December 2009
Volume 50, Issue 12
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Immunology and Microbiology  |   December 2009
Regulation of Interphotoreceptor Retinoid-Binding Protein (IRBP)-Specific Th1 and Th17 Cells in Anterior Chamber-Associated Immune Deviation (ACAID)
Author Affiliations & Notes
  • Yan Cui
    From the Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky; and
    the Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California.
  • Hui Shao
    From the Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky; and
  • Deming Sun
    From the Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky; and
    the Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California.
  • Henry J. Kaplan
    From the Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky; and
  • Corresponding author: Henry J. Kaplan, Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202; hank.kaplan@louisville.edu
Investigative Ophthalmology & Visual Science December 2009, Vol.50, 5811-5817. doi:10.1167/iovs.09-3389
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      Yan Cui, Hui Shao, Deming Sun, Henry J. Kaplan; Regulation of Interphotoreceptor Retinoid-Binding Protein (IRBP)-Specific Th1 and Th17 Cells in Anterior Chamber-Associated Immune Deviation (ACAID). Invest. Ophthalmol. Vis. Sci. 2009;50(12):5811-5817. doi: 10.1167/iovs.09-3389.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Intracameral (anterior chamber) injection of antigen inhibits the development of delayed-type hypersensitivity, a phenomenon known as anterior chamber-associated immune deviation (ACAID). The authors investigated the effect of intracameral injection of interphotoreceptor retinoid-binding protein (IRPB) peptides on the development of IFN-γ+ and IL-17+ pathogenic T cells.

Methods.: A uveitogenic (IRBP1–20) or nonuveitogenic (IRBP161–180) peptide was injected into the anterior chamber (AC) of B6 mice. Seven days later, the mice were primed with a pathogenic dose of IRBP1–20 in adjuvant. Thirteen days later, the pathogenic activity of the T cells isolated from the spleens of treated and untreated mice were compared, and the numbers of Th1 and Th17 T cells were assessed by intracellular staining. Regulatory T-cell activity was assessed by antibody staining and functional assays. The authors also compared the effect of inhibition on EAU of ocular injection to various sites, including the AC, the vitreous cavity, and the subretinal space.

Results.: Intraocular injection of the uveitogenic peptide (IRBP1–20), but not the nonuveitogenic peptide (IRBP161–180), inhibited the generation of IFN-γ+ and IL-17+ uveitogenic T cells and the development of experimental autoimmune uveitis (EAU). AC administration of IRBP1–20, but not IRBP161–180, significantly decreased the number of circulating γδ T cells after subsequent systemic immunization with IRBP1–20. Absence of the γδ T-cell population prohibited the development of ACAID.

Conclusions.: Injection of a uveitogenic peptide into the AC inhibited the development of EAU by regulation of Th1 and Th17 IRBP-specific T cells. The circulating γδ T-cell population was reduced and was associated with decreased activation of IL-17+ uveitogenic T cells.

