Immune Cell Infiltration into the Eye Is Controlled by IL-10 in Recoverin-Induced Autoimmune Retinopathy

This information is current as Enayat Nikoopour, Cheng-mao Lin, Sarah Sheskey, John R. of September 28, 2021. Heckenlively and Steven K. Lundy J Immunol 2019; 202:1057-1068; Prepublished online 11 January 2019; doi: 10.4049/jimmunol.1800574

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immune Cell Infiltration into the Eye Is Controlled by IL-10 in Recoverin-Induced Autoimmune Retinopathy

Enayat Nikoopour,*,†,‡ Cheng-mao Lin,* Sarah Sheskey,* John R. Heckenlively,* and Steven K. Lundy†,‡

Autoimmune retinopathy (AIR) is a treatable condition that manifests in acute and progressive vision loss in patients. It has recently been determined that AIR is associated with an imbalance of TH1 versus regulatory T cell immunity toward the retinal protein, recoverin. This study describes a new murine model to understand the immunopathology of AIR and its association with T cell responses toward recoverin. Immunization of C57BL/6 mice with recoverin resulted in ocular inflammation including infiltration of CD4+ and CD8+ T lymphocytes, B cells, and CD11b+Ly6C+ inflammatory monocytes in the eyes. Production of IFN-g and IL-17 from T cells was exacerbated in IL-10 knockout (KO) mice and kinetics of disease development was accelerated.

Infiltration of T cells and inflammatory monocytes into the eyes dramatically increased in recoverin-immunized IL-10 KO mice. Downloaded from An immunodominant peptide of recoverin, AG-16, was capable of inducing disease in IL-10 KO mice and resulted in expansion of AG-16 tetramer-specific CD4+ T cells in lymphoid organs and eyes. Adoptive transfer of recoverin-stimulated cells into naive mice was sufficient to induce AIR, and immunization of B cell–deficient mice led to a milder form of the disease. This model supports the hypothesis that recoverin-specific T cell responses are major drivers of AIR pathogenesis and that IL-10 is an important factor in protection. The Journal of Immunology, 2019, 202: 1057–1068. http://www.jimmunol.org/

utoimmune retinopathy (AIR) is characterized by sudden The diagnosis of AIR is supported by the detection of ARA and progressive loss of vision in association with cir- of the IgG isotype in patients’ serum. Abs against proteins A culating anti-retinal autoantibodies (ARA) (1–3). The involved in the glycolytic pathway, a-Enolase and Aldolase C, condition may occur as a primary autoimmune condition or in have been reported in sera of AIR patients. However, the association with retinitis pigmentosa (RP) or various expression of these proteins is not restricted to , and (1–4). The clinical manifestations of AIR and -associated autoantibodies against them have been detected in various retinopathies (CAR) are acute retinal degeneration leading to re- autoimmune diseases (5). One of the ARA most correlated with ductions in the visual field and altered electrical conductance in AIR is anti-recoverin Ab (6). Recoverin, a 23-kDa retina-specific by guest on September 28, 2021 response to light stimulation in electroretinogram (ERG). Visual calcium-binding protein in photoreceptors, was first identified as defects in CAR and AIR patients are similar but more acute than an immune target in patients with CAR (7, 8). Despite association changes observed in patients with RP, but in a subgroup of RP with disease, there is only indirect evidence showing autoanti- patients, an underlying defect in immune regulation may contribute bodies may have a pathogenic role. The presumed direct role of to acceleration of retinal degeneration. AIR is most often dis- the Abs in pathogenesis of AIR is mostly based on induction of tinguished from nonautoimmune RP based on differential clini- apoptosis of retinal cells in vitro and intravitreal injection of Abs cal findings, including lack of pigment deposits, later disease in animal models (9). Anti-recoverin Abs, for example, have been onset, more rapid progression, and unique visual field defects. shown to induce apoptosis of photoreceptor cells in