HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McPherson, S. W. Articles by Gregerson, D. S. Search for Related Content PubMed PubMed Citation Articles by McPherson, S. W. Articles by Gregerson, D. S.

Lymphopenia-Induced Proliferation Is a Potent Activator for CD4⁺ T Cell-Mediated Autoimmune Disease in the Retina¹

Department of Ophthalmology, University of Minnesota, Minneapolis, MN 55455

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
To study retinal immunity in a defined system, a CD4⁺ TCR transgenic mouse line (βgalTCR) specific for β-galactosidase (βgal) was created and used with transgenic mice that expressed βgal in retinal photoreceptor cells (arrβgal mice). Adoptive transfer of resting βgalTCR T cells, whether naive or Ag-experienced, into arrβgal mice did not induce retinal autoimmune disease (experimental autoimmune uveoretinitis, EAU) and gave no evidence of Ag recognition. Generation of βgalTCR T cells in arrβgal mice by use of bone marrow grafts, or double-transgenic mice, also gave no retinal disease or signs of Ag recognition. Arrβgal mice were also resistant to EAU induction by adoptive transfer of in vitro-activated βgalTCR T cells, even though the T cells were pathogenic if the βgal was expressed elsewhere. In vitro manipulations to increase T cell pathogenicity before transfer did not result in EAU. The only strategy that induced a high frequency of severe EAU was transfer of naive, CD25-depleted, βgalTCR T cells into lymphopenic arrβgal recipients, implicating regulatory T cells in the T cell inoculum, as well as in the recipients, in the resistance to EAU. Surprisingly, activation of the CD25-depleted βgalTCR T cells before transfer into the lymphopenic recipients reduced EAU. Taken together, the results suggest that endogenous regulatory mechanisms, as well as peripheral induction of regulatory T cells, play a role in the protection from EAU.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
T cell recognition of CNS Ags induces both tolerance that reinforces CNS immune privilege as well as immunopathogenesis when immune privilege fails to protect from autoimmune disease. The mechanisms that control the outcome of T cell recognition of CNS Ags form the basis for maintenance of peripheral tolerance in CNS tissues (1). The retina is a distinct subset of CNS tissue with an extraordinary concentration of tissue-specific proteins for visual transduction, the absence of meninges, and a lack of lymphatic drainage, which distinguish it from brain and spinal cord (2, 3).

Retinal immune privilege is maintained by several activities. Aire-related mechanisms are important, as Aire-driven expression of retinal interphotoreceptor retinoid-binding protein affected pathogenesis in murine experimental autoimmune uveoretinitis (EAU)³ (4, 5). A positive correlation between mRNA transcripts of photoreceptor cell Ags in the thymus and resistance to EAU has been reported for rodents (6, 7). Negative thymic selection of T cells specific for retinal Ag has been observed (8, 9, 10), as has positive selection of retinal-protective regulatory T cells (Tregs) (11). Induction of EAU was most potent when heterologous Ags or non-immunodominant epitopes of retinal Ags were used for immunizations (12), suggesting that endogenous photoreceptor cell Ags induced partial self-tolerance. Immunological ignorance of retinal Ags has been suggested from several studies (13, 14), and it has been proposed that the lack of retinal self-Ag in draining lymph nodes (LN) can compromise the generation of peripheral tolerance against autoreactive T cells, making the retina more vulnerable to autoimmune disease (10). This concept is supported by studies testing the consequences of peripheral expression of retinal Ag via retroviral transduction of bone marrow grafts (15, 16), by extraocular transgenic (Tg) expression using a class II MHC promoter or collagen promoter (13, 17), and by extraocular DNA vaccination (18). These studies showed that even exceptionally low levels of extra-retinal Ag expression yielded animals whose susceptibility to EAU was lost, while other measures of the immune response were little changed.

Given the breadth of activities working to maintain immune privilege, T cells from TCR Tg mice that recognize either endogenous or neo-self-Ags will be crucial in advancing our understanding of the multifaceted processes of self-tolerance and autoimmunity. Of particular relevance are TCR Tg mice specific to endogenous or Tg Ags expressed within the retina associated with EAU (9, 10). Using mice that express hen egg lysozyme (HEL) on retina-specific promoters, in conjunction with HEL-specific 3A9 TCR Tg T cells, a high incidence of severe, spontaneous EAU was found in the double-Tg mice, despite extensive thymic deletion (9, 10). To further understand the mechanisms and conditions of T cell recognition of retinal Ags leading to either tolerance or immunopathology, we created TCR Tg mice (βgalTCR mice) using the TCR from our CD4⁺, Escherichia coli β-galactosidase (βgal)-specific, 3E9 T cell clone (19) for use in experiments with Tg mice that express βgal as a neo-self-Ag specifically in photoreceptor cells (arrβgal mice). Unlike the HEL/3A9 model, we found the retina to be highly resistant to disease mediated by the βgalTCR T cells, even in arrβgal x βgalTCR double-Tg mice, but susceptible if the βgal was expressed in brain astrocytes. The only potent enhancer of retinal photoreceptor autoimmune disease was lymphopenia-induced proliferation (LIP) induced by adoptive transfer of CD25-depleted, naive βgalTCR T cells into arrβgal mice on the Rag^–/– background. Tregs present in, or generated from, the T cell inoculum contributed to the resistance to EAU in arrβgal mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation and analysis of βgalTCR Tg mice

Generation of the CD4⁺ 3E9 T cell clone specific for the βgal peptide (YVVDEANIETHGMV) has been described (19). Founder mice carrying transgenes for 3E9 TCR - and β-chains were generated (our unpublished data) and crossed to normal B10.A mice. The transgene-positive F₁ mice were then crossed to generate stable 3E9- or 3E9-β Tg mouse lines. Functional 3E9-TCR Tg mice (βgalTCR mice) were generated by crossing the 3E9- and 3E9-β Tg mice. F₁ mice were analyzed by PCR for both the and β transgenes. Mice positive for both transgenes were assayed for proliferative responses in PBMC to either βgal peptide or whole βgal protein. Mice whose PBMCs responded to βgal Ag were crossed to propagate the βgalTCR Tg mouse line. All subsequent βgalTCR Tg mice were assayed for the ability of PBMCs to respond in vitro to βgal Ag before any other analysis.

Other mice

The βgal-expressing arrβgal (previously named hi-arr-βgal), GFAPβgal, and ROSA26 Tg mice on the B10.A background have been described in detail elsewhere (13, 19, 20, 21). βgal expression in rod photoreceptor cells of arrβgal mice mimics expression of endogenous arrestin (22, 23), producing 150 ng βgal per retina and <0.5 ng per pineal gland. Rare, unidentified βgal-positive brain cells were seen. No other sites of expression were found. GFAPβgal mice express βgal in astrocytes of brain (175 ng/brain), retina, and optic nerve, based on activity of the GFAP promoter (24). Expression was not found in thymus of arrβgal or GFAPβgal mice. Expression of βgal in adult ROSA26 mice was low, but present in many tissues, including the thymus. Mice were handled in accordance with the Association for Research in Vision and Ophthalmology "Statement for the Use of Animals in Ophthalmic and Vision Research," as well as with the University of Minnesota animal use and care guidelines. Mice were housed under specific pathogen-free conditions on lactose-free chow.