Antigen injection into the anterior chamber (AC) of the eye, before systemic administration of an immunogenic dose, inhibits the development of delayed-type hypersensitivity (DTH) to the immunizing antigen. 19 This immunologic phenomenon has been termed anterior chamber-associated immune deviation (ACAID). The characteristics of ACAID are the suppression of DTH by antigen-specific CD4+ Th1 cells, the inhibition of priming of antigen-specific CD8+ cytotoxic T-lymphocytes (CTLs), and the preservation of noncomplement fixing antigen-specific antibody responses. 10 Previous studies have shown that AC injection of an encephalitogenic or uveitogenic peptide can impair the development of autoimmune disease in the relevant organ. 1,2  
A newly identified autoreactive T cell subset expressing IL-17 has been identified as a major pathogenic T-cell subset. 1114 Thus, we wanted to determine whether ACAID induced by an immunogenic peptide would affect the Th1- and Th17- autoreactive T-cell subsets. We pretreated EAU-prone B6 mice with the uveitogenic peptide IRBP1–20 to determine whether ACAID is associated with decreased function of IFN-γ+ and IL-17+ pathogenic T cells. We also explored whether the generation of IL-17+ uveitogenic T cells was different in mice pretreated with the nonuveitogenic peptide IRBP161–180. We observed that the AC injection of the uveitogenic, but not the nonuveitogenic, antigen inhibited the development of EAU. We also observed that ACAID induced in mice with the uveitogenic peptide had decreased function of IFN-γ+ and IL-17+ uveitogenic T cells, with the latter affected more significantly. The inhibition of these effector T cells was mediated by increased regulatory T-cell activity. 
Methods
Animals and Reagents
Pathogen-free female C57BL/6 (B6) mice (age range, 12–14 weeks) were purchased from Jackson Laboratory (Bar Harbor, ME) and were housed and maintained in the animal facilities of the University of Louisville. All animal studies conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Institutional approval was obtained, and institutional guidelines regarding animal experimentation were followed. Recombinant murine IL-2 and IL-23 were purchased from R&D Systems (Minneapolis, MN). FITC-conjugated anti–IFN-γ and anti–IL-17 antibody was purchased from Biolegend (San Diego, CA); anti–mouse FoxP3 antibody was obtained from eBioscience (San Diego, CA); all other antibodies were from BD Bioscience (La Jolla, CA). IRBP1–20 and IRBP161–180 peptides and complete Freund's adjuvant were obtained from Sigma (St. Louis, MO). IRBP1–20, IRBP161–180, and myelin oligodendrocyte glycoprotein (MOG)35–55 were synthesized by Sigma. The buffered aqueous glycerol solution of pertussis toxin from Bordetella was purchased from Sigma-Aldrich Inc. (St. Louis, MO). GL3 is a monoclonal antibody specific for the mouse TCR δ chain. The hybridoma cell line producing GL3 antibody was kindly provided by Leo Lefrançois (University of Connecticut Health Center, Farmington, CT). 
Intraocular Inoculations
Mice were anesthetized by intraperitoneal injection of 100 μL of a mixture containing 10 mg/mL ketamine (Sigma) and 2 mg/mL xylazine (Bayer Corp., Shawnee Mission, KS). One drop of proparacaine HCl (Alcon Inc., Humacao, Puerto Rico) was applied topically on the eye before injection. Under a dissecting microscope, 50 μg IRBP1–20, IRBP161–180, or MOG35–55 in 2 μL phosphate-buffered saline (PBS) was injected into the AC, vitreous cavity, or subretinal space of one eye with a microliter syringe and a 33-gauge needle (Hamilton, Reno, NV). 
Preparation of IRBP1–20–Specific T Cells
Briefly, B6 mice were immunized subcutaneously with 150 μL emulsion containing 200 μg IRBP1–20 in complete Freund's adjuvant (CFA), distributed over six spots at the tail base and on the flank with a single injection of PTX (200 ng/mouse, intraperitoneally). At day 13 after immunization, T cells were isolated from lymph node cells and spleen cells by passage through a nylon wool column. Then 1 × 107 cells in 2 mL RPMI medium in a six-well plate (Costar; Corning, Corning, NY) were stimulated for 48 hours with 10 μg/mL IRBP1–20 in the presence of 1 × 107 irradiated syngeneic spleen cells (antigen-presenting cells [APCs]) in the presence of IL-2 or IL-23 (10 ng/mL). The activated T-cell blasts were separated by Ficoll gradient centrifugation and cultured for another 72 hours in the same medium used for stimulation minus the peptide. 
Detection of Regulatory T-Cell Activity That Inhibits EAU
B6 mice were separated into three groups. EAU was induced by adoptive transfer of 2 × 106 IRBP-specific T cells. Splenic T cells (3 × 107) harvested from mice injected 7 days earlier in the AC with IRBP1–20 or IRBP161–180, or left untreated, were injected intraperitoneally together with the uveitogenic T cells. EAU was scored using clinical examination on days 9, 12, and 15 after EAU induction. 
Scoring of EAU
The mice were examined three times a week for clinical signs of EAU by indirect funduscopy. Pupils were dilated with 0.5% tropicamide and 1.25% phenylephrine hydrochloride ophthalmic solutions. Grading of disease was performed according to the scoring system described previously. 3 For histopathologic evaluation, whole eyes were collected at the end of the experiment and were immersed for 1 hour in 4% glutaraldehyde in phosphate buffer, pH 7.4, then transferred to 10% formaldehyde in phosphate buffer until processed. The fixed and dehydrated tissues were embedded in methacrylate, and 5-μm sections were cut through the pupillary-optic nerve plane and stained with hematoxylin and eosin. The presence or absence of disease was evaluated in a masked fashion by examination of six sections cut at different levels for each eye. Disease was graded depending on the cellular infiltration and structural changes. 4  
Immunofluorescence Flow Cytometry
Aliquots of 2 × 105 cells were double-stained with combinations of FITC- or PE-conjugated monoclonal antibodies against mouse αβTCR, γδTCR, CD4, CD8, CD25, and CD122. Data collection and analysis were performed on a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ) with companion software (CellQuest; BD Biosciences). 
Intracellular Cytokine Flow Cytometry
IRBP1–20–specific T cells were stimulated in vitro with 50 ng/mL phorbol 12-myristatet 13-acetate, 1 μg/mL ionomycin, and 1 μg/mL brefeldin A (Sigma) for 4 hours and then were washed, fixed, permeabilized overnight with buffer (Cytofix/Cytoperm; eBioscience, San Diego, CA), intracellularly stained with antibodies against IFN-γ and IL-17, and analyzed on a flow cytometer (FACSCalibur; BD Biosciences). 
ELISA
IL-17 and IFN-γ were measured with commercially available ELISA kits (R&D Systems). 
Statistical Analysis
Data are expressed as the mean ± SD of the results for at least three separate experiments. Statistical analyses were performed using Student's t-test. P ≤ 0.05 was considered statistically significant. 
Results
IRBP1–20 Peptide Inhibited the Development of Experimental Autoimmune Uveitis
First, we wanted to confirm that the AC injection of a uveitogenic antigen would inhibit the development of experimental autoimmune uveitis (EAU) to IRBP. B6 mice were divided into three groups and injected in the AC with the uveitogenic peptide IRBP1–20, the nonuveitogenic peptide IRBP161–180, or the encephalitogenic peptide MOG35–55. Seven days later the mice were immunized with a pathogenic dose (200 μg/mouse) of the IRBP1–20 peptide in CFA. To test the pathogenic activity of the IRBP-specific T cells, in vivo–primed T cells were stimulated with the immunizing antigen and APC, and 2 × 106 in vitro activated IRBP-specific T cells from each group were adoptively transferred to naive B6 mice. The development of EAU was monitored by clinical examination and histology. As we previously reported, 5 most IRBP-specific T cells isolated from the B6 mouse immunized with the uveitogenic peptide IRBP1–20 expressed IFN-γ; only a small portion of the cells expressed IL-17 when the in vivo–primed T cells were cultured in IL-2–containing medium (Th1 polarized) after in vitro stimulation with the immunizing peptide. However, the IFN-γIL-17+ IRBP-specific T cells became dominant when the in vivo–primed T cells were stimulated in vitro in the presence of IL-23 (Th17 polarized). As noted in Figure 1(A–E), the pathogenic activity of the IRBP-specific T cells was significantly decreased only in mice injected in the AC with the uveitogenic IRBP1–20 peptide but not with the nonuveitogenic IRBP161–180 or irrelevant MOG 35–55 peptides. Furthermore, in vitro proliferation of the IRBP-specific T cells was significantly inhibited only after AC injection of IRBP1–20 (Fig. 1F). 
Figure 1.
 