vitro (10). The role of the many different pathways of the in the pathogenesis and progression of AIR is not well *Department of Ophthalmology and Visual Sciences–Kellogg Eye Center, University of Michigan Medical School, Ann Arbor, MI 48105; †Division of Rheumatology, understood. The strongest evidence that the immune system is Department of Internal Medicine, University of Michigan Medical School, Ann involved in AIR pathogenesis is that modulation of the immune ‡ Arbor, MI 48109; and Graduate Program in Immunology, University of Michigan system with generally immunosuppressive drugs (11) and rit- Medical School, Ann Arbor, MI 48109 uximab (12) have been used successfully for treatment of AIR ORCIDs: 0000-0003-2409-9423 (C.-m.L.); 0000-0002-3655-2216 (S.K.L.). patients. These drugs have broad specificities but primarily Received for publication April 25, 2018. Accepted for publication December 1, 2018. target T cell–based immunity rather than humoral immunity. This work was supported by Laura and Edmond Opler. The imaging and Indeed, we have recently found that new onset AIR patients optical coherence tomography were performed in the Molecular Biology Core Facil- ity of the Kellogg Eye Center, which is supported by National Institutes of Health have a strong TH1-biased immune response toward recoverin National Eye Institute Grant P30 EY007003. (13). Our main objective in the current study was to establish a Address correspondence and reprint requests to Dr. Steven K. Lundy, Division of Rheu- mouse model to discover the specific mechanisms underlying matology, Department of Internal Medicine, University of Michigan Medical School, 4043 the pathogenesis of AIR. We found that breaking peripheral Biomedical Sciences Research Building, Ann Arbor, MI 48109-2200. E-mail address: [email protected] immune tolerance of recoverin resulted in significant infiltra- The online version of this article contains supplemental material. tion of immune cells and inflammation of the retina. Recoverin- Abbreviations used in this article: AIR, autoimmune retinopathy; ARA, anti-retinal specific TH1andTH17 cell responses and disease progression autoantibody; CAR, cancer-associated retinopathy; ERG, electroretinogram; KO, were greatly enhanced in IL-10–deficient mice. Adoptive knockout; MHC II, MHC class II; PTX, pertussis toxin; RP, retinitis pigmentosa; transfer and recoverin-peptide immunization methods were WT, wild-type. also developed to further define the role of T cells in the disease Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 progression. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800574 1058 RECOVERIN-INDUCED RETINAL AUTOIMMUNITY

Materials and Methods supernatants were collected for ELISA assays for IFN-g,IL-17,and/or Mice IL-10 cytokines (R&D Systems). C57BL/6 mice were purchased from The Jackson Laboratories. Breeders for Detection of recoverin-specific Abs tm1Cgn B6.129P2-Il10 /J (IL-10 knockout [KO]) mouse strain were kindly pro- Peripheral blood was collected at the time of sacrifice and sera were an- vided by Dr. G. Chen, University of Michigan, and B cell–deficient B6.129S2- alyzed for Ab measurement as follows. EIA plates were coated with 2 mg/ml Ighmtm1Cgn/J (mMT) mice were purchased from The Jackson Laboratories. recombinant mouse recoverin in carbonate buffer (pH 9.5) overnight at 4˚C. Mice were bred and maintained in a specific pathogen-free environment. The Excess protein was removed and plates were blocked with 1% BSA in animal protocols used in this study were approved by the University of Mich- PBS. Sera were diluted to 1:400 or 1:1600, and 100 ml was added into igan Unit for Laboratory Animal Medicine in accordance with the guidelines set wells in triplicate and incubated for 2 h at room temperature. For negative forth by the National Institutes of Health and the State of Michigan. control, sera were added to wells without Ag coating. Plates were washed Recombinant recoverin and peptides three times, and anti-mouse biotinylated IgG (1:500), and streptavidin– horse radish peroxidase (1:500) was added for 30 