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Trans-Endocytosis of CD80 and CD86: A Molecular Basis for the Cell-Extrinsic Function of CTLA-4

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Science  29 Apr 2011:
Vol. 332, Issue 6029, pp. 600-603
DOI: 10.1126/science.1202947

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.

The T cell protein cytotoxic T lymphocyte antigen 4 (CTLA-4) is essential to the prevention of autoimmune disease (13). Although the molecular basis for CTLA-4 action has been suggested to be a cell-intrinsic inhibitory signal (4) possibly mediated by the cytoplasmic domain (5), a cell-extrinsic function for CTLA-4 is clearly evident from in vivo models (613). Therefore, a molecular explanation of CTLA-4 function compatible with such a cell-extrinsic mechanism is needed. The intercellular transfer of a ligand from one cell to its receptor on a different cell is observed in both immune settings and elsewhere (1419). Because of its highly endocytic behavior, we tested whether CTLA-4 could potentially act in such a manner in order to deplete its ligands and extrinsically inhibit T cell activation via CD28. We cultured CTLA-4–expressing (CTLA-4+) CHO cells with donor CHO cells expressing a C-terminally tagged CD86 protein (CD86-GFP). Using a flow cytometric assay, we observed substantial transfer of CD86 into CTLA-4+ cells (Fig. 1A and fig. S1). This finding was confirmed by using confocal microscopy, in which acquisition of ligand by CTLA-4 caused the appearance of CD86-containing vesicles within the CTLA-4+ cell (Fig. 1B). Treatment with the lysosomal inhibitor bafilomycin A caused an increase in detectable CD86–green fluorescent protein (GFP) after transfer (Fig. 1A), which indicated degradation of the transferred ligand inside the CTLA-4+ cell. Accordingly, CD86 did not appear on the cell surface of the recipient CTLA-4+ cell (fig. S2). Bafilomycin A treatment did not result in an increase in CTLA-4 expression, which suggested that although CTLA-4 could capture and deliver ligand for degradation, CTLA-4 itself was not degraded (fig. S3). Overall, this process appeared different to the generalized intercellular exchange or “trogocytosis” reported for other receptors (2022), in which transferred proteins are detected at the cell surface.

Fig. 1

CTLA-4 mediated acquisition of costimulatory molecules. (A) Flow cytometric analysis of CD86-GFP transfer into CTLA-4–expressing cells. CHO cells expressing CD86-GFP (Far Red–labeled) were cocultured with CHO controls or with CTLA-4+ CHO cells in the presence or absence of 10 nM Bafilomycin A (BafA). Singlet CTLA-4–expressing cells were analyzed for GFP acquisition by excluding Far Red+ donor cells from analysis (fig. S1). (B) Projection of a confocal z-stack showing a (blue) CTLA-4+ CHO cell in contact with a (green) CD86-GFP–expressing CHO cell in the presence of BafA. GFP inside the CTLA-4 cell appears as light blue 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 through antibody staining. Colocalization of CD86 and CTLA-4 is shown in yellow. (Bottom) 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 CTLA-4 transfectants (red) expressing wild-type (wt) CTLA-4 or CTLA-4 lacking the cytoplasmic domain (del36) incubated for 2 hours with CD86-GFP–expressing cells (green). (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 percent of CTLA-4+ cells in the coculture). Surface CD86 was detected through antibody staining. (F) Response of CFSE-labeled CD4+CD25 T cells stimulated in the presence of antibody to CD3 with CD86-expressing cells fixed after coculture with wild-type CTLA-4 or CTLA-4 del36. All data are single representatives of three or more independent experiments. Scale bars, 10 μm.

To investigate the time course of CD86 acquisition, we performed live-cell imaging of CTLA-4+ CHO cells interacting with CHO cells expressing CD86-GFP (fig. S4A and movies S1 and S2) (23). Within minutes of cell contact, we observed a marked concentration of CD86-GFP at the site of cell-cell contact, from which CD86-positive vesicles emanated into the CTLA-4–expressing cell (movies S1 to S4). Quantitation of this process revealed a substantial depletion of GFP fluorescence from the plasma membrane of the CD86 donor cell and a corresponding increase in GFP inside the CTLA4+ recipient cell (fig. S4B). Kinetic analysis by means of flow cytometry also revealed that over 50% of CTLA-4+ cells acquired CD80 or CD86 within 3 hours (fig. S5). Furthermore, we estimated the number of CTLA-4 molecules and CD86 molecules expressed by our cell lines to determine the stoichiometry of CD86 depletion (fig. S6). This showed that a ratio of approximately 1:8 (CTLA-4:CD86) molecules was sufficient for functionally relevant depletion.