Injection of the uveitogenic peptide IRBP1–20 into the AC of B6 mice inhibited the generation and expansion of IRBP-specific T cells. (AC) Groups (n = 3) of B6 mice were injected in the AC with a tolerogenic dose (50 μg) of a uveitogenic (IRBP1–20), a nonuveitogenic (IRBP161–180), or an irrelevant peptide (MOG35–55). Seven days later all the animals were subcutaneously immunized with the uveitogenic peptide IRBP1–20. Thirteen days after immunization, IRBP-specific T cells were isolated from each group of mice and tested for pathogenic activity by adoptive transfer into naive B6 mice. Results showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 failed to induce EAU, whereas those from donor mice injected in the AC with either the nonuveitogenic (IRBP161–180) or the encephalitogenic (MOG35–55) peptide retained the ability to transfer EAU. (D, E) Results of clinical examination agreed with pathology findings and showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased ability to induce EAU. This was observed whether the uveitogenic IRBP-specific T cells were activated in the presence of IL-2 (D), which favors the activation of Th1 uveitogenic T cells, or in the presence of IL-23 (E), which favors the activation of Th17 uveitogenic T cells. (F) Proliferation assay showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased proliferative ability in vitro when exposed to the immunizing peptide IRBP1–20. Results shown are representative of those in three experiments.
Figure 1.
 
Injection of the uveitogenic peptide IRBP1–20 into the AC of B6 mice inhibited the generation and expansion of IRBP-specific T cells. (AC) Groups (n = 3) of B6 mice were injected in the AC with a tolerogenic dose (50 μg) of a uveitogenic (IRBP1–20), a nonuveitogenic (IRBP161–180), or an irrelevant peptide (MOG35–55). Seven days later all the animals were subcutaneously immunized with the uveitogenic peptide IRBP1–20. Thirteen days after immunization, IRBP-specific T cells were isolated from each group of mice and tested for pathogenic activity by adoptive transfer into naive B6 mice. Results showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 failed to induce EAU, whereas those from donor mice injected in the AC with either the nonuveitogenic (IRBP161–180) or the encephalitogenic (MOG35–55) peptide retained the ability to transfer EAU. (D, E) Results of clinical examination agreed with pathology findings and showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased ability to induce EAU. This was observed whether the uveitogenic IRBP-specific T cells were activated in the presence of IL-2 (D), which favors the activation of Th1 uveitogenic T cells, or in the presence of IL-23 (E), which favors the activation of Th17 uveitogenic T cells. (F) Proliferation assay showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased proliferative ability in vitro when exposed to the immunizing peptide IRBP1–20. Results shown are representative of those in three experiments.
IRBP1–20 Inhibited the Generation of IFN-γ+ and IL-17+ IRBP-Specific T Cells
We have previously shown that IRPB-specific T cells expressing IFN-γ or IL-17 are uveitogenic. 5 In this study, we investigated whether one of the uveitogenic T-cell subsets was preferentially affected by AC injection of the uveitogenic peptide. A protocol identical with that described was used to harvest lymph node and spleen T cells 13 days after immunization. The T cells were cocultured with syngeneic APC (irradiated spleen cells) in the presence of the immunizing peptide and either 10 ng/mL exogenous IL-2 for activation and expansion of the Th1-type uveitogenic T cells or IL-23 for the Th17-type uveitogenic T cells. 5 As demonstrated in Figure 2, mice injected in the AC with the uveitogenic IRBP1–20 peptide generated significantly fewer (<50%) IL-17+ IRBP-specific T cells than mice that only received systemic immunization with IRBP1–20. The number of IFN-γ+ T cells was also decreased, but to a lesser extent. 
Figure 2.
 
AC injection of the uveitogenic peptide IRBP1–20 inhibited the generation of IFN-γ+ and IL-17+ IRBP-specific T cells. IRBP-specific T cells isolated from ACAID or control donor mice (without AC injection) were assessed for IFN-γ or IL-17 expression by intracellular staining (A, B), production of IFN-γ or IL-17 (C, D), and ability to induce EAU (E, F). Results, which are representative of three experiments, showed that IRBP-specific T cells isolated from the donor mice injected in the AC with IRBP1–20 generated significantly lower numbers of IL-17+ and IFN-γ+ IRBP-specific T cells (A, B), produced decreased amounts of IFN-γ and IL-17 as assessed by ELISA (C, D), and failed to induce EAU on transfer to naive recipients (E) compared with IRBP-specific T cells isolated from control mice (F).
Figure 2.
 