In vitro T cell, PBMC, and cytokine analysis

Spleen or LN cell suspensions were filtered through a 70-µm cell strainer, and the RBC were lysed in 0.17 M NH₄Cl. The cells were then washed twice in PBS and resuspended in RPMI 1640 supplemented with 10% FCS. PBMCs were prepared from heparinized blood. Routine in vitro Ag stimulation was performed with 1 µM βgal peptide using βgalTCR splenocytes or purified T cells plus irradiated (3000 rad) B10.A splenocytes as APC at an APC/T cell ratio of 10:1. Proliferation and cytokine assays were performed in 96-well plates using 5 x 10⁵ βgalTCR splenocytes or 5 x 10⁵ APC with either 5 x 10⁴ purified βgalTCR T cells or 5–25 x 10⁴ PBMCs, and Ag as indicated in a final volume of 200 µl. For proliferation assays the cells were pulsed with [³H]thymidine at 48 h and counted at 72 h poststimulation. Cytokines were assayed at 48 h poststimulation by ELISA using Abs and reagents from R&D Systems or by cytometric bead array (BD Biosciences) per the manufacturer’s instructions. When indicated, mice were immunized by s.c. injection of 100 µg βgal peptide emulsified in CFA (Sigma-Aldrich).

T cell and bone marrow transfers

Purified βgalTCR T cells were prepared from spleen and LN using anti-CD90.2 (Thy1.2) and/or anti-CD25 microbeads (Miltenyi Biotec) per the manufacturer’s protocol. The cells were washed and resuspended to a concentration of 5 x 10⁷ cells/ml in PBS. Where indicated, the cells were labeled with 4 µM CFSE (Molecular Probes) for 10 min at 37°C with mixing. Labeling was terminated by addition of 10 ml RPMI 1640 media supplemented with 10% FCS. The cells were washed and resuspended to 2 x 10⁷ cells/ml in saline. Recipient mice received the indicated number (10 x 10⁶) of βgalTCR T cells via i.p. injection. βgalTCR bone marrow (BM) was extruded by femoral and tibial lavage with PBS. BM cells were PBS washed, RBC lysed, washed again, and resuspended in saline. BM recipient mice were irradiated (2 x 600 rad, 4-h interval) and received 4–5 x 10⁷ cells in 100 µl i.p.

In vivo treatment of βgalTCR T cell recipients

Groups of arrβgal and B10.A control mice were given an i.v. inoculation of 5 x 10⁶ in vitro Ag-experienced, CFSE-labeled, resting βgalTCR T cells on day 0. Recipient mice were treated with 0.5 µg/mouse of pertussis toxin (PTx; Sigma-Aldrich) on days 12 and 19, or given 20,000 U/mouse of IFN- (R&D Systems) on days 12, 13, 14, 17, and 18. Control mice received saline only. Spleens were harvested for flow cytometry on day 28.

Flow cytometry

Spleen, thymus, or LN cell suspensions were prepared as described above except that the final resuspension was made in FACS buffer (PBS with 2% FCS and 0.02% sodium azide). Cells (0.25–2.0 µl/10⁶ cells) of the appropriate fluorescent-labeled Abs (BD Biosciences or eBioscience) were added and the suspensions incubated on ice for 30 min. The cells were then washed and resuspended in FACS buffer and analyzed on a FACSCalibur flow cytometer using CellQuest (BD Biosciences) or FlowJo (Tree Star) software.

Induction of autoimmune disease by adoptive transfer of T cells

Adoptive transfer for induction of EAU or brain inflammation was done with pooled, unfractionated βgalTCR splenocytes and LN cells stimulated with βgal peptide (0.5 µM), with or without IL-12 (5.0 ng/ml) or the combination of IL-1 (5 ng/ml), IL-6 (10 ng/ml), and TGFβ1 (2 ng/ml) to induce IL-17 production. At 48 h poststimulation, IL-2 was added (10 U/ml) and the βgalTCR T cells were cultured for an additional 6 h. The cultures were washed, resuspended in PBS to 2 x 10⁷ cells/ml, and inoculated i.p. at 5–20 x 10⁶ βgalTCR T cells or splenocyte/lymphocyte cells per mouse. Where indicated, T cells were isolated by Thy1.2 selection and activated with βgal peptide and irradiated B10.A x Rag^–/– splenocytes at a splenocyte/T cell ratio of 10:1 for 48 h. At the indicated times posttransfer, tissues were harvested, fixed in 10% buffered formalin, paraffin embedded, sectioned (5 µm), and stained with H&E. The slides were examined in a masked fashion with EAU scores of 0 (no disease) to 4 (complete loss of photoreceptor cells) based on histopathological changes to the retina (25). Brain inflammation was scored from 0 (none) to 4 (severe) based on histopathology (26).

Induction of autoimmune disease in double-Tg mice

Arrβgal or arrβgal x βgalTCR double-Tg mice were given a single s.c. inoculation of 100 µg of βgal protein emulsified in CFA with or without 0.5 µg of PTx per mouse via i.p. injection. Eyes were harvested at 21 days postimmunization and analyzed for histopathology. The ability of soluble Ag and PTx to induce disease was assayed by injecting mice i.p. with 200 µg of βgal protein mixed with 0.5 µg of PTx. Eyes (from arrβgal x βgalTCR mice) and brains (from GFAPβgal x βgalTCR mice used as controls) were harvested at 35 and 21 days postimmunization, respectively, and analyzed for histopathology.

Induction and analysis of autoimmune disease in lymphopenic mice

CD25⁺ and CD25^– fractions were isolated from βgalTCR spleen and LN cell suspensions using a CD25⁺ selection kit and then enriched for CD4⁺ cells by negative selection (Miltenyi Biotec). The cells were washed and resuspended in PBS to 5 x 10⁶/ml, and 3 x 10⁵ cells were injected i.v. into Rag^–/– or arrβgal x Rag^–/– mice. The eyes were harvested at the indicated time points and analyzed as described above. Analysis of the delayed-type hypersensitivity response to βgal was done by injection of βgal (50 µg in 10 µl) into the ear pinna as described previously (20).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Analysis of βgalTCR T cells