min. Plates were washed Recombinant mouse recoverin (1) was prepared at the Center for Structural three times and TMB substrate was added, color reaction was stopped with Biology Protein Core Facility of the University of Michigan. A pET11a vector 2N sulfuric acid, and then the absorbance at OD450 was measured. containing full-length transcript of mouse recoverin was transfected into Rosetta 2(DE3)Escherichia coli strain. The protein product was purified using fast Intracellular cytokine staining protein liquid chromatography and screened with ToxinSensor Chromogenic Cells were isolated either directly from draining lymph nodes of immunized LAL Endotoxin Assay Kit (GenScript) to rule out the possibility of en- mice or from cultures of lymph node cells or splenocytes as indicated and treated dotoxin contamination. A recoverin peptide spanning aa 64–79 (AG-16) with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 h, with GolgiPlug (BD AYAQHVFRSFDANSDG was identified as a likely binding sequence to Biosciences) added at the last 2 h. Cells were stained with Abs against surface mouse H-2b MHC using Immune Epitope Database T cell Epitope pre- proteins CD3, CD4 and CD8 and then fixed and permeabilized with BD Downloaded from diction tools. AG-16 and a series of truncated peptides (outlined in Cytofix/Cytoperm kit. Cells were then stained intracellularly with Abs Supplemental Fig. 3) were synthesized and HPLC was purified to more against IFN-g and IL-17 purchased from BioLegend. than 98% purity and checked with mass spectrometry by Biomatik. Tetramer staining Induction of AIR AG-16 peptide/I-Ab tetramer (1.5 mg/ml) was made at the National Institutes Theimmunizationprotocolusedtoinduce AIR was adapted from studies per- of Health Tetramer Core Facility. For negative control, human CLIP 87–101 formed in the experimental autoimmune uveitis model (14). Male or female mice b peptide (PVSKMRMATPILMQA)/I-A tetramer (1.5 mg/ml) was obtained. http://www.jimmunol.org/ 7 to 10 wk old were immunized s.c. in the upper back with an emulsion of CFA Cells from mice immunized with AG-16 peptide were stained with CD3, supplemented with 4 mg heat-killed H37Ra Mycobacterium tuberculosis and CD4, CD44, AG-16/I-Ab (1/800 dilution), or control tetramer (1/800 mixed with either recombinant mouse recoverin (200 mg/mouse) or recoverin dilution) for 2 h at 37˚C prior to analysis by flow cytometry. AG-16 peptide (100 mg/mouse). Except where specified, mice were injected i.p. with 200 ng of pertussis toxin (PTX) on days 0 and 2 after immunization to Adoptive transfer facilitate cellular infiltration of the retina. Retina pathology was examined at 3–8 wk after immunization in the Functional Assessment Core Facility of the Kel- Donor IL-10 KO mice on the C57BL/6 background were immunized with logg Eye Center. Fundus imaging was performed using a Micron III Retinal recoverin (200 mg) emulsified in CFA, followed by i.p. injection of 200 ng Microscope (Phoenix Research Labs, Pleasanton, CA). The field of view for all PTX on days 0 and 2. After 3 to 6 wk, mice were euthanized and single- fundus images was 50˚, allowing analysis of a 1.8-mm-diameter section of the cell suspensions from draining lymph nodes were pooled and stimulated 3 mouse retina. Optical coherence tomography was performed using the Envisu in vitro with recoverin (2.5 mg/ml) for 4 d. Cells were harvested, and 10 by guest on September 28, 2021 6 R2200 Preclinical Spectral Domain Optical Coherence Imaging System (Bio- 10 cells were transferred into each recipient naive C57BL/6 mouse. After ptigen, Durham, NC) with images showing a 1.4-mm cross section of the mouse 3 wk, the recipient mice were examined for pathological changes in fundus retina. A group of unimmunized IL-10–deficient control mice was similarly imaging. For adoptive transfer of CFSE-labeled cells, donor cells were examined from 4 to 36 wk of age. A scoring system for AIR was established stained with 5 mM of CellTrace CFSE (Invitrogen) in PBS for 20 min and with the following criteria: 0 = no change, 0.5 = a slight change in fundus washed with 