AC injection of the uveitogenic peptide IRBP1–20 inhibited the generation of IFN-γ+ and IL-17+ IRBP-specific T cells. IRBP-specific T cells isolated from ACAID or control donor mice (without AC injection) were assessed for IFN-γ or IL-17 expression by intracellular staining (A, B), production of IFN-γ or IL-17 (C, D), and ability to induce EAU (E, F). Results, which are representative of three experiments, showed that IRBP-specific T cells isolated from the donor mice injected in the AC with IRBP1–20 generated significantly lower numbers of IL-17+ and IFN-γ+ IRBP-specific T cells (A, B), produced decreased amounts of IFN-γ and IL-17 as assessed by ELISA (C, D), and failed to induce EAU on transfer to naive recipients (E) compared with IRBP-specific T cells isolated from control mice (F).
Induction of Ocular Tolerance Was Most Effective after Injection into the Anterior Chamber
Although the eye is an immunologically privileged site and ocular tolerance has been observed after the intraocular injection of antigen into the AC, the vitreous cavity, or the subretinal space, we wanted to compare the effect of inhibition on EAU in the various sites. As observed in Figure 3A, only AC injection of antigen resulted in the marked inhibition of EAU. This was associated with a more significant decrease in the IL-17+ than the IFN-γ+ IRBP-specific T-cell subset (Fig. 3B). However, the T-cell proliferative response to the peptide was reduced by injection into each of the three compartments (Fig. 3C). 
Figure 3.
 
AC injection of IRBP1–20 was more effective than intravitreal or subretinal injection. Four groups of B6 mice (n = 3) were injected with tolerogenic doses of IRBP1–20 in the AC, intravitreally (VCAID) or subretinally. Mice in the control group were untreated. Seven days after intraocular injection, all the mice were immunized with the uveitogenic peptide IRBP1–20 emulsified in CFA. Thirteen days after immunization, IRBP-specific T cells were isolated from the mice in each group and were activated in vitro by stimulation with the immunizing peptide and APCs. Pathogenic activity of the IRPB-specific T cells was determined by (A) adoptive transfer to naive syngeneic recipients, (B) number of IFN-γ+ and IL-17+ IRPB-specific T cells by intracellular staining, and (C) proliferative response of the T cells by in vitro proliferation to the immunizing antigen. Results showed that the AC injection of the tolerogenic antigen IRBP1–20 was most effective in the inhibition of EAU, in decreasing the number of IL-17+ IRBP-specific T cells, and in decreasing the proliferative response of such T cells.
Figure 3.
 
AC injection of IRBP1–20 was more effective than intravitreal or subretinal injection. Four groups of B6 mice (n = 3) were injected with tolerogenic doses of IRBP1–20 in the AC, intravitreally (VCAID) or subretinally. Mice in the control group were untreated. Seven days after intraocular injection, all the mice were immunized with the uveitogenic peptide IRBP1–20 emulsified in CFA. Thirteen days after immunization, IRBP-specific T cells were isolated from the mice in each group and were activated in vitro by stimulation with the immunizing peptide and APCs. Pathogenic activity of the IRPB-specific T cells was determined by (A) adoptive transfer to naive syngeneic recipients, (B) number of IFN-γ+ and IL-17+ IRPB-specific T cells by intracellular staining, and (C) proliferative response of the T cells by in vitro proliferation to the immunizing antigen. Results showed that the AC injection of the tolerogenic antigen IRBP1–20 was most effective in the inhibition of EAU, in decreasing the number of IL-17+ IRBP-specific T cells, and in decreasing the proliferative response of such T cells.
Antigen-Specific Regulatory T Cells Mediated the Inhibition of EAU
ACAID after intraocular injection of antigen can be caused by a decreased number of effector T cells, increased regulatory T-cell activity, or both. Therefore, we separated B6 mice into two groups. Mice in group 1 were AC injected with PBS, and mice in group 2 were injected with the uveitogenic peptide IRBP1–20. Seven days later, all mice were immunized with IRBP1–20. T cells were isolated from the immunized mice on day 13 and stimulated in vitro with IRBP1–20 and APCs (irradiated spleen cells) for 3 days. The cells were then separated by Ficoll gradient centrifugation and were stained intracellularly with Foxp3 or surface stained with antibodies to CD8, CD4, CD25, and CD122. As seen in Fig. 4A, in mice injected in the AC with IRBP1–20, the number of CD4+CD25+, CD4+Foxp3+, and CD8+CD122+ T cells was slightly increased compared to the controls. Additionally, the difference in regulatory T-cell subsets between the groups of mice injected in the AC with specific (IRBP1–20) and nonspecific (MOG and IRBP161–180) uveitogenic antigen was not statistically significant (data not shown). Only the splenic T cells of mice that were AC injected with the uveitogenic, but not the nonuveitogenic, peptide inhibited the development of EAU when adoptively transferred to naive mice (Fig. 4B). 
Figure 4.
 