Construction and initial identification of βgalTCR mice was made by PCR showing the presence of both the and β TCR genes in genomic DNA (our unpublished data). Unprimed βgalTCR mice exhibited a positive delayed-type hypersensitivity response while single-chain 3E9 and β Tg mice did not (our unpublished data). Analysis of βgalTCR splenocytes by flow cytometry showed 55–60% of CD3⁺ cells were CD4⁺Vβ10.1⁺, and >85% of CD4⁺ cells expressed Vβ10.1 (Fig. 1A). Furthermore, comparison of naive B10.A and βgalTCR CD4⁺VB10.1⁺ T cells showed similar expression of the activation markers CD25, CD44, CD45RB, and CD69, at levels consistent with the cells being phenotypically naive (Fig. 1B). The number of CD62L^low cells from spleen was modestly increased in βgalTCR mice (Fig. 1B). In vitro proliferation assays demonstrated that βgalTCR splenocytes and/or lymphocytes responded to both βgal peptide and native βgal protein in a dose-dependent manner (Fig. 1C). Ag stimulation of unfractionated βgalTCR splenocytes produced significant amounts of IL-2 and IFN-, consistent with Th1 cells (27, 28) (Table I), but they produced little IL-4, IL-5, IL-10, and TNF-. IL-17 was also produced, consistent with the Th17 phenotype of autoimmune disease-inducing T cells (29, 30). The Ag specificity of βgalTCR T cells was confirmed by the lack of cytokine production when stimulated with OVA.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Analysis of phenotype and Ag response of T cells from βgalTCR Tg mice. A, FACS analysis of CD3+ splenocytes from B10.A control (right) and βgalTCR Tg (left) mice. Percentage of cells with a specific phenotype is indicated. B, FACS analysis of CD3+CD4+Vβ10+ splenocytes from naive B10.A control (right) and βgalTCR Tg (left) mice for cell surface activation markers. C, Proliferative response of control B10.A and TCRβgal Tg mouse lymph node cells and/or splenocytes to βgal peptide (left) or native βgal protein (right). Values shown are the mean of three animals, each done in triplicate, with error bars indicating SEM.

Interactions in vivo between naive βgal-specific T cells and retinal βgal were tested using purified, resting βgalTCR T cells from pooled spleen and LN. T cells were labeled with CFSE and transferred into βgal Tg and control mice. Labeled βgalTCR T cells were recovered from recipient LN and spleens 10 days posttransfer and analyzed for evidence of Ag recognition. βgalTCR T cells transferred into ROSA26 mice, or control B10.A mice immunized with βgal peptide after transfer, showed clear evidence of Ag recognition compared with βgalTCR T cells transferred into normal B10.A recipients. Proliferation of βgalTCR T cells was indicated by dilution of CFSE in Vβ10⁺ lymphocytes from ROSA26 and B10.A/peptide-inoculated mice (Fig. 2A) and in B10.A mice inoculated with whole βgal (Fig. 2B). Their expression of cell surface molecules associated with Ag recognition by T cells was also skewed toward the activated phenotype (increased CD44 and CD69 and decreased CD45RB and CD62L; Fig. 2A). Conversely, βgalTCR T cells recovered from normal B10.A recipients or from arrβgal mice did not dilute their CFSE and maintained the naive phenotype (Fig. 2A). Ag specificity was further shown by inoculation with OVA, which did not induce CFSE dilution (Fig. 2B). βgalTCR T cells recovered from submandibular and cervical LN of recipient arrβgal mice were not phenotypically different than those recovered from inguinal and popliteal LN (our unpublished data).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Analysis of Ag recognition in vivo by adoptively transferred, CFSE-labeled naive βgalTCR T cells. A, Representative FACS analysis of Vβ10+ lymphocytes recovered from recipient mice 10 days posttransfer for CFSE dilution and the indicated cell surface molecules. The percentage of lymphocytes having a resting/naive (R) or an activated (A) phenotype is indicated. B10.A + Ag = 100 µg of βgal peptide given i.p. to B10.A recipient mice 3 days after T cell transfer. B, Analysis of Ag specificity. FACS analysis of Vβ10+ lymphocytes from spleens of B10.A mice that received 100 rad irradiation before transfer of naive, CFSE-labeled βgalTCR T cells. Cells were harvested 14 days posttransfer. Recipients were given i.p. injections of saline (No Ag), 1 mg of OVA, or 300 µg of native βgal 3 days after T cell transfer.

Since short-term residence of naive βgalTCR T cells in arrβgal Tg mice offered little evidence of recognition of endogenous βgal, we asked if βgalTCR T cells maturing in arrβgal mice were altered in their development or ability to respond to βgal as measures of Ag recognition. Introduction of a more physiologically relevant number of developing βgal-specific T cells was achieved using BM grafting. βgalTCR mouse BM was engrafted into irradiated B10.A or arrβgal mice, and T cell development and function were analyzed at 40 days posttransfer. FACS analysis of PBMC showed similar levels of βgalTCR T cell engraftment in both arrβgal and B10.A recipients (Fig. 3A). CD4⁺Vβ10⁺ T cells represented an average of 26.6% and 29.9% of all CD3⁺ lymphocytes in the recipient B10.A and arrβgal mice, respectively. A vast majority of these T cells are likely to be bona fide βgalTCR T cells since the endogenous Vβ10 TCR gene is expressed on only 5% of all T cells in control mice. βgalTCR T cells did not repopulate well in ROSA26 mice transferred with βgalTCR BM as shown by the reduced percentage of CD4⁺Vβ10⁺ in CD3⁺ lymphocytes (5.4%). Unlike arrβgal mice, ROSA26 mice express βgal in the thymus, and therefore this result is consistent with central tolerance and negative selection of maturing βgalTCR T cells. Analysis of CD3⁺Vβ10⁺ thymocytes from βgalTCR BM-engrafted arrβgal vs B10.A mice showed nearly identical distributions of T cells into either CD4 or CD8 single-positive or CD4/CD8 double-positive or double-negative subsets (Fig. 3B), suggesting little or no central tolerance to βgal in arrβgal mice. Arrβgal mice receiving βgalTCR BM did not develop EAU. Under these conditions, the results indicate that expression of βgal from the rod photoreceptor cell arrestin promoter had little effect on βgalTCR T cell development.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Analysis of βgalTCR T cells derived from βgalTCR BM-engrafted mice. A, Representative FACS analysis of CD3+ lymphocytes from peripheral blood for percentage of CD4+Vβ10+ cells 40 days after BM transfer. The normal B10.A mice were not irradiated or grafted; all other groups were irradiated and given βgalTCR BM where indicated. B, FACS analysis of CD3+Vβ10+ thymocytes from control (B10.A) and βgalTCR BM-engrafted mice for T cell subset distribution. Also shown is the percentage of CD3+ thymocytes expressing Vβ10. Assays performed 40 days after BM transfer. C, FACS analysis of CD4+Vβ10+ splenocytes for the indicated cell surface activation marker from B10.A, βgalTCR, and βgalTCR BM-engrafted mice 60 days after BM transfer. D, Proliferative response of splenocytes from βgalTCR BM-engrafted mice to βgal peptide. Values represent the mean of three animals, each done in triplicate, with error bars indicating SEM. E, FACS analysis of CD4+Vβ10+ splenocytes for cell surface markers of memory phenotype in immunized and nonimmunized βgalTCR BM-engrafted recipient mice. For B, C, and E, bars indicate the mean for all animals in each group (n 3), with error bars indicating SEM.

