1,25(OH)2D3 Promotes the Efficacy of CD28 Costimulation Blockade by Abatacept

Inhibition of the CD28:CD80/CD86 T cell costimulatory pathway has emerged as an effective strategy for the treatment of T cell–mediated inflammatory diseases. However, patient responses to CD28-ligand blockade by abatacept (CTLA-4-Ig) in conditions such as rheumatoid arthritis are variable and often suboptimal. In this study, we show that the extent to which abatacept suppresses T cell activation is influenced by the strength of TCR stimulation, with high-strength TCR stimulation being associated with relative abatacept insensitivity. Accordingly, cyclosporin A, an inhibitor of T cell stimulation via the TCR, synergized with abatacept to inhibit T cell activation. We also observed that 1,25-dihydroxyvitamin D3 enhanced the inhibition of T cell activation by abatacept, strongly inhibiting T cell activation driven by cross-linked anti-CD3, but with no effect upon anti-CD28 driven stimulation. Thus, like cyclosporin A, 1,25-dihydroxyvitamin D3 inhibits TCR-driven activation, thereby promoting abatacept sensitivity. Vitamin D3 supplementation may therefore be a useful adjunct for the treatment of conditions such as rheumatoid arthritis in combination with abatacept to promote the efficacy of treatment

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity

Genetic variants associated with psychiatric disorders are enriched at epigenetically active sites in lymphoid cells

Multiple psychiatric disorders have been associated with abnormalities in both the innate and adaptive immune systems. The role of these abnormalities in pathogenesis, and whether they are driven by psychiatric risk variants, remains unclear. We test for enrichment of GWAS variants associated with multiple psychiatric disorders (cross-disorder or trans-diagnostic risk), or 5 specific disorders (cis-diagnostic risk), in regulatory elements in immune cells. We use three independent epigenetic datasets representing multiple organ systems and immune cell subsets. Trans-diagnostic and cis-diagnostic risk variants (for schizophrenia and depression) are enriched at epigenetically active sites in brain tissues and in lymphoid cells, especially stimulated CD4+ T cells. There is no evidence for enrichment of either trans-risk or cis-risk variants for schizophrenia or depression in myeloid cells. This suggests a possible model where environmental stimuli activate T cells to unmask the effects of psychiatric risk variants, contributing to the pathogenesis of mental health disorders.This work was funded by an Medical Research Council award MR/S006257/1 (M.E.L.); NIHR Senior Investigator award (E.T.B); Open Targets grant OTAR040 (B.S., G.T.). Wellcome Trust grant WT206194 (G.T.); US Veterans Affairs Career Development Award (D.F.L.); NIHR Research Professorship RP-2017-08-ST2-002 (M.R.C.). This work was additionally supported by the NIHR Cambridge Biomedical Research Centre

Suppression of CTLA-4 by Th17 polarising cytokines is not specific to IL-17+ T cells.

<p>CD4+CD25- T cells were stimulated in the presence of Th17 polarising cytokines for four days and assessed for IL-17, IFNγ, IL-21, TNFα, IL-2 or Foxp3 in combination with CTLA-4 by flow cytometry. <b>A)</b> Frequency of total CTLA-4+ cells. <b>B)</b> Representative bivariate FACS plot of CTLA-4 versus IL-17 for cells cultured under Th17 polarising conditions. <b>C)</b> CTLA-4 expression in CTLA-4+ T cells gated according to IL-17 expression. <b>D)</b> CTLA-4 expression by CTLA-4+ T cells that expressed IL-17, IFNγ, IL-21, TNFα or IL-2. <b>E)</b> CTLA-4 expression in CTLA-4+ T cells defined by FoxP3 expression. In C, D and E expression under Th17 conditions is expressed relative to the level under Th0 conditions. Data are summarised for n≥7 donors. Bars indicate median values and error bars show the semi interquartile range. Significance with respect to cells expressing the marker under Th0 conditions was tested by Wilcoxon matched paired tests. (* = P<0.05, ** = P<0.01, *** = P<0.001).</p

Th17 polarising cytokines reduce CTLA-4 expression.