To confirm that trans-endocytosis of ligand was not an artifact of using GFP-fusion proteins, we also performed experiments with wild-type CD86 expressed in CHO cells. CHO-CD86 cells were cultured alone or with CHO–CTLA-4 cells and then stained for CD86 and CTLA-4 by using antibodies (Fig. 1C). In the absence of CTLA-4, CD86-expressing cells display a characteristic plasma membrane–staining pattern (fig. S7A); however, in the presence of CTLA-4, CD86-containing vesicles were observed inside CTLA-4 recipient cells (Fig. 1C and fig. S7A). Moreover, because CD86 was stained by using an antibody against the cytoplasmic domain this indicated that the whole ligand had been transferred into the CTLA-4+ cell. Analysis of immunofluorescence images showed no acquisition of CTLA-4 by the CD86-expressing cells (fig. S7B). In addition, studies using the membrane dye PKH26 revealed that trans-endocytosis was associated with transfer of small amounts of membrane lipids (fig. S7C). Taken together, these data indicated that protein transfer was unidirectional and appeared to involve transfer of membrane lipid. Because the C terminus of CTLA-4 is required for endocytosis (fig. S8) (24, 25) and shows a remarkable degree of evolutionary conservation, we tested the contribution of this region to CD86 trans-endocytosis by deleting the C terminus of CTLA-4 (CTLA-4 del36). Although CD86 still localized to regions of cell-cell contact (Fig. 1D), wild-type CTLA-4 was much more effective at depleting CD86 than was CTLA-4 del36 (Fig. 1E). As expected, when the CD86-depleted cells from these experiments were used to stimulate T cells, those exposed to wild-type CTLA-4 had impaired stimulatory capacity as compared with those exposed to nonendocytic CTLA-4 (Fig. 1F). Together, these results indicate that by a process of trans-endocytosis, CTLA-4 removes CD86 from neighboring cells, resulting in impaired T cell responses.

To test these observations in a more physiological setting, we activated human CD4+CD25 T cells in the presence of monocyte-derived dendritic cells (DCs) so as to allow the induction of CTLA-4 and looked for evidence of trans-endocytosis. In the absence of T cells, CD86 was robustly expressed on the surface of DCs (Fig. 2A). In the presence of activated T cells, however, CD86 on the plasma membrane of DCs was reduced and instead found in a punctate pattern that colocalized with CTLA-4 (Fig. 2B). Incubation with a blocking antibody to CTLA-4 prevented the down-regulation of CD86 on the DC as well as the appearance of CD86 in CTLA-4+ vesicles in the T cell (Fig. 2, B and C, and fig. S9). Although CD86 was down-regulated in a CTLA-4–dependent manner, expression of other molecules on the DC such as CD40 was unaffected (fig. S9). These results indicated that CTLA-4–mediated trans-endocytosis was specific to CD80 and CD86. To establish whether CTLA-4 was sufficient to confer this function to T cells, we also generated a Jurkat cell line expressing CTLA-4 (fig. S10A). Jurkat cells transduced with CTLA-4 acquired the ability to capture CD86-GFP (fig. S10B). Analysis by means of electron microscopy also showed acquired CD86 in distinct intracellular vesicles within CTLA-4+ Jurkat cells (fig. S10C). The use of a number of blocking reagents, including CTLA-4–immunoglobulin and antibody to CD28, confirmed the specificity of CD86-GFP transfer to Jurkat cells (fig. S11 and fig. S12) and demonstrated that CD28 was not capable of trans-endocytosis. We have previously shown that transfection of resting human T cells with CTLA-4 confers suppressive activity (8). We therefore tested whether this approach also conferred the ability to capture CD86 from antigen-presenting cells (APCs). CTLA-4–transfected (but not mock-transfected) resting T cells exhibited specific sequestration and internalization of CD86, from the DC (Fig. 2, D and E), but had no effect on human leukocyte antigen (HLA)–DR expression. Taken together, these data demonstrated CTLA-4 expression by T cells was sufficient to confer the ability to remove CD86 from DCs.

