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. 2011 Apr 29;332(6029):600-3.
doi: 10.1126/science.1202947. Epub 2011 Apr 7.

Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4

Omar S Qureshi  1 , Yong Zheng, Kyoko Nakamura, Kesley Attridge, Claire Manzotti, Emily M Schmidt, Jennifer Baker, Louisa E Jeffery, Satdip Kaur, Zoe Briggs, Tie Z Hou, Clare E Futter, Graham Anderson, Lucy S K Walker, David M Sansom
Affiliations

Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4

Omar S Qureshi et al. Science. .

Abstract

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 shares two ligands (CD80 and CD86) with a stimulatory receptor, CD28. Here, we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. After removal, these costimulatory ligands are degraded inside CTLA-4-expressing cells, resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen-presenting cells is stimulated by T cell receptor engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation in which CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28-CTLA-4 system.

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Figures

Fig. 1
Fig. 1
CTLA-4 mediated acquisition of co-stimulatory molecules. (A) Flow cytometric analysis of CD86-GFP transfer into CTLA-4 expressing cells. CHO cells expressing CD86-GFP (Far Red labeled) were co-cultured with CHO controls or with CTLA-4+ CHO cells in the presence or absence of 10 nM Bafilomycin A. Singlet CTLA-4 expressing cells were analyzed for GFP acquisition by excluding Far Red+ donor cells from analysis (see fig. S1) (B) Projection of a confocal z-stack showing a CTLA-4+ CHO cell (blue) in contact with a CD86 GFP-expressing CHO cell (green) in the presence of Bafilomycin A (BafA). GFP inside the CTLA-4 cell appears as cyan puncta. (C) Confocal micrographs of adherent CD86-expressing CHO cells and CTLA-4+ CHO cells after overnight incubation. CD86 (green) and CTLA-4 (red) were detected by antibody staining. Co-localization of CD86 and CTLA-4 is shown in yellow. Lower panels show an enlargement of the boxed area, with single color images shown in white for equal contrast. CHO-CD86 cultured alone are shown in fig. S7A. (D) Confocal images of cells expressing wild type (wt) CTLA-4 or CTLA-4 lacking the cytoplasmic domain (del36) (red) incubated for 2 hours with CD86-GFP (green) expressing cells. (E) Flow cytometric analysis of CD86 surface expression on CHO-CD86 cells co-incubated with increasing numbers of untransfected (control), wild-type CTLA-4 or CTLA-4 del36 cells (expressed as % CTLA-4+ cells in the co-culture). Surface CD86 was detected by antibody staining. (F) Response of CFSE-labelled CD4+CD25−T cells stimulated in the presence of anti-CD3 antibody with CD86-expressing cells fixed after co-culture with CTLA-4 WT or CTLA-4 del36. All data are single representatives of 3 or more independent experiments, Scale bars are 10 μm.
Fig. 2
Fig. 2
Human T cells use CTLA-4 to remove CD86 from dendritic cells. (A) Typical CD86 expression on a human monocyte-derived dendritic cell (DC) cultured in the absence of T cells. (B) DC cultured for 72h with anti-CD3-activated CD4+CD25−T cells (outlined in white) stained with anti-CD86 (green) and anti-CTLA-4 (red). Single staining is shown as white for equal contrast. Cells were co-cultured in the absence or presence of blocking anti-CTLA-4. (C) Quantitation of surface CD80 and CD86 expression on DCs after co-culture with T cells in the presence or absence of anti-CTLA-4 determined by flow cytometry. Data show MFI change pooled from >5 experiments with SEM. (D) Confocal micrographs of allogeneic DCs co-cultured overnight with CTLA-4-transfected (CTLA-4 TF) or control resting CD4+CD25−human T cells. Cultures were fixed and stained with anti-CD86 (green) or anti-HLA-DR (red). White arrowsheads highlight position of T cells, green arrowsheads highlight DCs. White images show single color staining for contrast. (E) Mean fluorescence intensity of surface CD86 on DCs after incubation with either CTLA-4-transfected or control T cells as determined by flow cytometry. All data are representative of at least 3 independent experiments. Error bars represent the SEM. Scale bars = 10 μM.
Fig. 3
Fig. 3
TCR stimulation promotes CTLA-4 trafficking and trans-endocytosis of CD86. (A) Acquisition of CD86-GFP from CHO cells by human CD4+ T cell blasts in the presence or absence of anti-CD3 stimulation. Right hand panels show the effect of anti-CTLA-4 antibody on GFP uptake. (B) SEB-specific CD4+ T cell blasts were incubated with either unpulsed or SEB-pulsed DCs. Cells were fixed and stained with anti-CD86 (green) and anti-CTLA-4 (red). Yellow indicates colocalization. Single colors are shown in white for equal contrast. (C) Surface levels of CD86 on DCs incubated with SEB-specific T cell blasts for 16h as determined by flow cytometry. (D) CD4+CD25+ (Treg) or CD4+CD25−T cells were incubated with DCs and anti-CD3 overnight, fixed, stained using anti-CD86 (green), anti-CD3 for T cells (blue), and visualized by confocal microscopy. Yellow arrow indicates CD86 puncta within T cells. (E) Surface levels of CD86 on DCs incubated with CD4+CD25+ (Treg) and CD4+CD25−T cells overnight determined by flow cytometry. All data are representative of at least 3 independent experiments. Error bars represent the SEM. Scale bars = 10 μM.
Fig. 4
Fig. 4
In vivo capture of CD86 by CTLA-4. Balbc Rag2−/− mice were reconstituted with CD86-GFP transduced Balbc Rag2−/− bone marrow to permit the development of APC expressing CD86-GFP. 3wk later mice were injected with DO11.10 CD4+ T cells and immunized as described in figure S16. (A) 6h after i.v. OVA peptide re-challenge, splenocytes were harvested, labeled at 4°C for CD4 (blue) and CD25 (red) and immediately imaged by confocal microscopy. Representative images of T cells from OVA peptide challenged or unchallenged mice are shown. (B) Representative images of CD4+ T cells purified from spleen after treatment in vivo with peptide showing CD4 and CD25 staining. (C) CD4+ T cells from either Ctla-4+/+ or Ctla-4−/− (Rag2−/− DO11.10 Rip-mOVA) mice were injected into mice that previously received CD86-GFP Rag2−/− bone marrow cells. Cells were re-challenged with OVA in vivo as above. Splenocytes were isolated and immediately analyzed for CD86-GFP (green), CD25 (red) CD4 (blue) by confocal microscopy. Two representative panels are shown for both Ctla-4+/+ and Ctla-4−/− conditions. Data are representative of 3 independent experiments. Scale bars = 10 μM.
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Comment in

  • Immunology. Damping by depletion.
    Sakaguchi S, Wing K. Sakaguchi S, et al. Science. 2011 Apr 29;332(6029):542-3. doi: 10.1126/science.1206122. Science. 2011. PMID: 21527700 No abstract available.

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References

    1. Tivol EA, et al. Immunity. 1995;3:541. - PubMed
    1. Chambers CA, Sullivan TJ, Allison JP. Immunity. 1997;7:885. - PubMed
    1. Waterhouse P, et al. Science. 1995;270:985. - PubMed
    1. Rudd CE, Taylor A, Schneider H. Immunol Rev. 2009 May;229:12. - PMC - PubMed
    1. Choi JM, et al. Nat Med. 2006 May;12:574. - PubMed

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