To compare the development of memory βgalTCR T cells in mice whose naive T cells matured with or without βgal expressed in photoreceptor cells, βgalTCR BM recipient mice or control mice were immunized with βgal peptide, and splenocytes were analyzed 21 days postimmunization. Immunized βgalTCR BM-recipient arrβgal mice and B10.A mice showed similar increases in CD44 levels and decreases in CD62L levels (Fig. 3E), indicating that the development of the effector memory T cell phenotype was not affected by the presence of βgal in photoreceptor cells. In summary, these results suggested that the presence of βgal in photoreceptor cells had little effect on the phenotype, response, or effector functions of naive or memory βgalTCR T cells. Furthermore, no spontaneous EAU or EAU induction by immunization with βgal/CFA was observed in any of the βgal Tg mice grafted with βgalTCR BM.

Manipulation of antigenic ignorance

It has been proposed that a lack of Ag presentation of retina-derived Ags in peripheral LN limits the generation of peripheral tolerance, leading to increased susceptibility to EAU (10). Since Ag-experienced T cells are widely thought to have increased ability to detect and respond to Ag, adoptive transfer of Ag-experienced, CFSE-labeled, resting βgalTCR T cells and treatments to promote Ag presentation of retinal βgal were used to probe for evidence of peripheral retinal Ag recognition (Fig. 4). Although none of the mice receiving the βgalTCR T cells along with IFN- or PTx treatment developed EAU, there were small, but significant, Ag-dependent changes in: 1) recovery of CFSE-labeled βgalTCR T cells from spleen, 2) their percentage of all CD4⁺Vβ10⁺ cells, and 3) the level of CFSE fluorescence in CFSE⁺ cells recovered from IFN--treated or PTx-treated arrβgal vs B10.A recipients. These parameters were unchanged in control comparisons of saline-treated arrβgal vs B10.A mice and IFN--treated vs saline-treated B10.A mice. These results suggest that manipulations designed to increase Ag presentation produced a limited, nonpathogenic T cell recognition of retinal βgal.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. In vivo administration of IFN- or PTx leads to Ag-dependent changes in βgalTCR T cells. Resting, CFSE-labeled βgalTCR T cells were transferred into mice that were treated with IFN- (left) or PTx (right) as described in Materials and Methods. Bars indicate mean value for all animals in each group, with error bars indicating SEM. The p values were determined by Student’s t test.

Several TCR Tg mouse models of autoimmune disease have used double-Tg mice in which both the target Ag and Ag-specific T cells are present (9, 10, 31, 32). Accordingly, we generated arrβgal x βgalTCR double-Tg mice and compared the ability to induce EAU in these double-Tg mice vs single-Tg arrβgal mice. Arrβgal mice were moderately susceptible to induction of EAU by immunization with βgal in CFA, followed by PTx given i.v. (Table II). Conversely, double-Tg arrβgal x βgalTCR mice were highly resistant to the same immunization protocol, and they did not develop spontaneous EAU when aged up to 14 mo in specific pathogen-free conditions.

Using βgal peptide to activate pooled spleen and LN cells from βgalTCR mice in vitro led to potent activation as shown by cytokine production (Table I). After 2 days, 5–20 x 10⁶ viable cells were transferred into the βgal Tg mice. Preliminary studies showed that GFAPβgal mice, which express βgal in CNS astrocytes, developed CNS infiltrates and lesions following adoptive transfer of βgalTCR T cells (our unpublished data). Therefore, they were used as positive controls for the activity of the various preparations of βgalTCR T cells that follow. None of 18 arrβgal mice developed EAU, but 4 of 8 GFAPβgal mice developed inflammatory lesions in the brain (Table III). The large difference in susceptibility of the retina as compared with brain was unexpected, as the concentration of Ag in retina is much higher than in brain; that is, the 150 ng of βgal/retina is concentrated in 3 µl of retinal tissue, whereas the 175 ng in brain is contained in 500 µl of tissue.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Induction of IL-17 production by βgalTCR T cells. Production of IL-17 (A) and IL-2 (B) following one or two rounds of Ag/APC stimulation with indicated cytokine treatments as described in Materials and Methods. Error bars indicate SEM.

View larger version (61K): [in this window] [in a new window]   FIGURE 6. Representative photomicrographs of autoimmune pathology in arrβgal retina induced by βgalTCR T cells. A, Normal retina. B, Retina 21 days posttransfer of 5 x 106 βgalTCR T cells after four stimulation cycles under IL-17-inducing culture conditions. Modest loss of peripheral photoreceptor cells was evident after active inflammation subsided (arrow). C and D, Retinas from an arrβgal x βgalTCR double-Tg mouse 35 days after i.v. inoculation of 0.5 µg of PTx and 200 µg of βgal showing modest infiltration of the optic nerve head (C, arrow) and substantial active infiltration of the inner layers of the retina plus disruption of the photoreceptor cell layer (D, arrows). E–G, Arrβgal x Rag–/– retinas at 49 (E), 62 (F), and 75 (G) days posttransfer of 3 x 105 naive CD25– βgalTCR T cells showing progressive loss of photoreceptor cells. A–G, x100 magnification.

Since a recent study reported that soluble Ag along with PTx, in conjunction with transferred, Ag-specific CD4⁺ T cells, was a potent inducer of autoimmune disease in mice that expressed the Ag in the eye (38), the ability of PTx and soluble βgal to induce EAU in the arrβgal x βgalTCR double-Tg mice was analyzed. Only 1 of 15 double-Tg mice developed disease using this protocol (Fig. 6, C and D, and Table IV). To confirm the function of the βgalTCR Tg T cells in double-Tg mice, GFAPβgal x βgalTCR mice were tested concurrently and found to develop significant inflammatory infiltrates (Table IV). PTx alone did not induce autoimmune disease in the susceptible GFAPβgal x βgalTCR mice, suggesting that Ag availability was a limiting factor.

LIP has been associated with acquisition of an activated T cell phenotype and inflammatory disease in murine models (39, 40, 41, 42). A small number (3 x 10⁵) of CD25^– T cells isolated from βgalTCR x Rag^–/– mice were transferred into arrβgal x Rag^–/– mice and a control group of Rag^–/– mice. Eyes harvested between 2 and 11 wk posttransfer showed a slowly progressive but relentless destruction of the entire photoreceptor layer (Fig. 7) by a process that was minimally inflammatory but highly destructive (Fig. 6E–G). Transfer into Rag^–/– mice lacking retinal βgal expression gave no EAU (0 of 12), nor did transfer of an equivalent number of CD25⁺ T cells from βgalTCR x Rag^–/– donors into arrβgal x Rag^–/– recipients (0 of 4).

View larger version (7K): [in this window] [in a new window]   FIGURE 7. LIP leads to EAU in arrβgal mice. CD25– βgalTCR T cells (3 x 105) were transferred into arrβgal x Rag–/– recipients, and the eyes were harvested at the indicated times posttransfer and scored for EAU. Each dot represents one eye.

In addition to the retinal destruction associated with LIP by naive CD25-depleted βgalTCR T cells in the arrβgal x Rag^–/– recipients, evidence for other Ag-dependent effects on the number, phenotype, and function of the recovered cells was sought. Approximately twice as many βgalTCR T cells were recovered from Rag^–/– recipients compared with arrβgal x Rag^–/– recipients (Table V). Each of the populations contained similar portions of CD25⁺Foxp3⁺ T cells (4%), but the number of cells with a CD25⁺Foxp3⁺ Treg phenotype was greater in the Rag^–/– recipients without βgal. CD25⁺Foxp3⁺ T cells could be detected at 3 wk posttransfer (our unpublished data) and were similarly present in both types of recipients at 10 wk posttransfer (Fig. 8A).