Fig. 2

Human T cells use CTLA-4 to remove CD86 from DCs. (A) Typical CD86 expression on a human monocyte–derived DC cultured in the absence of T cells. (B) DC cultured for 72 hours with antibody-to-CD3–activated CD4+CD25 T cells (outlined in white) stained with (green) antibody to CD86 and (red) antibody to CTLA-4. Single-color staining is shown as white for equal contrast. Cells were cocultured in the absence or presence of blocking antibody to CTLA-4. (C) Quantitation of surface CD80 and CD86 expression on DCs after coculture with T cells in the presence or absence of antibody to CTLA-4 determined by means of flow cytometry. Data show mean fluorescence intensity (MFI) change pooled from greater than five experiments with SEM. (D) Confocal micrographs of allogeneic DCs cocultured overnight with CTLA-4–transfected (CTLA-4 TF) or control resting CD4+CD25 human T cells. Cultures were fixed and stained with (green) antibody to CD86 or (red) antibody to HLA-DR. White arrowheads highlight position of T cells, and green arrowheads highlight DCs. White images show single-color staining for contrast. (E) MFI of surface CD86 on DCs after incubation with either CTLA-4–transfected or control T cells as determined by means of flow cytometry. All data are representative of at least three independent experiments. Error bars represent the SEM. Scale bars, 10 μM.

Because T cell receptor (TCR) engagement leads to enhanced trafficking of CTLA-4 to and from the plasma membrane (fig. S13, A and B) (2628), we predicted that TCR stimulation should enhance CD86 acquisition. To test this, human CTLA-4+ T cell blasts were incubated in the presence of CD86-GFP–expressing CHO cells with or without antibody to CD3. TCR stimulation increased the acquisition of CD86-GFP in a manner that was blocked by antibody to CTLA-4 and enhanced by bafilomycin (Fig. 3A). Similarly, staphylococcal enterotoxin B (SEB)–reactive T cell blasts incubated with SEB-pulsed DCs also showed enhanced acquisition of CD86 (Fig. 3, B and C).

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 antibody-to-CD3 stimulation. (Right) The effect of antibody to CTLA-4 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 (green) antibody to CD86 and (red) antibody to CTLA-4. 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 16 hours as determined by means of flow cytometry. (D) CD4+CD25+ (Treg cells) or CD4+CD25 T cells were incubated with DCs and antibody to CD3 overnight, fixed, stained with (green) antibody to CD86 and (blue) antibody to CD3 for T cells, and visualized with confocal microscopy. The yellow arrow indicates CD86 puncta within T cells. (E) Surface levels of CD86 on DCs incubated with CD4+CD25+ (Treg cells) and CD4+CD25 T cells overnight determined by means of flow cytometry. All data are representative of at least three independent experiments. Error bars represent the SEM. Scale bars, 10 μM.

We next determined whether CD4+CD25+ regulatory T cells (Treg cells) could acquire CD86 from APCs because CTLA-4 is constitutively expressed on Treg cells and is recycled upon stimulation (fig. S13, C and D). DCs were incubated overnight with purified human CD4+CD25 T cells or CD4+CD25+ Treg cells and antibody to CD3. In the presence of CTLA-4+ Treg cells, CD86 was down-regulated from the APC surface and observed in intracellular puncta inside the Treg cells. In contrast, CD86 remained on the plasma membrane of DC in the presence of CD4+CD25 T cells that lacked CTLA-4 (Fig. 3, D and E). To test whether down-regulation of CD86 by Treg cells affected T cell stimulation, we stimulated Treg cells with DCs in the presence or absence of a blocking antibody to CTLA-4. We then repurified these DCs and used them to stimulate carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled responder T cells. This revealed that blocking CTLA-4 on Treg cells maintained the stimulatory capacity of the DC as compared with where CTLA-4 was available to deplete ligands (fig. S14 A). Moreover, suppression could be overcome by restoring co-stimulation by using CD86-expressing transfectants (fig. S14 B). We also observed that T cell blasts could act as suppressor cells in a CTLA-4–dependent manner (fig. S14 C), again indicating that depletion of costimulatory molecules by CTLA-4 has functional consequences.