Assessment of regulatory T-cell activity. (A) Regulatory T cells were increased in mice injected in the AC with IRBP1–20. Control B6 mice and mice injected in the AC with the uveitogenic peptide IRBP1–20 were analyzed for the number of CD25+, Foxp3+, and CD8+CD122+ cells in splenic T cells by FACS. (B) Regulatory T cells from mice injected with IRBP1–20 inhibited the induction of EAU. B6 mice were separated into three groups (n = 3), with EAU induced by adoptive transfer of 2 × 106 IRBP-specific T cells. Splenic T cells (3 × 107) harvested from mice injected in the AC with IRBP1–20 (group 1), IRBP161–180 (group 2), or untreated (group 3) were then injected intraperitoneally. EAU was scored using clinical examination on days 9, 12, and 15 after EAU induction. *P < 0.05; **P < 0.01.
Figure 4.
 
Assessment of regulatory T-cell activity. (A) Regulatory T cells were increased in mice injected in the AC with IRBP1–20. Control B6 mice and mice injected in the AC with the uveitogenic peptide IRBP1–20 were analyzed for the number of CD25+, Foxp3+, and CD8+CD122+ cells in splenic T cells by FACS. (B) Regulatory T cells from mice injected with IRBP1–20 inhibited the induction of EAU. B6 mice were separated into three groups (n = 3), with EAU induced by adoptive transfer of 2 × 106 IRBP-specific T cells. Splenic T cells (3 × 107) harvested from mice injected in the AC with IRBP1–20 (group 1), IRBP161–180 (group 2), or untreated (group 3) were then injected intraperitoneally. EAU was scored using clinical examination on days 9, 12, and 15 after EAU induction. *P < 0.05; **P < 0.01.
Induction of ACAID Was Dependent on the γδ T Cell
Our recent studies suggest that the γδ T cell plays a major role in regulating the autoreactive T cells expressing IL-17 and in the intensity of the induced uveitis (DS, manuscript in preparation). Therefore, we studied whether the induction of ACAID was dependent on γδ T-cell function. We injected the uveitogenic peptide IRBP1–20 into the AC of three groups of mice: wt-B6 mice, γδ TCR−/− mice, and B6 mice preinjected with an antibody specific for the TCR δ chain segments. All the mice were immunized with IRBP1–20, and isolated T cells were tested for uveitogenic activity by adoptive transfer into naive syngeneic B6 mice (Fig. 5). As seen in Figure 5A, though IRBP-specific T cells isolated from control B6 mice induced EAU, those from mice injected in the AC with IRBP1–20 failed to develop disease (Fig. 5B). Furthermore, IRBP-specific T cells obtained from γδTCR−/− or B6 mice pretreated with the GL3 antibody also developed EAU (Figs. 5C, D), suggesting that absence of the γδ T-cell population prevented the development of ACAID after AC injection of IRBP1–20. We also observed that the AC administration of IRBP1–20 significantly decreased the number of circulating γδ T cells after subsequent systemic immunization with IRBP1–20 (Fig. 5E), compared with the nonuveitogenic peptide IRBP161–180 (Fig. 5F) and the control (Fig. 5G). Thus, it appeared that the development of ACAID was dependent on the participation of a functional γδ T-cell population. 
Figure 5.
 
ACAID did not develop in γδ T-cell deficient mice. (AD) Four groups of mice (n = 6) were studied for the induction of ACAID. Control (A), wt-B6 (B), γδ TCR-KO (C), and mice treated with GL3 (D). All mice, except the control, were injected in the AC with IRBP1–20 and then were systemically immunized 7 days later with IRBP1–20 in CFA. Control B6 mice developed EAU (A), ACAID-induced B6 mice resisted induction of EAU (B), γδTCR-KO mice (C) and B6 mice treated with a TCR δ chain-specific antibody (GL3) (D) did not inhibit the development of EAU. (EG) Anterior chamber injection resulted in a decreased number of γδ T cells. Groups of B6 mice (n = 3) were injected in the AC with the uveitogenic IRBP1–20 peptide (E), the nonuveitogenic IRBP161–180 peptide (50 μg/each) (F), or were untreated (G). Seven days later, all the mice were immunized with IRPB1–20. Thirteen days after immunization, splenic T cells were isolated, and the number of γδ T cells was identified by staining with specific antibody and FACS analysis.
Figure 5.
 