View larger version (13K): [in this window] [in a new window]   FIGURE 8. Analysis of T cells recovered from lymphopenic recipient mice reflects the contributions of Th1, Th2, and regulatory T cells. Spleen and LN cells were harvested 9–11 wk posttransfer of CD25– βgalTCR T cells into Rag–/– and arrβgal x Rag–/– mice. A, Representative FACS plots of CD4+Vβ10+ T cells recovered from transferred Rag–/– and arrβgal x Rag–/– mice showing similar proportions of cells that are CD25+Foxp3+. B, Analysis of cytokine production by 5 x 105 pooled, Ag-stimulated spleen and LN cells from normal βgalTCR Tg mice and lymphopenic mice transferred with CD25– βgalTCR T cells. Filled bar indicates with Ag; open bar, no Ag. C, IL-2 production by 1 x 105 CD25+ T cells and CD25– T cells recovered from Rag–/– and arrβgal x Rag–/– recipient mice, and 1 x 105 recovered CD25+ T cells mixed with 3.5 x 104 naive βgalTCR T cells upon stimulation with Ag and APC.

Tregs derived from the T cell inoculum

We previously demonstrated the presence of an uncharacterized, transferable regulatory activity in the arrβgal mice (20), and we showed above that the arrβgal mice were resistant to adoptive transfer of EAU by the βgalTCR T cells. Since Rag^–/– mice do not have conventional, endogenous CD4⁺25⁺Foxp3⁺ T cells, we tested the possibility that arrβgal mice on the Rag^–/– background might be more susceptible to EAU induced by adoptive transfer of in vitro-activated βgalTCR T cells. Thy1.2⁺ cells were isolated from pooled βgalTCR spleen and LN, activated for 48 h with Ag presented by irradiated splenic APC from Rag^–/– mice, and transferred to arrβgal and arrβgal x Rag^–/– mice. The modified activation protocol generated T cells that induced a low but significant incidence of EAU, with minimal severity. However, there was no difference in the incidence and severity of EAU between the two groups of recipients (Fig. 9, left), suggesting that endogenous Tregs had a limited role in preventing pathogenesis.

View larger version (15K): [in this window] [in a new window]   FIGURE 9. Induction of EAU by adoptive transfer of βgalTCR T cells. The T cells were activated and/or selected as indicated and transferred into indicated recipient mice. *, p < 0.005 compared with all other groups for both incidence (Fisher’s exact test) and severity (Mann-Whitney U test).

View larger version (35K): [in this window] [in a new window]   FIGURE 10. Production of T cells with the Treg phenotype in Ag-stimulated cultures. Normal and selected βgalTCR T cells were analyzed for CD25+Foxp3+ cells before culturing and after 48 h in culture with or without Ag. FACS plots are gated on CD4+ lymphocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

T cells from TCR Tg mice that recognize either endogenous self-Ags or neo-self-Ags have been instrumental in advancing the understanding of the multifaceted processes of self-tolerance and autoimmunity (31). Both regulatory and immunopathogenic mechanisms were recently reported in two related models of retinal Ag-specific, TCR Tg mice. Membrane-bound retinal expression of HEL under control of the interphotoreceptor retinoid-binding protein promoter, in conjunction with the 3A9 TCR Tg T cells specific for HEL, gave a high incidence of spontaneous, severe EAU (10). The absence of Ag presentation, due to the sequestration of Ag in a neural environment, was proposed to limit the generation of peripheral T cell anergy, and Tregs were not found. These conditions were proposed to be responsible for the high degree of spontaneous HEL-targeted retinal autoimmune disease in the HEL/3A9 double-Tg mice, despite the significant level of negative selection due to Aire-dependent thymic expression of HEL (10). In a related model, HEL with a membrane anchor was expressed on the retinal rhodopsin promoter and studied in double-Tg mice also made with 3A9 T cells (9). In this model, severe, spontaneous disease was also found in the double-Tg mice, but there was also clear evidence of substantial negative selection due to thymic HEL expression. The authors proposed that the robust disease was due to low-affinity, HEL-specific T cells that escaped thymic deletion, although the source of these cells in the TCR Tg T cell population was not identified. The tolerance induced by HEL expression in single-Tg mice was proposed to be largely due to negative selection. It is possible that the photoreceptor cell damage associated with this form of HEL Tg expression in the photoreceptor cells altered the local environment and subsequent immunological response to the Ag (9).

The results from both of these models differ from ours in several ways. First, we found no evidence for thymic βgal expression. There were minimal differences in the number, survival, phenotype, or qualitative responses of βgalTCR T cells that resided and matured in B10.A and arrβgal hosts that would be associated with thymic expression. Conversely, the low-level systemic and thymic expression of βgal in ROSA26 mice strongly affected the development and function of βgalTCR T cells. Furthermore, whether by βgalTCR BM transfer, or transfer of mature, naive βgalTCR T cells, or analysis of T cells from double-Tg mice, there was little evidence of recognition by T cells of photoreceptor cell-derived βgal in untreated recipients, as no reliable differences in cell surface phenotype or function were detected. Second, the double-Tg mice were highly resistant to spontaneous EAU, even though the TCRβgal T cells were highly pathogenic under LIP conditions.

Findings from CNS studies consistent with our results were found using double-Tg mice that express OVA in brain oligodendrocytes and generate OVA-specific OT-II TCR Tg T cells. There was no evidence for recognition of cognate Ag by the T cells and no spontaneous autoimmune disease (43). A naive phenotype was also observed in influenza virus hemagglutinin Ag-specific CD4⁺ T cells derived from hemagglutinin-specific TCR Tg mice crossed with mice expressing hemagglutinin in glial cells (44). The lack of spontaneous autoimmune disease in arrβgal x βgalTCR double-Tg mice also bears a superficial resemblance to experimental autoimmune encephalomyelitis models using various well-known myelin basic protein (MBP)-specific TCR Tg mice, especially those whose T cells are specific for the MBP1–11 epitope in which administration of adjuvants is required for disease onset (32). Several additional important factors further distinguish our results from those. First, the use of adjuvants did not reverse the resistance to disease induction in arrβgal mice. Second, compared with the widely expressed MBP, there is very little total βgal in arrβgal mice (300 ng/mouse), and its expression is highly restricted to photoreceptor cells of the retina and to subnanogram levels in the pineal gland. Third, the MBP epitopes are also contained in the golli form of the protein that is highly expressed, even in cells of the immune system including dendritic cells (45, 46). This leads to selection of the T cell repertoire, resulting in the generation of pathogenic and regulatory T cells that recognize MBP (47, 48, 49). Fourth, the βgal epitope is immunodominant, implying the efficient generation and persistence of stable peptide-MHC class II complexes. It has been reported that MBP1–11-MHC class II complexes are unstable (50) and that there is competition for MHC class II binding between MBP1–11 and flanking golli and MBP sequences (51). Finally, the CNS has clear pathways of lymphatic drainage (2, 3), whereas the retina contains only occasional LYVE-1⁺ microglia (52), which does not constitute draining lymphatics in the normal retina.

