and cytokine production, and lost their ability to induce airway inflammation on adoptive transfer. Furthermore, intranasal administration of Ca(v)1AS suppressed airway inflammation and hyperreactivity in an active model of asthma. CONCLUSIONS: These results indicate that Th2 cells selectively express Ca(v)1 channels that may be efficiently targeted in T lymphocytes to prevent experimental asthma.

Calcium channel blocker prevents T helper type 2 cell-mediated airway inflammation.

RATIONALE: Ca(2+) signaling controls the production of T helper (Th) type 2 cytokines known to be deleterious in asthma. Recently, we showed that Ca(2+) signaling was dihydropyridine (DHP)-sensitive in Th2 lymphocytes and that the DHP derivate, nicardipine, used in the treatment of cardiovascular pathologies, prevents Th2-dependent B cell polyclonal activation. OBJECTIVES: We tested the effect of nicardipine in experimental allergic asthma. METHODS: BALB/c mice immunized with ovalbumin (OVA) in alum and challenged with intranasal OVA were treated with nicardipine once the Th2 response, or even airway inflammation, was induced. We also tested the effect of nicardipine in asthma induced by transferring OVA-specific Th2 cells in BALB/c mice exposed to intranasal OVA. We checked the impact of nicardipine on T-cell responses and airway inflammation. MEASUREMENTS AND MAIN RESULTS: Nicardipine inhibited in vitro Ca(2+) response in Th2 cells. In vivo, it impeded the development of Th2-mediated airway inflammation and reduced the capacity of lymphocytes from lung-draining lymph nodes to secrete Th2, but not Th1, cytokines. Nicardipine did not affect antigen presentation to CD4(+) T lymphocytes, nor the initial localization of Th2 cells into the lungs of mice exposed to intranasal OVA; however, it reduced the production of type 2 cytokines and the amplification of the Th2 response in mice with asthma. Conversely, nicardipine had no effect on Th1-mediated airway inflammation. CONCLUSIONS: Nicardipine improves experimental asthma by impairing Th2-dependent inflammation. This study could provide a rationale for developing drugs selectively targeting DHP receptors of Th2 lymphocytes, potentially beneficial in the treatment of asthma.

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes.

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.

Cutting edge: T lymphocyte activation by repeated immunological synapse formation and intermittent signaling.

The activation of biological T cell responses requires prolonged contact with APCs and sustained signaling. We investigated whether signaling must be uninterrupted to commit T cells to cytokine production or whether T cell activation may also result from summation of interrupted signals. Upon periodic addition and removal of a src kinase inhibitor, human CD4(+) T cells destroyed and re-formed immunological synapses while aborting and restarting signal transduction. Remarkably, under these conditions, T cells were eventually activated to IFN-gamma production and the amount of IFN-gamma produced was directly related to the total signaling time despite the repeated interruptions. Our results illustrate that T cell activation does not require a stable immunological synapse and can be achieved by interrupted signaling. It is implied that T cells can add activation signals, possibly collected on multiple APCs.

Dihydropyridine receptors are selective markers of Th2 cells and can be targeted to prevent Th2-dependent immunopathological disorders.

Th1 cells that produce IFN-gamma are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4(+) T cells from BN rats into (LEW x BN)F(1) hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.