ACAID did not develop in γδ T-cell deficient mice. (AD) Four groups of mice (n = 6) were studied for the induction of ACAID. Control (A), wt-B6 (B), γδ TCR-KO (C), and mice treated with GL3 (D). All mice, except the control, were injected in the AC with IRBP1–20 and then were systemically immunized 7 days later with IRBP1–20 in CFA. Control B6 mice developed EAU (A), ACAID-induced B6 mice resisted induction of EAU (B), γδTCR-KO mice (C) and B6 mice treated with a TCR δ chain-specific antibody (GL3) (D) did not inhibit the development of EAU. (EG) Anterior chamber injection resulted in a decreased number of γδ T cells. Groups of B6 mice (n = 3) were injected in the AC with the uveitogenic IRBP1–20 peptide (E), the nonuveitogenic IRBP161–180 peptide (50 μg/each) (F), or were untreated (G). Seven days later, all the mice were immunized with IRPB1–20. Thirteen days after immunization, splenic T cells were isolated, and the number of γδ T cells was identified by staining with specific antibody and FACS analysis.
Discussion
The intracameral injection of an antigen, before systemic immunization with an immunogenic dose, results in a deviant immune response termed ACAID. 68 The immunologic characteristics of ACAID include an inhibited antigen-specific DTH response, a suppressed cytotoxic T-cell response, and an intact noncomplement fixing antibody response. 8 In previous studies ACAID has been induced by the intracameral injection of many antigens, including soluble proteins (e.g., ovalbumin), 811 surface alloantigens, hapten-derivatized cell surface molecules, 7,12 and virus-encoded antigens. In this study we explored the mechanism by which the intraocular injection of a uveitogenic autoantigen (IRBP1–20) induced immune suppression. 
Recent studies have discovered a new subset of autoreactive T cells that secrete IL-17. 1315 Although the immunologic mechanisms responsible for ACAID have been studied in detail, the recent recognition that the pathogenesis of autoimmune uveitis involves a subset of T cells producing IL-17 3 has raised the question whether intraocular tolerance affected both IFN-γ+ and IL-17+ uveitogenic T cells or whether there was a preference for one of the pathogenic T-cell subsets. Our results showed that ACAID to IRBP1–20 significantly suppressed IFN-γ+ and IL-17+ IRBP-specific T cells but that the latter were suppressed more severely. Furthermore, the IRBP-specific T cells isolated from mice injected in the AC with IRPB1–20 had greatly decreased pathogenic activity. 
We also observed that only mice injected with a uveitogenic peptide in the AC generated a sufficient number of regulatory T cells to protect the recipients from EAU and that they had increased numbers of CD25+, FoxP3+, and CD8+CD122+ regulatory T cells. Functional tests demonstrated that only T cells obtained from mice injected in the AC with the uveitogenic peptide suppressed EAU. One plausible interpretation of these results is that many different antigens injected into the anterior chamber can induce regulatory T-cell activity; however, most of these regulatory T cells are functionally nonspecific. Only mice injected in the AC with an immunogenic peptide, and subsequently systemically immunized with the same antigen, generate a strong antigen-specific regulatory T-cell response. These T cells are functionally more effective than antigen-nonspecific regulatory T cells in inhibiting autoimmune disease. It is likely that these antigen-specific regulatory T cells constitute only a small fraction of the total regulatory T-cell population. Thus, it is difficult to experimentally distinguish between antigen-specific and antigen-nonspecific regulatory T cells other than by disease inhibitory activity. 
Our previous studies have shown that the activation and expansion of IL-17+ uveitogenic T cells in B6 mice is associated with an expansion of the γδ T-cell population and that the depletion of γδ T cells significantly suppressed the response of IL-17+ uveitogenic T cells (unpublished observations, 2008). In this study, we observed that intraocular injection of the uveitogenic peptide significantly decreased γδ T-cell expansion and activation after systemic immunization with the same peptide. Furthermore, this decrease in the γδ T-cell population was associated with decreased activation of IL-17+ uveitogenic T cells. Other investigators 16,17 have reported that the number of circulating γδ T cells was increased in ACAID, whereas we have observed the opposite effect. It is likely that difference in our experimental systems is responsible for the different results. We studied the number of γδ T cells after intraocular injection, followed by systemic immunization with the same antigen, whereas the previous studies examined the γδ T-cell population solely after intraocular injection. Nevertheless, studies suggest that intraocular immunization induces systemic tolerance through involvement of innate and adaptive immunity. 
Finally, we compared the effect of injecting the uveitogenic peptide in the various intraocular compartments on the subsequent inhibition of EAU. We observed that injection into the AC was more effective than into either the vitreous cavity or the subretinal space in the suppression of EAU. However, all three compartments did result in a decreased proliferative response to the immunogenic peptide. 
The control and regulation of autoimmune uveitis may involve cells of the innate and adaptive immune response. We have previously reported that there is an increased infiltration of NKT cells in the eye-infiltrating cells in EAU. 18 More recently, Grajewski et al. 19 showed that iNKT cells suppressed EAU by innate IFN-γ production and dampening of the TH1 and TH17 responses. Given that iNKT cells are required for ACAID induction, they may very well complement the regulatory activity of T cells in protection from autoimmune uveitis. 
In summary, our studies have demonstrated that the systemic immune response after intraocular injection of a uveitogenic peptide, compared with a nonuveitogenic peptide, is distinctly different. The induction of EAU is markedly inhibited after intracameral inoculation of the uveitogenic peptide. Although IFN-γ+ and IL-17+ uveitogenic T cells are inhibited, the latter are inhibited more effectively. Finally, the inhibition of autoimmune disease after intracameral inoculation is associated with a significant depletion of γδ T cells. We suggest that the induction of an increased number of antigen-specific regulatory T cells after intraocular inoculation of the uveitogenic peptide contributed to the inhibition of EAU. 
Footnotes
 Supported in part by National Institutes of Health Grants NEI-EY014366 (DS) and EY017373, EY12974, and EY14599 (HS); Research to Prevent Blindness; Kentucky Research Challenge Trust Fund (HJK); and National Eye Institute Core Grant R24 EY015636 (HJK).
Footnotes
 Disclosure: Y. Cui, None; H. Shao, None; D. Sun, None; H.J. Kaplan, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Figure 1.
 