The mechanisms that yield the remarkable resistance to autoimmune disease to an Ag expressed in photoreceptor cells appear to be multiple. Evidence supporting the antigenic ignorance hypothesis by Lambe et al. (10) is found from results showing no differences between naive βgalTCR T cells existing in βgal Tg vs B10.A mice, suggesting diminished peripheral access to the Ag. However, βgalTCR T cells clearly crossed the blood-retinal barrier, gained access to their target Ag in photoreceptor cells, and created the severe retinal pathology shown in lymphopenic recipients. Activated T cells, even with no particular specificity for an Ag within the retina or brain, penetrate the blood-brain barrier and the blood-retinal barrier (53, 54). The outcome of that penetration appears to result from an important, but undefined, local factor in susceptibility. The partial reversal of this state of ignorance by systemic treatment with IFN- and PTx found in the arrβgal mice is further consistent with maintenance of antigenic ignorance. However, this state should leave the mice, as proposed by Lambe et al. (10), hypersensitive to attack by adoptive transfer of activated, autoreactive T cells. This is clearly not the case with the arrβgal Tg mice. While attack on βgal⁺ astrocytes by the T cells was promoted by in vitro activation protocols, no significant susceptibility was observed in the arrβgal mice. Obviously, there remains an important local barrier to pathogenesis in these mice that is unrelated to sequestration, as the retina appears to pose no barrier to activated T cells (54).

In a recent report using the MBP model for experimental autoimmune encephalomyelitis, transfer of naive, TCR Tg, MBP-specific CD4 T cells into Rag^–/– or TCR^(–/–) deficient mice was found to rapidly produce severe signs of experimental autoimmune encephalomyelitis within 5 days of transfer (42). Pre-transfer of normal spleen cells provided sufficient regulatory T cells to prevent disease. In our LIP strategy, note that disease required 8–9 wk to develop in the arrβgal x Rag^–/– mice, but then progressed to total destruction of the target cells in the retina. Recovery of the T cells from Rag^–/– and arrβgal x Rag^–/– recipient mice showed that their Ag-stimulated cytokine production was similar, but it was much different from naive βgalTCR T cells that had not undergone LIP.

While adoptive transfer of activated, autoreactive CD4⁺ T cells is a common strategy to induce experimental autoimmune disease in the retina, use of Tg mice that express a target Ag in photoreceptor cells, in conjuction with CD4⁺ TCR Tg T cells, revealed the presence of multiple mechanisms of resistance to EAU. Transfer of naive, CD25-depleted, βgalTCR T cells into arrβgal mice on the Rag^–/– background was the only procedure we have found to date that induced a high frequency of severe retinal disease. Since Rag^–/– recipients have no endogenous Tregs that can limit autoimmunity, this suggests that such Tregs may be another source of resistance to EAU in arrβgal mice. Surprisingly, CD25 depletion of the donor βgalTCR mice before harvesting the T cells, and then depleting the CD25⁺ cells again before in vitro activation with APC and Ag still did not confer significant pathogenicity, even in lymphopenic recipients. Since the activation of CD25-depleted T cells before transfer induced Foxp3⁺ expression, additional evidence for peripheral induction of Tregs was found. In light of these results, we propose that the limitation on retinal disease acted via at least two possible mechanisms: 1) the CD25-depleted donor T cell population was still able to give rise to regulatory cells, and 2) interaction of the transferred T cells with a local retinal cell population limited disease induction and/or progression. LIP-induced activation and in vitro Ag/APC stimulation both resulted in the appearance of CD4⁺CD25⁺Foxp3⁺ T cells, but significantly more disease was found in the lymphopenic recipients. We are pursuing the possibility that regulatory cells induced by Ag stimulation are more protective than those that appear during LIP.

	Acknowledgments

 
The authors thank Heidi Roehrich for histology, Thien N. Sam for technical assistance, and Kathleen M. Pierson for animal care and genetic analysis.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflicts of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by National Institutes of Health Grants EY011542 and EY016376; Research to Prevent Blindness, The Anna Heilmaier Foundation (St. Paul, MN), and the Minnesota Lions and Lionesses Clubs.

² Address correspondence and reprint requests to Dr. Scott W. McPherson, Department of Ophthalmology, University of Minnesota, 2001 6th Street SE, Minneapolis, MN 55455-3007. E-mail address: mcphe003{at}umn.edu

³ Abbreviations used in this paper: EAU, experimental autoimmune uveoretinitis; arrβgal, photoreceptor expression of βgal; BM, bone marrow; βgal, Escherichia coli β-galactosidase; βgalTCR, βgal-specific TCR Tg T cells; GFAPβgal, astrocyte expression of βgal; HEL, hen egg lysozyme; LIP, lymphopenia-induced proliferation; LN, lymph node; MBP, myelin basic protein; PTx, pertussis toxin; Tg, transgenic; Treg, regulatory T cell.

