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. 2003 Jul 7;198(1):161-71.
doi: 10.1084/jem.20030097. Epub 2003 Jun 30.

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

Sunao Sugita  1 , J Wayne Streilein
Affiliations

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

Sunao Sugita et al. J Exp Med. .

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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Figures

Figure 1.
Figure 1.
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.
Figure 2.
Figure 2.
Survey of gene expression of candidate-suppressive cell surface molecules on PE cells cultured from iris, ciliary body, and retina. mRNA was extracted from IPE, CBPE, and RPE cells after 14 d of culture, reverse transcribed, and amplified by PCR. PCR products were electrophoresed in 1, 1.5, and 2% agarose gel and visualized by staining with ethidium bromide.
Figure 3.
Figure 3.
Capacity of neutralizing antibodies to CD80 and CD86 to prevent suppression of T cell activation by cultured PE cells. Anti-CD3 stimulation of purified C57BL/6 T cells was performed as described in the legend to Fig. 1. Cultured IPE (A), CBPE (B), or RPE cells (C) were added in the presence or absence of 1 μg/ml anti-CD80 and/or anti-CD86 antibodies. As isotype controls, purified hamster IgG for CD80 and purified rat IgG for CD86 were used. After 72 h of incubation, the cultures were assayed for uptake of [3H]thymidine. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence of PE cells with and without anti-CD80 or anti-CD86 antibodies are compared. *, P < 0.05; **, P < 0.005.
Figure 4.
Figure 4.
Detection of B7 molecules on cultured PE cells by flow cytometry. IPE (BALB/c and C57BL/6), CBPE (C57BL/6), and RPE cells (C57BL/6) were cultured on 6-well plates for 14 d. Thereafter, the confluent cells were triturated several times through 21-gauge and then 23-gauge needles to create a single cell suspension. PE cells were incubated with FITC-conjugated antibodies: anti-CD80, anti-CD86, or purified hamster IgG as isotype control for 1 h at room temperature. After washing and fixation, the cells were analyzed by flow cytometry. Percentages in upper right corners indicate positive cells. Number in parenthesis indicate mean fluorescence index.
Figure 5.
Figure 5.
Detection of mRNA expression of costimulatory molecules in ocular PE. mRNA, extracted from cultured IPE, CBPE, and RPE cells with or without 100 U/ml recombinant IFN-γ (samples 1–6), and from freshly obtained mouse iris, ciliary body, and retina (samples 7–9), was reverse transcribed and amplified by PCR using primers for (A) CD80 (B7-1), (B) CD86 (B7-2), (C) PD-L1 (B7-H1), (D) CD40, and (E) GAPDH. PCR products were electrophoresed in 1.5% agarose gel and visualized by staining with ethidium bromide.
Figure 6.
Figure 6.
Capacity of cultured PE cells from donors deficient in CD80 plus CD86 expression to inhibit T cell proliferation. Purified C57BL/6 T cells (2.5 × 105 cells/well) were stimulated with 1 μg/ml anti-CD3 antibody and cultured for 72 h in the presence or absence of cultured IPE (A), CBPE (B), or RPE (C) obtained from eyes of CD80/CD86 KO or wild-type (C57BL/6) mice. After 72 h, the cultures were assayed for uptake of [3H]thymidine. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence of wild-type or CD80/CD86 KO PE cells are compared. **, P < 0.005, compared with two groups. NS, not significant.
Figure 7.
Figure 7.
Influence of CTLA-4–Ig on suppression of T cell activation by ocular PE. PE cells were cultured from iris, ciliary body, and retina of C57BL/6 mice. IPE served as substrates for purified T cells stimulated with anti-CD3 antibodies in the presence of varying concentrations of CTLA-4–Ig. Cultures were terminated at 72 h and [3H]thymidine incorporation was assessed. Mean counts per minute for triplicate cultures are presented ± SEM. Differences of means of T cell proliferation in the presence or absence of CTLA-4–Ig are compared. *, P < 0.05.
Figure 8.
Figure 8.
Capacity of ocular PE to suppress activation of T cells from mice with disrupted CD28 and CTLA-4 (CD152) genes. PE were cultured from iris, ciliary body, and retina of C57BL/6 mice and used as substrates for purified T cells prepared from the spleens of 22-d-old wild-type mice, 22-d-old CTLA-4−/− mice, and 6-wk-old CD28−/− mice. The T cells were stimulated with anti-CD3 antibodies at concentrations of (A) 0.5 and (B) 1.0 μg/ml. Cultures were terminated at 72 h and [3H]thymidine incorporation was assessed. 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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