Injection of the uveitogenic peptide IRBP1–20 into the AC of B6 mice inhibited the generation and expansion of IRBP-specific T cells. (AC) Groups (n = 3) of B6 mice were injected in the AC with a tolerogenic dose (50 μg) of a uveitogenic (IRBP1–20), a nonuveitogenic (IRBP161–180), or an irrelevant peptide (MOG35–55). Seven days later all the animals were subcutaneously immunized with the uveitogenic peptide IRBP1–20. Thirteen days after immunization, IRBP-specific T cells were isolated from each group of mice and tested for pathogenic activity by adoptive transfer into naive B6 mice. Results showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 failed to induce EAU, whereas those from donor mice injected in the AC with either the nonuveitogenic (IRBP161–180) or the encephalitogenic (MOG35–55) peptide retained the ability to transfer EAU. (D, E) Results of clinical examination agreed with pathology findings and showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased ability to induce EAU. This was observed whether the uveitogenic IRBP-specific T cells were activated in the presence of IL-2 (D), which favors the activation of Th1 uveitogenic T cells, or in the presence of IL-23 (E), which favors the activation of Th17 uveitogenic T cells. (F) Proliferation assay showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased proliferative ability in vitro when exposed to the immunizing peptide IRBP1–20. Results shown are representative of those in three experiments.
Figure 1.
 
Injection of the uveitogenic peptide IRBP1–20 into the AC of B6 mice inhibited the generation and expansion of IRBP-specific T cells. (AC) Groups (n = 3) of B6 mice were injected in the AC with a tolerogenic dose (50 μg) of a uveitogenic (IRBP1–20), a nonuveitogenic (IRBP161–180), or an irrelevant peptide (MOG35–55). Seven days later all the animals were subcutaneously immunized with the uveitogenic peptide IRBP1–20. Thirteen days after immunization, IRBP-specific T cells were isolated from each group of mice and tested for pathogenic activity by adoptive transfer into naive B6 mice. Results showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 failed to induce EAU, whereas those from donor mice injected in the AC with either the nonuveitogenic (IRBP161–180) or the encephalitogenic (MOG35–55) peptide retained the ability to transfer EAU. (D, E) Results of clinical examination agreed with pathology findings and showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased ability to induce EAU. This was observed whether the uveitogenic IRBP-specific T cells were activated in the presence of IL-2 (D), which favors the activation of Th1 uveitogenic T cells, or in the presence of IL-23 (E), which favors the activation of Th17 uveitogenic T cells. (F) Proliferation assay showed that IRBP-specific T cells from donor mice intraocularly injected with IRBP1–20 had a greatly decreased proliferative ability in vitro when exposed to the immunizing peptide IRBP1–20. Results shown are representative of those in three experiments.
Figure 2.
 
AC injection of the uveitogenic peptide IRBP1–20 inhibited the generation of IFN-γ+ and IL-17+ IRBP-specific T cells. IRBP-specific T cells isolated from ACAID or control donor mice (without AC injection) were assessed for IFN-γ or IL-17 expression by intracellular staining (A, B), production of IFN-γ or IL-17 (C, D), and ability to induce EAU (E, F). Results, which are representative of three experiments, showed that IRBP-specific T cells isolated from the donor mice injected in the AC with IRBP1–20 generated significantly lower numbers of IL-17+ and IFN-γ+ IRBP-specific T cells (A, B), produced decreased amounts of IFN-γ and IL-17 as assessed by ELISA (C, D), and failed to induce EAU on transfer to naive recipients (E) compared with IRBP-specific T cells isolated from control mice (F).
Figure 2.
 
AC injection of the uveitogenic peptide IRBP1–20 inhibited the generation of IFN-γ+ and IL-17+ IRBP-specific T cells. IRBP-specific T cells isolated from ACAID or control donor mice (without AC injection) were assessed for IFN-γ or IL-17 expression by intracellular staining (A, B), production of IFN-γ or IL-17 (C, D), and ability to induce EAU (E, F). Results, which are representative of three experiments, showed that IRBP-specific T cells isolated from the donor mice injected in the AC with IRBP1–20 generated significantly lower numbers of IL-17+ and IFN-γ+ IRBP-specific T cells (A, B), produced decreased amounts of IFN-γ and IL-17 as assessed by ELISA (C, D), and failed to induce EAU on transfer to naive recipients (E) compared with IRBP-specific T cells isolated from control mice (F).
Figure 3.
 
AC injection of IRBP1–20 was more effective than intravitreal or subretinal injection. Four groups of B6 mice (n = 3) were injected with tolerogenic doses of IRBP1–20 in the AC, intravitreally (VCAID) or subretinally. Mice in the control group were untreated. Seven days after intraocular injection, all the mice were immunized with the uveitogenic peptide IRBP1–20 emulsified in CFA. Thirteen days after immunization, IRBP-specific T cells were isolated from the mice in each group and were activated in vitro by stimulation with the immunizing peptide and APCs. Pathogenic activity of the IRPB-specific T cells was determined by (A) adoptive transfer to naive syngeneic recipients, (B) number of IFN-γ+ and IL-17+ IRPB-specific T cells by intracellular staining, and (C) proliferative response of the T cells by in vitro proliferation to the immunizing antigen. Results showed that the AC injection of the tolerogenic antigen IRBP1–20 was most effective in the inhibition of EAU, in decreasing the number of IL-17+ IRBP-specific T cells, and in decreasing the proliferative response of such T cells.
Figure 3.
 