53 complete RPMI media. Cells were resuspended in sterile imaging with one or two spots, 1 = several degenerative spots in examination of PBS and injected i.p. into recipient mice. The mice were analyzed for fundus, 2 = intermediate number of spots with no infiltration of the vitreous, 3 = presence of CFSE-labeled cells 7 to 10 d after adoptive transfer. high number of spots and infiltration of some cells into vitreous in optical co- herence tomography scans, and 4 = extension of lesions and merging of de- Statistical analysis generative spots with infiltration of many cells into the vitreous. Data shown in bar graphs are mean 6 SEM of triplicate samples unless Isolation of lymphocytes from eyes and flow cytometry otherwise indicated. Scatter plots depict data from individual mouse eyes (two per mouse) with lines indicating mean 6 SEM. Data were analyzed Eyes were harvested and pooled for each experimental group. After removal of for difference between means by Mann–Whitney U test using GraphPad lens, eyes were minced and digested with 1 mg/ml collagenase D from Prism 7 software and p values , 0.05 were considered statistically sig- Clostridium histolyticum (Roche Diagnostics, Mannheim, Germany) in culture nificant (for all graphs: *p # 0.05, **p # 0.01, ***p # 0.001, ****p # medium consisting of RPMI 1640 supplemented with 10% FCS, 1% penicillin/ 0.0001, and ns, not significant). 25 streptomycin, 2% L-glutamine, and 5 3 10 M of 2-ME for 30 min at 37˚C. Cells were extracted through a 40-mm cell strainer, and live cells were counted using a hemocytometer and trypan dye exclusion, washed in flow cytometry Results buffer (0.2% BSA, 0.1% sodium azide in PBS), and stained with Recoverin immunization induces AIR fluorochrome-conjugated Abs against mouse CD45.2 (Clone 104), CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD19 (6D5), CD11b (M1/70), CD11c AIR has been reported and studied as primarily a humoral immune (N418), I-Ab (AF6-120.1), and Ly6C (HK1.4). Abs were purchased from mediated disease, with the retinal protein recoverin being one of BioLegend. Flow cytometry was performed on an LSR II cytometer in the several antigenic targets. Ab responses against proteins are dependent Molecular Biology Core Facility of the Kellogg Eye Center, and data were analyzed using FlowJo v10.0.7 software (Tree Star). Cells were gated on T cell help, yet very little is known about the immunogenicity of for lymphocytes (low side scatter and intermediate forward scatter) and then recoverin in terms of driving cellular responses, in part because of the live singlet cells that are positive for CD45.2 marker. For calculation of total lack of a good animal model. Previous attempts at immunizing number of cells, equal number of cells in lymphocyte gate were considered. C57BL/6 mice with recoverin using commercial grade CFA had failed In vitro stimulation and cytokine assays to induce retinal pathology (data not shown). In the current study, damage to the retina did occur when C57BL/6 mice were immunized Mice were euthanized at the time points indicated in each figure, and with the recombinant recoverin (100 to 200 mg), emulsified in CFA single-cell suspensions were made from spleen and draining lymph nodes in culture medium (see above). Cells were added to round-bottom 96-well that was supplemented with heat-killed H37Ra M. tuberculosis bac- plates (2 to 4 3 106/ml) in triplicate and stimulated with recoverin (2.5 teria,andthengiventwoi.p.injectionsofPTXondays0and2to to 5 mg/ml) or recoverin peptides (0–10 mM) for 4 d, at which time facilitate breakdown of the blood/retina barrier. The Journal of Immunology 1059

