Availability of 25-hydroxyvitamin D(3) to APCs controls the balance between regulatory and inflammatory T cell responses

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, exerts potent effects on several tissues including cells of the immune system, where it affects T cell activation, differentiation and migration. The circulating, inactive form of vitamin D, 25(OH)D(3), is generally used as an indication of “vitamin D status”. However, utilization of this precursor depends on its uptake by cells and subsequent conversion by the enzyme 25(OH)D(3)-1α-hydroxylase (CYP27B1) into active 1,25(OH)(2)D(3). Using human T cells, we now show that addition of inactive 25(OH)D(3) is sufficient to alter T cell responses only when dendritic cells (DCs) are present. Mechanistically, CYP27B1 is induced in DCs upon maturation with LPS or upon T cell contact resulting in the generation and release of 1,25(OH)(2)D(3) which subsequently affects T cell responses. In most tissues, vitamin D binding protein (DBP) acts as a carrier to enhance the utilization of vitamin D. However, we show that DBP modulates T cell responses by restricting the availability of inactive 25(OH)D(3) to DC. These data indicate that the level of “free” 25(OH)D(3) available to DCs determines the inflammatory/regulatory balance of ensuing T cell responses

Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM) within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system

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

Role of the CTLA-4 C-terminus in regulating its intracellular trafficking

CTLA-4 is an important inhibitor of T cell immune responses. The location of CTLA-4 in intracellular vesicles is the most dominating aspect of its biology, yet the significance of this at the functional level remains to be completely understood. I have therefore investigated the role of the CTLA-4 cytoplasmic domain in the intracellular trafficking of the receptor with particular emphasis on sorting signals encoded within this domain. We found that CTLA-4 was located in punctate intracellular vesicles in transfected cells, activated T cells and in regulatory T cells. CTLA-4 internalisation from the cell surface was clathrin dependent and was driven by the YVKM motif encoded within the cytoplasmic domain. Post-internalisation CTLA-4 colocalised with markers of late endosomes. Since the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression we investigated this further and found that ubiquitination of intracellular lysine residues targets CTLA-4 to lysosomes. The ability of CTLA-4 to recycle was dependent on the YVKM motif and subtle changes in this motif reduced recycling efficiency. Moreover, in the absence of lysine residues CTLA-4 recycling was enhanced. CTLA-4 transendocytosis was conserved through evolution but the exact sorting signals required for this function remain to be identified. Overall this thesis emphasises the importance of the CTLA-4 cytoplasmic domain in regulating its intracellular trafficking

Recycling rates of CTLA-4 chimeras.

<p><b>A</b>. CHO cells expressing WT human CTLA-4 were labeled with mouse anti-CTLA-4 PE at 37°C to detect cycling CTLA-4, washed and any recycling CTLA-4-PE antibody detected by addition of Alexa647 anti-mouse IgG at either 4°C or 37°C. Representative FACS plots are shown for PE-label (cycling CTLA-4) vs Alexa647 label (re-cycling CTLA-4) at the indicated time points. <b>B</b>. Recycling rates are plotted for the chimeras as normalised to the 4°C control.</p

Cellular localisation of CTLA-4 chimeras.

<p><b>A.</b> CHO cells expressing CTLA-4 chimeras were incubated with unlabeled anti-CTLA-4 Ab at 37°C for 1 hour, cooled to 4°C and surface CTLA-4 stained red with anti-mouse Alexa 555. Cells were subsequently fixed, permeabilised and stained with Alexa488 anti-mouse IgG (green) and imaged by confocal microscopy. <b>B.</b> The ratio of plasma membrane to internalised CTLA-4 fluorescence (PM/I) was calculated by outlining cells in ImageJ. <b>C.</b> CHO cells expressing human CTLA-4 were labeled with anti-CTLA-4 PE at 37°C for 30 minutes followed by labeling surface CTLA-4 on ice (4°C) with Alexa647 anti-mouse IgG. Cells were analysed by flow cytometry and data are plotted as cycling CTLA-4 (37°C label) vs surface CTLA-4 (4°C label). <b>D.</b> CHO cells expressing the CTLA-4 chimeras were labeled as described in <b>C</b> and analysed by flow cytometry. Dotted line provides a standard gradient for reference purposes.</p

Generation and localisation of CTLA-4 chimeras.

<p><b>A</b>. C-terminal sequence alignments of selected mammalian CTLA-4 based on sequence data from Ensembl and in ref 12. <b>B.</b> Diagram of human CTLA-4 chimeras containing the extracellular and transmembrane domain of human CTLA-4 and the C-terminus of species shown. C-terminal amino acid sequence alignments of human, chicken, <i>xenopus</i> and trout CTLA-4 are shown below, based on alignments using Clustal W. <b>C</b>. CHO cells expressing CTLA-4 chimeras were incubated with WGA-tetramethylrhodamine at 4°C for 45minutes. Cells were subsequently fixed, permeabilised, and stained with an unlabeled anti-CTLA-4 Ab followed by Alexa488 anti-human IgG (green) to stain total CTLA-4 protein. Cells were analysed by confocal microscopy. <b>D</b> Relative expression of surface (4°C) and total CTLA-4 for each chimera as determined by flow cytometry.</p

Degradation of CTLA-4 chimeras.

<p><b>A</b>. CHO cells expressing CTLA-4 chimeras were incubated in medium or medium supplemented with cycloheximide (CHX) at 37°C for 3 hours. Cells were fixed, permeabilised and stained for total CTLA-4 with an unlabeled anti-CTLA-4 Ab followed by Alexa488 anti-mouse IgG (green) and nuclei counterstained using DAPI (blue). Cells were analysed by confocal microscopy. <b>B</b> Total CTLA-4 was quantified by outlining cells in ImageJ and MFI plotted (left column). For flow cytometric quantitation (right hand column) cells were stained for total CTLA-4 using anti-CTLA-4 PE after 3 hours of CHX or NH₄Cl treatment at 37°C and MFI plotted as a percentage of initial fluorescence. <b>C</b>. CHO cells expressing CTLA-4 chimeras were transfected with CD63-GFP. Cells were incubated in medium supplemented with NH₄Cl at 37°C for 3 hours. Cells were fixed, permeabilised, and stained with an unlabeled anti-CTLA-4 Ab and Alexa565 anti-human IgG (red) to stain total CTLA-4 protein and analysed by confocal microscopy. The red arrows indicate co-localisation.</p

Endocytosis rates of CTLA-4 chimeras.

<p><b>A.</b> CHO cells expressing CTLA-4 chimeras were labeled at 4°C with anti-CTLA-4 to label surface CTLA-4. Cells were then warmed to 37°C to allow endocytosis for the times indicated. Cells were then placed on ice and any remaining surface CTLA-4 detected with Alexa647 anti-mouse IgG. The 647 signal was plotted against time as the fraction remaining compared to 4°C. <b>B.</b> CHO cells expressing CTLA-4 chimeras were labeled as in A but in medium supplemented with sucrose (0.45 M) to prevent endocytosis. <b>C.</b> CHO cells expressing the chimeric CTLA-4 constructs were incubated with a transferrin (Tf) Alexa633 conjugate (Invitrogen) and anti-CTLA-4 PE at 37°C for 45 minutes. Cells were subsequently fixed and analysed by confocal microscopy. The red arrows indicate co-localisation. <b>D.</b> Rate of endocytosis of VGNF mutant was performed as described in A. <b>E.</b> Transferrin uptake of VGNF mutant was performed as described in C and analysed by confocal microscopy.</p