Received for publication June 2, 2008. Accepted for publication November 12, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Carson, M. J., J. M. Doose, B. Melchior, C. D. Schmid, C. C. Ploix. 2006. CNS immune privilege: hiding in plain sight. Immunol. Rev. 213: 48-65. [Medline]
2. Cserr, H. F., C. J. Harling-Berg, P. M. Knopf. 1992. Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance. Brain Pathol. 2: 269-276. [Medline]
3. Yamada, S., M. DePasquale, S. C. Patlak, H. F. Cserr. 1991. Albumin outflow into deep cervical lymph from different regions of rabbit brain. Am. J. Physiol. 261: H1197-H1204. [Medline]
4. Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. Berzins, S. J. Turley, H. Von Boehmer, R. Bronson, A. Dierich, C. Benoist, D. Mathis. 2002. Projection of an immunological self-shadow within the thymus by the aire protein. Science 298: 1395-1401. [Abstract/Free Full Text]
5. DeVoss, J., Y. Hou, K. Johannes, W. Lu, G. I. Liou, J. Rinn, H. Chang, R. Caspi, L. Fong, M. S. Anderson. 2006. Spontaneous autoimmunity prevented by thymic expression of a single self-antigen. J. Exp. Med. 203: 2727-2735. [Abstract/Free Full Text]
6. Egwuagu, C. E., P. Charukamnoetkanok, I. Gery. 1997. Thymic expression of autoantigens correlates with resistance to autoimmune disease. J. Immunol. 159: 3109-3112. [Abstract]
7. McPherson, S. W., D. S. Gregerson. 1994. Nucleotide sequences homologous to retinal S-antigen in the rat thymus lack a known EAU inducing epitope. R. B. Nussenblatt, and S. M. Whitcup, and R. R. Caspi, and I. Gery, eds. Sixth International Symposium on the Immunology and Immunopathology of the Eye 67-70. Elsevier Sciences, Bethesda, MD.
8. Avichezer, D., R. S. Grajewski, C. C. Chan, M. J. Mattapallil, P. B. Silver, J. A. Raber, G. I. Liou, B. Wiggert, G. M. Lewis, L. A. Donoso, R. R. Caspi. 2003. An immunologically privileged retinal antigen elicits tolerance: major role for central selection mechanisms. J. Exp. Med. 198: 1665-1676. [Abstract/Free Full Text]
9. Ham, D. I., S. J. Kim, J. Chen, B. P. Vistica, R. N. Fariss, R. S. Lee, E. F. Wawrousek, H. Takase, C. R. Yu, C. E. Egwuagu, et al 2004. Central immunotolerance in transgenic mice expressing a foreign antigen under control of the rhodopsin promoter. Invest. Ophthalmol. Visual Sci. 45: 857-862. [Abstract/Free Full Text]
10. Lambe, T., J. C. Leung, H. Ferry, T. Bouriez-Jones, K. Makinen, T. L. Crockford, H. R. Jiang, J. M. Nickerson, L. Peltonen, J. V. Forrester, R. J. Cornall. 2007. Limited peripheral T cell anergy predisposes to retinal autoimmunity. J. Immunol. 178: 4276-4283. [Abstract/Free Full Text]
11. Grajewski, R. S., P. B. Silver, R. K. Agarwal, S. B. Su, C. C. Chan, G. I. Liou, R. R. Caspi. 2006. Endogenous IRBP can be dispensable for generation of natural CD4⁺CD25⁺ regulatory T cells that protect from IRBP-induced retinal autoimmunity. J. Exp. Med. 203: 851-856. [Abstract/Free Full Text]
12. Fling, S. P., L. A. Donoso, D. S. Gregerson. 1991. In vitro unresponsiveness to autologous sequences of the immunopathogenic autoantigen, S-antigen. J. Immunol. 147: 483-489. [Abstract]
13. Gregerson, D. S.. 2002. Peripheral expression of ocular antigens in regulation and therapy of ocular autoimmunity. Int. Rev. Immunol. 21: 101-121. [Medline]
14. Gregerson, D. S., J. Xiao. 2001. Failure of memory (CD44^high) CD4 T cells to recognize their target antigen in retina. J. Neuroimmunol. 120: 34-41. [Medline]
15. Agarwal, R. K., Y. Kang, E. Zambidis, D. W. Scott, C. C. Chan, R. R. Caspi. 2000. Retroviral gene therapy with an immunoglobulin-antigen fusion construct protects from experimental autoimmune uveitis. [See comments.]. J. Clin. Invest. 106: 245-252. [Medline]
16. McPherson, S. W., J. P. Roberts, D. S. Gregerson. 1999. Systemic expression of rat soluble retinal antigen induces resistance to experimental autoimmune uveoretinitis. J. Immunol. 163: 4269-4276. [Abstract/Free Full Text]
17. Xu, H., E. F. Wawrousek, T. M. Redmond, J. M. Nickerson, B. Wiggert, C. C. Chan, R. R. Caspi. 2000. Transgenic expression of an immunologically privileged retinal antigen extraocularly enhances self tolerance and abrogates susceptibility to autoimmune uveitis. Eur. J. Immunol. 30: 272-278. [Medline]
18. Silver, P. B., R. K. Agarwal, S. B. Su, I. Suffia, R. S. Grajewski, D. Luger, C. C. Chan, R. M. Mahdi, J. M. Nickerson, R. R. Caspi. 2007. Hydrodynamic vaccination with DNA encoding an immunologically privileged retinal antigen protects from autoimmunity through induction of regulatory T cells. J. Immunol. 179: 5146-5158. [Abstract/Free Full Text]
19. Gregerson, D. S., J. W. Torseth, S. W. McPherson, J. P. Roberts, T. Shinohara, D. J. Zack. 1999. Retinal expression of a neo-self antigen, β-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis. J. Immunol. 163: 1073-1080. [Abstract/Free Full Text]
20. Gregerson, D. S., C. Dou. 2002. Spontaneous induction of immunoregulation by an endogenous retinal antigen. Invest. Ophthalmol. Visual Sci. 43: 2984-2991. [Abstract/Free Full Text]
21. McPherson, S. W., J. Yang, C. C. Chan, C. Dou, D. S. Gregerson. 2003. Resting CD8 T cells recognize β-galactosidase expressed in the immune-privileged retina and mediate autoimmune disease when activated. Immunology 110: 386-396. [Medline]
22. Kimura, A., D. Singh, E. F. Wawrousek, M. Kikuchi, M. Nakamura, T. Shinohara. 2000. Both PCE-1/RX and OTX/CRX interactions are necessary for photoreceptor- specific gene expression. J. Biol. Chem. 275: 1152-1160. [Abstract/Free Full Text]
23. Kikuchi, T., K. Raju, M. L. Breitman, T. Shinohara. 1993. The proximal promoter of the mouse arrestin gene directs gene expression in photoreceptor cells and contains an evolutionarily conserved retinal factor-binding site. Mol. Cell. Biol. 13: 4400-4408. [Abstract/Free Full Text]
24. Johnson, W. B., M. D. Ruppe, E. M. Rockenstein, J. Price, V. P. Sarthy, L. C. Verderber, L. Mucke. 1995. Indicator expression directed by regulatory sequences of the glial fibrillary acidic protein (GFAP) gene: in vivo comparison of distinct GFAP-lacZ transgenes. Glia 13: 174-184. [Medline]
25. Gregerson, D. S., W. F. Obritsch, L. A. Donoso. 1993. Oral tolerance in experimental autoimmune uveoretinitis: distinct mechanisms of resistance are induced by low dose vs high dose feeding protocols. J. Immunol. 151: 5751-5761. [Abstract]
26. McPherson, S. W., N. D. Heuss, H. Roehrich, D. S. Gregerson. 2006. Bystander killing of neurons by cytotoxic T cells specific for a glial antigen. Glia 53: 457-466. [Medline]
27. Caspi, R. R.. 2002. Th1 and Th2 responses in pathogenesis and regulation of experimental autoimmune uveoretinitis. Int. Rev. Immunol. 21: 197-208. [Medline]