Mice immunized with recoverin showed pathological changes in IL-10 regulates the progression and severity of AIR retina in fundus examination after 6 wk (Fig. 1A, 1B, Supplemental To further investigate the role of IL-10 cytokine in regulating the anti- Fig. 1). As shown in Fig. 1A, C57BL/6 mouse appear nor- recoverin immune response, we compared immunized IL-10 deficient mal 3 wk after immunization but begin to show significant depig- mice with wild-type (WT) C57BL/6 mice. Although inflammation in mentation in spots after 6 wk. Clinical scoring, as exemplified in the eyes of WT mice occurred in time points longer than 6 wk after Supplemental Fig. 1, of all C57BL/6 mice in the study after 6 wk immunization, the kinetics of disease development in IL-10 KO mice revealed a range of pathology, with some mice having very severely was much faster and happened within 3 wk (Fig. 3A). Observation of affected eyes (Fig. 1B). Clinical scoring was based primarily on eyes by fundus imaging often showed an alteration of retinal structure fundus imaging, with lower scores indicating the presence of few in a ring form, possibly due to an atrophy of retinal pigmented epi- isolated degenerative spots. Increasing scores were based on higher thelial cells or degeneration of photoreceptors (Fig. 3B, Supplemental numbers of spots that coalesced to form degenerative patches in Fig. 2B). Interestingly, we found some abnormalities in the form of fundus (Fig. 1B, Supplemental Fig. 1). Also, higher scores ($3) tiny degenerated spots in the retinas of unimmunized IL-10 KO mice were often associated with the presence of aggregated cells that that may have increased with age (Fig. 3C, Supplemental Fig. 2A). infiltrated into vitreous in optical coherence tomography scans Similar to the later time points in WT mice, clinical scores in (Supplemental Fig. 2C). recoverin-immunized IL-10 KO mice spanned mild to severe eye Characterization of the immune cells infiltrating the eyes disease (Fig. 3C) and were often accompanied by infiltration of ag- The data in Fig. 1C demonstrate significant infiltration into the eyes of gregated cells into the vitreous humor (Fig. 3B, Supplemental Fig. + recoverin-immunized mice consisting of high numbers of CD45.2 2C). These changes observed by optical coherence tomography were Downloaded from hematopoietic cells. These included CD4+ and CD8+ T lymphocytes present in mice with high scores of disease irrespective of being WT and MHC class II (MHC II) expressing B lymphocytes. Frequency or IL-10 KO but were seen earlier and more often in IL-10 KO mice. and absolute cell counts of CD45.2+ T cells in the eyes of recoverin- The anti-recoverin IgG response did not show a significant dif- immunized mice and control mice were 29.07 6 4.90% versus ference between IL-10 KO and WT mice for 1:400 dilution of serum, 2.01 6 0.21% (p # 0.002) and 28377 6 5544 cells per eye versus but the difference was revealed upon further dilution of serum to 1:1600 or higher (data not shown). In recall response, recoverin- 1282 6 515 cells per eye (p # 0.004), respectively (Fig. 1C, 1E). http://www.jimmunol.org/ There were more CD4+ and CD8+ T cells among CD45.2+ cells. specific T cells from draining lymph nodes of IL-10 KO mice secreted Absolute cell count for CD3+CD4+ and CD3+CD8+ T cell subsets in much higher levels of inflammatory cytokines than T cells from WT the eye for recoverin-immunized and control mice was 4824 6 1752 mice, IFN-g (IL-10 KO versus WT, p # 0.05), and IL-17 (IL-10 KO versus 85 6 48 (p # 0.02) and 1271 6 413 versus 107 6 67 versus WT, p # 0.05) (Fig. 3D). Using intracellular staining of cells (p # 0.02), respectively (Fig. 1E). In control mice, a population of from draining lymph nodes, in freshly isolated cells stained ex vivo, + monocytes expressing CD11b, Ly6C, and CD11c was present at a IL-17 but not IFN-g production from CD4 T cells was observed relatively high frequency in the eye but had low expression of (Fig. 3E). However, in vitro stimulation of lymph node cells with + + MHC II, indicative of a noninflammatory phenotype (Fig. 1D). After recoverin led to increased IFN-g–producing CD4 and CD8 T cells immunization, this monocyte population increased in number in the (Fig. 3E, Supplemental Fig. 1B). The production of intracellular IFN- by guest on September 28, 2021 + eyes and showed a marked increase in the levels of MHC II expression g in CD4 T cells from the recoverin-immunized mice was 12 6 3% (Fig. 1D). Absolute cell counts for CD11b+Ly6C+ I-Ab+CD11c+ in- after