Our data suggest a model in which antigen stimulation of either T cells or Treg cells promotes the removal and degradation of CD80 and CD86 from APCs by CTLA-4. To test whether this process could also take place in vivo, we generated a system to study trans-endocytosis in mice. We first established that mouse CD4+ T cells stimulated in vitro could acquire CD86-GFP from CHO cell targets (fig. S15, A and B). We next developed an in vivo protocol (fig. S16) in which DO11.10 TCR transgenic T cells [specific for a peptide fragment of chicken ovalbumin (OVA) presented by I-Ad] were transferred into Balbc Rag2−/− mice that 3 weeks prior had been reconstituted with CD86-GFP–transduced Rag2−/− bone marrow. Recipient mice therefore lacked lymphocytes, except the adoptively transferred DO11.10 T cells, and expressed CD86-GFP on their APCs. Seven days after OVA immunization, mice were rechallenged with OVA peptide in the presence of the lysosomal inhibitor chloroquine. T cells were then harvested and immediately analyzed with confocal imaging. This revealed CD86-GFP in endosomal compartments of antigen-stimulated, but not unstimulated, T cells (Fig. 4A). Moreover, internalized CD86-GFP was restricted to CD4+CD25+ T cells (Fig. 4B), which is consistent with the expression of CTLA-4 by these cells. These cells did not express Foxp3 (fig. S17A, left). The amount of CD86-GFP transfer was extensive because the mean GFP fluorescence inside T cells approached the amounts on donor cells themselves (fig. S17B). To establish the requirement for CTLA-4, we tested CD4+ cells from Ctla4+/+ or Ctla4−/−,Rag2−/− DO11.10 mice bred to mice that express OVA under the control of the rat insulin promotor (Rip-mOVA). These mice were useful because they develop OVA-specific Treg cells, and we have shown that those deficient in CTLA-4 fail to regulate diabetes (13). After in vivo rechallenge with OVA peptide, CD86-GFP acquisition was only observed in CD4+CD25+ T cells capable of CTLA-4 expression but not those from Ctla4−/− mice (Fig. 4C). Moreover, CD4+CD25+ T cells from these mice were almost entirely Foxp3+ (fig. S17A, right). Overall, analysis indicated that approximately 25 to 40% of wild-type CD4+CD25+ T cells acquired ligand (fig. S17C). No transfer of control (unfused) GFP molecules was observed (fig. S17D). Together, these data indicate that both Foxp3+ and Foxp3 T cells are capable of CD86 acquisition in vivo.

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 APCs expressing CD86-GFP. Three weeks later, mice were injected with DO11.10 CD4+ T cells and immunized as described in fig. S16. (A) Six hours after intravenous OVA peptide rechallenge, splenocytes were harvested, labeled at 4°C for (blue) CD4 and (red) CD25, and immediately imaged with 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 rechallenged with OVA in vivo as above. Splenocytes were isolated and immediately analyzed for (green) CD86-GFP, (red) CD25, and (blue) CD4 by use of confocal microscopy. Two representative panels are shown for both Ctla-4+/+ and Ctla-4−/−conditions. Data are representative of three independent experiments. Scale bars, 10 μM.

The CTLA-4 molecule plays a critical role in suppressing autoimmunity and maintaining immune homeostasis; however, its precise mechanism of action has been a subject of debate. Recent data have provided evidence that CTLA-4 can perform a nonredundant effector function for Treg cells, requiring a cell-extrinsic mechanism of action (9, 13). We demonstrated a cell-extrinsic model of CTLA-4 function that operates by the removal of costimulatory ligands from APCs via trans-endocytosis. Using both human and mouse T cells, we established that trans-endocytosis of ligand occurs in precisely the same settings in which we have demonstrated CTLA-4–dependent regulation (8, 13). Moreover, this mechanism is specific for CTLA-4/CD28 ligands and operates in an antigen-dependent manner in vivo. Our data are also compatible with several studies that demonstrate reduced levels of costimulatory ligand expression in the presence of CTLA-4–expressing Treg cells (9, 29, 30) and are consistent with a role for CTLA-4 on both activated and regulatory T cells (9, 31, 32). Taken together, these results not only provide a widely applicable basis for CTLA-4 function but also offer cogent explanations for long-standing paradoxes in the field: namely, how CTLA-4 can function in a cell-extrinsic manner, why CTLA-4 shares ligands with the stimulatory receptor CD28, and why CTLA-4 exhibits endocytic behavior. Although not excluding other mechanisms of CTLA-4 action, we suggest that CTLA-4 carries out the same molecular functions, whether expressed by T cells or by Treg cells—a concept that has considerable implications for our understanding of immune homeostasis. Together, these data provide a new framework for studies of CTLA-4 and should facilitate our understanding of its immunoregulatory role in the key settings of cancer, HIV infection, and autoimmune disease (33, 34).

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1202947/DC1

Materials and Methods

Figs. S1 to S17

References 33 and 34

Movies S1 to S4

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We would like to thank A. Sharpe for the generous gift of Ctla4−/− mice, W. Heath for the rip-mOVA mice, and G. Freeman for the I-Ad–expressing CHO cells. This work was supported by the UK Biotechnology and Biological Sciences Research Council (O.Q.), MRC (L.S.K.W., Y.Z., K.A., E.S., and K.N.), Wellcome Trust (C.M., T.H., and Z.B.), and Arthritis Research UK (L.J.). L.S.K.W. is an MRC Senior Fellow. Cell lines expressing CD80, CD86, and CTLA-4 are available under a material transfer agreement for noncommercial use.
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