<p>Cell trace-labelled CD4+CD25- T cells were stimulated for four days with antiCD3CD28 beads under no cytokine supplement (Th0), with TGFβ alone or with the pro-Th17 cocktail (TGFβ with IL-1β, IL-6 and IL-23) as indicated and expression of total CTLA-4 and Foxp3 assessed by flow cytometry. <b>A)</b> Representative FACS plots showing CTLA-4 against Foxp3 expression and cell division, indicated by cell-trace dilution. <b>B)</b> Summary of CTLA-4 expression for 12 donor donors. Bars indicate mean values and error bars show standard deviation. Significance was tested by repeated measures, single factor within subject analysis (* = P<0.05, *** = P<0.001).</p

1,25(OH)₂D₃ maintains a regulatory T cell phenotype even under inflammatory, Th17 polarising conditions.

<p>CD4+CD25- T cells were stimulated in the presence of recombinant cytokines IL-1β, IL-6, IL-23 and TGFβ as indicated with or without 1,25(OH)₂D₃ and expression of regulatory-associated markers CTLA-4, Foxp3 and CD25 assessed at four days and cytokines IL-2, IL-17, IFNγ, IL-21 and IL-10 measured at five days by flow cytometry. Data are summarised for n≥5 donors. Bars indicate mean values and error bars show the standard deviation. Repeated measures, two factor within subject analysis was used to test interaction between 1,25(OH)₂D₃ and cytokine treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131539#pone.0131539.s002" target="_blank">S1 Table</a>). For markers that did not show interaction the two factor analysis was re-run in the absence of interaction and P values for each factor are shown (1,25(OH)₂D₃ = <i>P</i>_<i>D3</i> and cytokine treatment = <i>P</i>_<i>Cyt</i>. Where interaction was detected, single factor analysis was performed. P values are shown for the effect of cytokine treatment under control (<i>P</i>_{<i>cyt—D3</i>}) and 1,25(OH)₂D₃ (<i>P</i>_{<i>cyt + D3</i>}) conditions separately. Significant contrasts between cytokine treatments are indicated by stars (* = P<0.05, ** = P<0.01, *** = P<0.001).</p

1,25(OH)₂D₃ promotes CTLA-4-mediated B7 depletion from dendritic cells and suppression of T cell proliferation.

<p>CTLA-4 expressing ‘suppressor cells’ were prepared by stimulating CD4+CD25- T cells under Th0 or Th17 conditions in the presence or absence of 1,25(OH)₂D₃. <b>A)</b> Suppressor cells were cultured with autologous DCs and antiCD3 for 24 hours with or without CTLA-4 blocking antibody. Expression of CD80, CD86, CD11c and CD40 by DCs was measured by flow cytometry. Dot plots show the ratio of marker expression in control versus anti-CTLA-4-treated cultures for four donors. <b>B)</b> Suppressor cells were CFDA-SE labeled and added to autologous DC plus antiCD3 stimulations of allogeneic cell trace violet labeled CD4+CD25- T cells (responders) with or without anti-CTLA-4. Parallel stimulations were also prepared in which CFDA-SE-labeled CD4+CD25- were added in place of suppressor T cells as a control for cell number. At five days, proliferation of responder T cells was assessed by flow cytometry. Data are from one donor but representative of four. Shaded histograms show proliferation in the absence of suppressors. Dotted and solid lines indicate proliferation in the presence versus the absence of anti-CTLA-4 respectively.</p

Transendocytic function of CTLA-4 is promoted by 1,25(OH)₂D₃ and maintained under inflammatory conditions.

<p>CTLA-4 transendocytic function was tested as described in the methods. <b>A)</b> Gating strategy to ensure exclusion of CD86-GFP donor cells from the measurement of T cell GFP acquisition. <b>B)</b> Representative FACS plots of CD86-GFP acquisition versus trafficking CTLA-4. <b>C)</b> Total CTLA-4 mediated CD86 acquisition by T cells summarized for n = 5 donors. Bars indicate mean values and error bars show the standard deviation. P values for the separate effects of Th17 cytokines (P_cyt) and 1,25(OH)₂D₃ (P_D3) are shown as determined by the 2 factor without interaction model since no interaction was detected (P = 0.146).</p

Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations

Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency (CVID) with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T cell function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA-negative Foxp3+ fraction. CTLA-4 induction following stimulation, and the use of lysosomal blocking compounds, distinguished CTLA-4 from LRBA mutations. Short term T cell stimulation improved the capacity for discriminating the Foxp3+ Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene sequencing approaches