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Iris pigment epithelium expressing CD86 (B7-2) directly suppresses T cell activation in vitro via binding to cytotoxic T lymphocyte-associated antigen 4.

Sugita S, Streilein JW - J. Exp. Med. (2003)

Bottom Line: When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation.IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice.Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
A monolayer of pigment epithelium (PE) lines the iris PE (IPE), ciliary body PE, and retina PE of the inner eye, an immune-privileged site. These neural crest-derived epithelial cells participate in ocular immune privilege through poorly defined molecular mechanisms. Murine PE cells cultured from different ocular tissues suppress T cell activation by differing mechanisms. In particular, IPE cells suppress primarily via direct cell to cell contact. By examining surface expression of numerous candidate molecules (tumor necrosis factor receptor [TNFR]1, TNFR2, CD36, CD40, CD47, CD80, CD86, PD-L1, CD95 ligand, and type I interferon receptor), we report that IPE cells uniquely express on their surface the costimulatory molecule CD86. When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation. IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice. We conclude that iris pigment epithelial cells constitutively express cell surface CD86, which enables the cells to contact inhibit T cells via direct interaction with CTLA-4. Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation.

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Influence of transwell membranes on capacity of cultured ocular PE to suppress anti-CD3 T cell activation. PE from C57BL/6 iris (A), ciliary body (B), and retina (C) were grown in 24-well culture plates. Transwell cell culture inserts were placed in these wells, and syngeneic T cells plus anti-CD3 antibodies were placed therein. [3H]thymidine was added 8 h before the termination of culture at 72 h. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence or absence of PE cells are compared. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; NS, not significant.
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fig1: Influence of transwell membranes on capacity of cultured ocular PE to suppress anti-CD3 T cell activation. PE from C57BL/6 iris (A), ciliary body (B), and retina (C) were grown in 24-well culture plates. Transwell cell culture inserts were placed in these wells, and syngeneic T cells plus anti-CD3 antibodies were placed therein. [3H]thymidine was added 8 h before the termination of culture at 72 h. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence or absence of PE cells are compared. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; NS, not significant.

Mentions: The diverse ocular PE cells exist in components of the eye (iris, ciliary body, and retina) with distinct functions, and in each location T cells from the blood pass between adjacent PE cells in monolayers to enter the inner eye. To assay the role of cell to cell contact in PE-dependent suppression of T cell activation, ocular PE cells from iris, ciliary body, and retina of eyes of C57BL/6 mice were cultured separately for 14 d in 24-well culture plates. Transwell inserts were then placed in these wells and the transwell contained C57BL/6 T cells plus anti-CD3, or MLRs composed of responder C57BL/6 T cells and MMC-treated BALB/c spleen cells as stimulators. T cell proliferation was assessed by [3H]thymidine incorporation at 72 (anti-CD3) or 96 h (MLRs). As revealed in Fig. 1, RPE cells suppressed anti–CD3-driven T cell proliferation readily across a transwell membrane, indicating that soluble factors released from the PE cells in the insert were able to diffuse through the membrane and act on the responding T cells in the culture below (Fig. 1 C). By contrast, IPE cells largely failed to suppress T cell proliferation across the transwell membrane, indicating that cell to cell contact is important when IPE mediate suppression of T cell activation (Fig. 1 A). CBPE cells assumed an intermediate suppressing capacity, retaining a modest, but significant, capacity to suppress T cell activation across the transwell membrane (Fig. 1 B). The effects of PE cells on T cell proliferation was similar whether the T cell stimulation was mediated by anti-CD3 antibodies, or by exposure to allogeneic spleen cells (not depicted). Comparable results were obtained when PE cells were harvested from eyes of BALB/c mice (not depicted). These results strongly suggest that cultured IPE cells use a contact-dependent mechanism in suppressing T cell activation. The results do not exclude a similar mechanism for RPE and CBPE, however, because the capacity of these cells to secrete immunosuppressive soluble factors has the potential to eclipse our ability to detect a contact-dependent suppressive mechanism. The finding that CBPE suppressed less well when separated from target T cells by the transwell membrane suggests that CBPE probably use a cell contact inhibitory mechanism in addition to soluble immunosuppressive factors they secrete. The subsequent experiments describe our attempts to identify the interacting cell surface molecules that (a) are differentially expressed on IPE as they suppress T cell activation almost exclusively via a cell contact-dependent mechanism, and (b) are expressed on the inhibited T cells.


Iris pigment epithelium expressing CD86 (B7-2) directly suppresses T cell activation in vitro via binding to cytotoxic T lymphocyte-associated antigen 4.