AC injection of IRBP1–20 was more effective than intravitreal or subretinal injection. Four groups of B6 mice (n = 3) were injected with tolerogenic doses of IRBP1–20 in the AC, intravitreally (VCAID) or subretinally. Mice in the control group were untreated. Seven days after intraocular injection, all the mice were immunized with the uveitogenic peptide IRBP1–20 emulsified in CFA. Thirteen days after immunization, IRBP-specific T cells were isolated from the mice in each group and were activated in vitro by stimulation with the immunizing peptide and APCs. Pathogenic activity of the IRPB-specific T cells was determined by (A) adoptive transfer to naive syngeneic recipients, (B) number of IFN-γ+ and IL-17+ IRPB-specific T cells by intracellular staining, and (C) proliferative response of the T cells by in vitro proliferation to the immunizing antigen. Results showed that the AC injection of the tolerogenic antigen IRBP1–20 was most effective in the inhibition of EAU, in decreasing the number of IL-17+ IRBP-specific T cells, and in decreasing the proliferative response of such T cells.
Figure 4.
 
Assessment of regulatory T-cell activity. (A) Regulatory T cells were increased in mice injected in the AC with IRBP1–20. Control B6 mice and mice injected in the AC with the uveitogenic peptide IRBP1–20 were analyzed for the number of CD25+, Foxp3+, and CD8+CD122+ cells in splenic T cells by FACS. (B) Regulatory T cells from mice injected with IRBP1–20 inhibited the induction of EAU. B6 mice were separated into three groups (n = 3), with EAU induced by adoptive transfer of 2 × 106 IRBP-specific T cells. Splenic T cells (3 × 107) harvested from mice injected in the AC with IRBP1–20 (group 1), IRBP161–180 (group 2), or untreated (group 3) were then injected intraperitoneally. EAU was scored using clinical examination on days 9, 12, and 15 after EAU induction. *P < 0.05; **P < 0.01.
Figure 4.
 
Assessment of regulatory T-cell activity. (A) Regulatory T cells were increased in mice injected in the AC with IRBP1–20. Control B6 mice and mice injected in the AC with the uveitogenic peptide IRBP1–20 were analyzed for the number of CD25+, Foxp3+, and CD8+CD122+ cells in splenic T cells by FACS. (B) Regulatory T cells from mice injected with IRBP1–20 inhibited the induction of EAU. B6 mice were separated into three groups (n = 3), with EAU induced by adoptive transfer of 2 × 106 IRBP-specific T cells. Splenic T cells (3 × 107) harvested from mice injected in the AC with IRBP1–20 (group 1), IRBP161–180 (group 2), or untreated (group 3) were then injected intraperitoneally. EAU was scored using clinical examination on days 9, 12, and 15 after EAU induction. *P < 0.05; **P < 0.01.
Figure 5.
 
ACAID did not develop in γδ T-cell deficient mice. (AD) Four groups of mice (n = 6) were studied for the induction of ACAID. Control (A), wt-B6 (B), γδ TCR-KO (C), and mice treated with GL3 (D). All mice, except the control, were injected in the AC with IRBP1–20 and then were systemically immunized 7 days later with IRBP1–20 in CFA. Control B6 mice developed EAU (A), ACAID-induced B6 mice resisted induction of EAU (B), γδTCR-KO mice (C) and B6 mice treated with a TCR δ chain-specific antibody (GL3) (D) did not inhibit the development of EAU. (EG) Anterior chamber injection resulted in a decreased number of γδ T cells. Groups of B6 mice (n = 3) were injected in the AC with the uveitogenic IRBP1–20 peptide (E), the nonuveitogenic IRBP161–180 peptide (50 μg/each) (F), or were untreated (G). Seven days later, all the mice were immunized with IRPB1–20. Thirteen days after immunization, splenic T cells were isolated, and the number of γδ T cells was identified by staining with specific antibody and FACS analysis.
Figure 5.
 
ACAID did not develop in γδ T-cell deficient mice. (AD) Four groups of mice (n = 6) were studied for the induction of ACAID. Control (A), wt-B6 (B), γδ TCR-KO (C), and mice treated with GL3 (D). All mice, except the control, were injected in the AC with IRBP1–20 and then were systemically immunized 7 days later with IRBP1–20 in CFA. Control B6 mice developed EAU (A), ACAID-induced B6 mice resisted induction of EAU (B), γδTCR-KO mice (C) and B6 mice treated with a TCR δ chain-specific antibody (GL3) (D) did not inhibit the development of EAU. (EG) Anterior chamber injection resulted in a decreased number of γδ T cells. Groups of B6 mice (n = 3) were injected in the AC with the uveitogenic IRBP1–20 peptide (E), the nonuveitogenic IRBP161–180 peptide (50 μg/each) (F), or were untreated (G). Seven days later, all the mice were immunized with IRPB1–20. Thirteen days after immunization, splenic T cells were isolated, and the number of γδ T cells was identified by staining with specific antibody and FACS analysis.
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