28. McPherson, S. W., J. P. Roberts, D. S. Gregerson. 2005. Peripheral expression of rod photoreceptor arrestin induces an epitope-specific, protective response against experimental autoimmune uveoretinitis. Curr. Eye Res. 30: 491-502. [Medline]
29. Tang, J., W. Zhu, P. B. Silver, S. B. Su, C. C. Chan, R. R. Caspi. 2007. Autoimmune uveitis elicited with antigen-pulsed dendritic cells has a distinct clinical signature and is driven by unique effector mechanisms: initial encounter with autoantigen defines disease phenotype. J. Immunol. 178: 5578-5587. [Abstract/Free Full Text]
30. Peng, Y., G. Han, H. Shao, Y. Wang, H. J. Kaplan, D. Sun. 2007. Characterization of IL-17⁺ interphotoreceptor retinoid-binding protein-specific T cells in experimental autoimmune uveitis. Invest. Ophthalmol. Visual Sci. 48: 4153-4161. [Abstract/Free Full Text]
31. Lafaille, J. J.. 2004. T-cell receptor transgenic mice in the study of autoimmune diseases. J. Autoimmun. 22: 95-106. [Medline]
32. Goverman, J., A. Woods, L. Larson, L. P. Weiner, L. Hood, D. M. Zaller. 1993. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell 72: 551-560. [Medline]
33. Caspi, R. R., P. B. Silver, C. C. Chan, B. Sun, R. K. Agarwal, J. Wells, S. Oddo, Y. Fujino, F. Najafian, R. L. Wilder. 1996. Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response. J. Immunol. 157: 2668-2675. [Abstract]
34. Amadi-Obi, A., C. R. Yu, X. Liu, R. M. Mahdi, G. L. Clarke, R. B. Nussenblatt, I. Gery, Y. S. Lee, C. E. Egwuagu. 2007. TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat. Med. 13: 711-718. [Medline]
35. Furuzawa-Carballeda, J., M. I. Vargas-Rojas, A. R. Cabral. 2007. Autoimmune inflammation from the Th17 perspective. Autoimmun. Rev. 6: 169-175. [Medline]
36. Dick, A. D., J. V. Forrester, J. Liversidge, A. P. Cope. 2004. The role of tumour necrosis factor (TNF-) in experimental autoimmune uveoretinitis (EAU). Prog. Retin. Eye Res. 23: 617-637. [Medline]
37. Su, S. B., R. S. Grajewski, D. Luger, R. K. Agarwal, P. B. Silver, J. Tang, J. Tuo, C. C. Chan, R. R. Caspi. 2007. Altered chemokine profile associated with exacerbated autoimmune pathology under conditions of genetic interferon- deficiency. Invest. Ophthalmol. Visual Sci. 48: 4616-4625. [Abstract/Free Full Text]
38. Fujimoto, C., C. R. Yu, G. Shi, B. P. Vistica, E. F. Wawrousek, D. M. Klinman, C. C. Chan, C. E. Egwuagu, I. Gery. 2006. Pertussis toxin is superior to TLR ligands in enhancing pathogenic autoimmunity, targeted at a neo-self antigen, by triggering robust expansion of Th1 cells and their cytokine production. J. Immunol. 177: 6896-6903. [Abstract/Free Full Text]
39. Krupica, T., Jr, T. J. Fry, C. L. Mackall. 2006. Autoimmunity during lymphopenia: a two-hit model. Clin. Immunol. 120: 121-128. [Medline]
40. Knoechel, B., J. Lohr, E. Kahn, J. A. Bluestone, A. K. Abbas. 2005. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J. Exp. Med. 202: 1375-1386. [Abstract/Free Full Text]
41. Abbas, A. K., J. Lohr, B. Knoechel. 2007. Balancing autoaggressive and protective T cell responses. J. Autoimmun. 28: 59-61. [Medline]
42. Cabbage, S. E., E. S. Huseby, B. D. Sather, T. Brabb, D. Liggitt, J. Goverman. 2007. Regulatory T cells maintain long-term tolerance to myelin basic protein by inducing a novel, dynamic state of T cell tolerance. J. Immunol. 178: 887-896. [Abstract/Free Full Text]
43. Cao, Y., C. Toben, S. Y. Na, K. Stark, L. Nitschke, A. Peterson, R. Gold, A. Schimpl, T. Hunig. 2006. Induction of experimental autoimmune encephalomyelitis in transgenic mice expressing ovalbumin in oligodendrocytes. Eur. J. Immunol. 36: 207-215. [Medline]
44. Cabarrocas, J., E. Piaggio, J. P. Zappulla, S. Desbois, L. T. Mars, H. Lassmann, R. S. Liblau. 2004. A transgenic mouse model for T-cell ignorance of a glial autoantigen. J. Autoimmun. 22: 179-189. [Medline]
45. Campagnoni, A. T., T. M. Pribyl, C. W. Campagnoni, K. Kampf, S. Amur-Umarjee, C. F. Landry, V. W. Handley, S. L. Newman, B. Garbay, K. Kitamura. 1993. Structure and developmental regulation of Golli-mbp, a 105-kilobase gene that encompasses the myelin basic protein gene and is expressed in cells in the oligodendrocyte lineage in the brain. J. Biol. Chem. 268: 4930-4938. [Abstract/Free Full Text]
46. Pribyl, T. M., C. W. Campagnoni, K. Kampf, T. Kashima, V. W. Handley, J. McMahon, A. T. Campagnoni. 1993. The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems. Proc. Natl. Acad. Sci. USA 90: 10695-10699. [Abstract/Free Full Text]
47. Clark, L., L. Otvos, Jr, P. L. Stein, X. M. Zhang, A. F. Skorupa, G. E. Lesh, F. A. McMorris, E. Heber-Katz. 1999. Golli-induced paralysis: a study in anergy and disease. J. Immunol. 162: 4300-4310. [Abstract/Free Full Text]
48. Targoni, O. S., P. V. Lehmann. 1998. Endogenous myelin basic protein inactivates the high avidity T cell repertoire. J. Exp. Med. 187: 2055-2063. [Abstract/Free Full Text]
49. Perchellet, A., I. Stromnes, J. M. Pang, J. Goverman. 2004. CD8⁺ T cells maintain tolerance to myelin basic protein by "epitope theft.". Nat. Immunol. 5: 606-614. [Medline]
50. Fairchild, P. J., R. Wildgoose, E. Atherton, S. Webb, D. C. Wraith. 1993. An autoantigenic T cell epitope forms unstable complexes with class II MHC: a novel route for escape from tolerance induction. Int. Immunol. 5: 1151-1158. [Abstract/Free Full Text]
51. Maverakis, E., J. Beech, D. B. Stevens, A. Ametani, L. Brossay, P. van den Elzen, R. Mendoza, Q. Thai, L. H. Macias, D. Ethell, et al 2003. Autoreactive T cells can be protected from tolerance induction through competition by flanking determinants for access to class II MHC. Proc. Natl. Acad. Sci. USA 100: 5342-5347. [Abstract/Free Full Text]
52. Xu, H., M. Chen, D. M. Reid, J. V. Forrester. 2007. LYVE-1-positive macrophages are present in normal murine eyes. Invest. Ophthalmol. Visual Sci. 48: 2162-2171. [Abstract/Free Full Text]
53. Hickey, W., B. Hsu, H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28: 254-260. [Medline]
54. Prendergast, R. A., C. E. Iliff, N. M. Coskuncan, R. R. Caspi, G. Sartani, T. K. Tarrant, G. A. Lutty, D. S. McLeod. 1998. T cell traffic and the inflammatory response in experimental autoimmune uveoretinitis. Invest. Ophthalmol. Visual Sci. 39: 754-762. [Abstract/Free Full Text]

This article has been cited by other articles:

				D. S. Gregerson, N. D. Heuss, U. Lehmann, and S. W. McPherson Peripheral Induction of Tolerance by Retinal Antigen Expression J. Immunol., July 15, 2009; 183(2): 814 - 822. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McPherson, S. W. Articles by Gregerson, D. S. Search for Related Content PubMed PubMed Citation Articles by McPherson, S. W. Articles by Gregerson, D. S.