Sugita S, Streilein JW - J. Exp. Med. (2003)

Influence of transwell membranes on capacity of cultured ocular PE to suppress anti-CD3 T cell activation. PE from C57BL/6 iris (A), ciliary body (B), and retina (C) were grown in 24-well culture plates. Transwell cell culture inserts were placed in these wells, and syngeneic T cells plus anti-CD3 antibodies were placed therein. [3H]thymidine was added 8 h before the termination of culture at 72 h. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence or absence of PE cells are compared. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; NS, not significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196085&req=5

fig1: Influence of transwell membranes on capacity of cultured ocular PE to suppress anti-CD3 T cell activation. PE from C57BL/6 iris (A), ciliary body (B), and retina (C) were grown in 24-well culture plates. Transwell cell culture inserts were placed in these wells, and syngeneic T cells plus anti-CD3 antibodies were placed therein. [3H]thymidine was added 8 h before the termination of culture at 72 h. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence or absence of PE cells are compared. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; NS, not significant.
Mentions: The diverse ocular PE cells exist in components of the eye (iris, ciliary body, and retina) with distinct functions, and in each location T cells from the blood pass between adjacent PE cells in monolayers to enter the inner eye. To assay the role of cell to cell contact in PE-dependent suppression of T cell activation, ocular PE cells from iris, ciliary body, and retina of eyes of C57BL/6 mice were cultured separately for 14 d in 24-well culture plates. Transwell inserts were then placed in these wells and the transwell contained C57BL/6 T cells plus anti-CD3, or MLRs composed of responder C57BL/6 T cells and MMC-treated BALB/c spleen cells as stimulators. T cell proliferation was assessed by [3H]thymidine incorporation at 72 (anti-CD3) or 96 h (MLRs). As revealed in Fig. 1, RPE cells suppressed anti–CD3-driven T cell proliferation readily across a transwell membrane, indicating that soluble factors released from the PE cells in the insert were able to diffuse through the membrane and act on the responding T cells in the culture below (Fig. 1 C). By contrast, IPE cells largely failed to suppress T cell proliferation across the transwell membrane, indicating that cell to cell contact is important when IPE mediate suppression of T cell activation (Fig. 1 A). CBPE cells assumed an intermediate suppressing capacity, retaining a modest, but significant, capacity to suppress T cell activation across the transwell membrane (Fig. 1 B). The effects of PE cells on T cell proliferation was similar whether the T cell stimulation was mediated by anti-CD3 antibodies, or by exposure to allogeneic spleen cells (not depicted). Comparable results were obtained when PE cells were harvested from eyes of BALB/c mice (not depicted). These results strongly suggest that cultured IPE cells use a contact-dependent mechanism in suppressing T cell activation. The results do not exclude a similar mechanism for RPE and CBPE, however, because the capacity of these cells to secrete immunosuppressive soluble factors has the potential to eclipse our ability to detect a contact-dependent suppressive mechanism. The finding that CBPE suppressed less well when separated from target T cells by the transwell membrane suggests that CBPE probably use a cell contact inhibitory mechanism in addition to soluble immunosuppressive factors they secrete. The subsequent experiments describe our attempts to identify the interacting cell surface molecules that (a) are differentially expressed on IPE as they suppress T cell activation almost exclusively via a cell contact-dependent mechanism, and (b) are expressed on the inhibited T cells.

Bottom Line: When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation.IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice.Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
A monolayer of pigment epithelium (PE) lines the iris PE (IPE), ciliary body PE, and retina PE of the inner eye, an immune-privileged site. These neural crest-derived epithelial cells participate in ocular immune privilege through poorly defined molecular mechanisms. Murine PE cells cultured from different ocular tissues suppress T cell activation by differing mechanisms. In particular, IPE cells suppress primarily via direct cell to cell contact. By examining surface expression of numerous candidate molecules (tumor necrosis factor receptor [TNFR]1, TNFR2, CD36, CD40, CD47, CD80, CD86, PD-L1, CD95 ligand, and type I interferon receptor), we report that IPE cells uniquely express on their surface the costimulatory molecule CD86. When IPE were blocked with anti-CD86 or were derived from CD80/CD86 (but not CD80) knockout (KO) mice, the cells displayed reduced capacity to suppress T cell activation. IPE also failed to suppress activation of T cells in the presence of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immunoglobulin or if the T cells were obtained from CTLA-4 (but not CD28) KO mice. We conclude that iris pigment epithelial cells constitutively express cell surface CD86, which enables the cells to contact inhibit T cells via direct interaction with CTLA-4. Thus, ocular immune privilege is achieved in part by subversion of molecules that are usually used for conventional immune costimulation.

Show MeSH
Related in: MedlinePlus