Monocyte:T cell interaction regulates human T cell activation through a CD28/CD46 crosstalk

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TCR-stimulated changes in cell surface CD46 expression generate type 1 regulatory T cells

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Calcitriol modulates the CD46 pathway in T cells

The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)2D3 or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNc ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS

The Dynamic Processing of CD46 Intracellular Domains Provides a Molecular Rheostat for T Cell Activation

Adequate termination of an immune response is as important as the induction of an appropriate response. CD46, a regulator of complement activity, promotes T cell activation and differentiation towards a regulatory Tr1 phenotype. This Tr1 differentiation pathway is defective in patients with MS, asthma and rheumatoid arthritis, underlying its importance in controlling T cell function and the need to understand its regulatory mechanisms. CD46 has two cytoplasmic tails, Cyt1 and Cyt2, derived from alternative splicing, which are co-expressed in all nucleated human cells. The regulation of their expression and precise functions in regulating human T cell activation has not been fully elucidated.Here, we first report the novel role of CD46 in terminating T cell activation. Second, we demonstrate that its functions as an activator and inhibitor of T cell responses are mediated through the temporal processing of its cytoplasmic tails. Cyt1 processing is required to turn T cell activation on, while processing of Cyt2 switches T cell activation off, as demonstrated by proliferation, CD25 expression and cytokine secretion. Both tails require processing by Presenilin/γSecretase (P/γS) to exert these functions. This was confirmed by expressing wild-type Cyt1 and Cyt2 tails and uncleavable mutant tails in primary T cells. The role of CD46 tails was also demonstrated with T cells expressing CD19 ectodomain-CD46 C-Terminal Fragment (CTF) fusions, which allowed specific triggering of each tail individually.We conclude that CD46 acts as a molecular rheostat to control human T cell activation through the regulation of processing of its cytoplasmic tails

CD46 Plasticity and Its Inflammatory Bias in Multiple Sclerosis

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Calcitriol allows the switch from Th1 to Tr1 in CD4+ T cells.

<p>(<b>A</b>) Purified CD4+ T cells from one healthy donor were left unstimulated or stimulated as indicated by immobilized anti-CD3 and anti-CD3/CD46 antibodies, in presence or absence of IL-2 and calcitriol. After 2 days, the production of IL-10 and IFNγ was assessed using the secretion catch assay (Miltenyi). (<b>B</b>) The production of IL-10 and IFNγ in the supernatants from the same wells was also determined by ELISA. The IL-10:IFNγ ratio is also represented (mean ± SEM; samples were analyzed using Student's t-test).</p

CD46 costimulation and calcitriol modulate CD8+ T cells.

<p>(<b>A</b>) Purified CD8+ T cells from 3 healthy donors were left unstimulated or stimulated as indicated by immobilized anti-CD3 and anti-CD3/CD46 antibodies, in presence or absence of calcitriol. (<b>A</b>) The expression levels of surface CD46, CD28, CD25, OX40, PD-1 and 4-1BB were determined by flow cytometry after 3 days. (<b>B</b>) The levels of CTLA-4 and Foxp3 were determined after intracellular staining. (<b>C</b>) Proliferation was determined by ³H incorporation. The levels of IFNγ in the supernatants were determined by ELISA (mean ± SEM). The data shown for this donor are representative of the three donors.</p

Calcitriol modulates CD46 expression in CD4+ T cells.

<p>Purified CD4+ T cells from healthy donors were left unstimulated (US), or were stimulated by immobilized anti-CD3, anti-CD3/CD28, or anti-CD3/CD46 antibodies as indicated in presence of calcitriol (10⁻⁷M) or ethanol as vehicle control. CD46 expression was monitored by flow cytometry. The representative expression of CD46 after 5 days of culture is shown in (<b>A</b>). The average expression of CD46 detected after 2 or 5 days for the different donors analyzed (mean ± SEM; n = 15) is shown in (<b>B</b>). Samples were analyzed using the Wilcoxon test.</p

Calcitriol-treated cell supernatants decrease proliferation of bystander CD4+ T cells.

<p>(<b>A</b>) Purified CD4+ T cells were CFSE pre-labeled and then activated by anti-CD3 or anti-CD3/CD28 antibodies, in presence or absence of culture supernatants from CD46-costimulated T cells with or without calcitriol from either a healthy donor or a patient with MS, as indicated. Proliferation was assessed by flow cytometry after 3 days. (<b>B</b>) The data obtained with the supernatants from CD46-costimulated T cells from 3 healthy donors are represented. (<b>C</b>) CFSE-labeled naïve T cells were activated by anti-CD3 antibodies (1 µg/ml), in presence of cell supernatants from CD46-costimulated T cells with or without calcitriol (n = 3). A blocking anti-IL-10 antibody or control IgG1 was added to the culture. Proliferation was assessed after 3 days.</p