recoverin stimulation in vitro versus 1.4 6 0.6% nonstimulated + flammatory monocytes in each eye increased 26-fold from 43 6 29 cells (p # 0.01), respectively. The production of IFN-g in CD8 for control mice to 1121 6 281 (p # 0.01) for recoverin-immunized T cells was 30 6 9% after recoverin stimulation in vitro versus 1.5 6 mice. Although total numbers of all of the hematopoietic cell pop- 0.8% in nonstimulated cells (p # 0.01). Similar cytokine and Ab ulations increased, the only subset that increased in percentage was the results were found with immunization of IL-10 KO mice on the CD4+ T cells (Fig. 1E). BALB/c strain background (data not shown), indicating that the re- sults were not exclusive to C57BL/6 mice. Thus, the increased Cytokine and Ab responses toward recoverin were altered clinical severity of retinopathy in IL-10 KO mice is less attributable by PTX to the marginal increase in Ab titer and much more likely due to In vitro recall responses toward recoverin were compared in cells activation of T cells. isolated from spleen or draining lymph nodes of C57BL/6 mice that The populations of hematopoietic cells infiltrating the eyes of had been immunized with emulsions of CFA + H37Ra M. tuberculosis immunized IL-10 KO mice showed a similar pattern as for WT mice mixed with or without recoverin and then injected i.p. with two doses (Fig. 4A) but appeared much sooner. Three weeks after immunization of PTX or PBS as a control. Secretion of the proinflammatory cy- with recoverin, the extent of cell infiltration in IL-10–deficient mice tokines IFN-g and IL-17 by splenocytes in response to recoverin was was much higher than WT mice in that time window (Fig. 4B). In- similar in the recoverin-immunized groups regardless of injections deed, the extent of cell infiltration in WT mice after 3 wk of recoverin with PTX (Fig. 2A). Recoverin immunization also resulted in 2- to immunization is very limited and not significantly different from 3-fold increase in production of the anti-inflammatory cytokine IL-10 nonimmunized mice (Fig. 4B). However, immunization of WT mice by splenocytes stimulated in vitro with recoverin in comparison with with recoverin did appear to cause an increase in CD11c and MHC II splenocytes from mice immunized without recoverin. However, IL-10 expression on the resident monocytes as early as 3 wk post- production was significantly inhibited in cultured splenocytes from immunization (Fig. 4B). In sum, our data indicate that IL-10 plays an the PTX-injected mice when recoverin was present in the CFA important role in decreasing T cell responsiveness to recoverin im- emulsion. Cytokine responses from cells derived from draining munization and in slowing immune cell infiltration into the eye and lymph nodes were less conclusive (Fig. 2A). Recoverin immunization the pathogenesis of AIR. also led to production of high titers of anti-recoverin IgG that were absent in the control mice immunized with CFA + PTX or CFA alone Identification of an antigenic peptide of recoverin (Fig. 2B). The addition of PTX injections led to a significant increase CD4+ T cell responses are mediated by recognition of peptides in anti-recoverin Abs in the serum of mice immunized with recoverin presented in the context of MHC II. Prediction of peptide binding (Fig. 2B). for the MHC II allele I-Ab expressed by C57BL/6 mice using the 1060 RECOVERIN-INDUCED RETINAL AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. AIR in recoverin-immunized mice. C57BL/6 mice were immunized s.c. in the upper back with an emulsion of CFA with mouse recoverin and i.p. injection of PTX. Mice in control group were not immunized. (A) Mice were examined for retinal changes in fundus of the eyes at indicated time points. Data are shown for individual mice to represent mild to severe disease in the eye. (B) Summary of clinical scores for three experiments (n = 13 mice per group). (C and D) After 6 to 8 wk, mice were euthanized and the eyes from each group were pooled. After removal of lens, eyes were minced and digested with collagenase 1 mg/ml for 30 min at 37˚C. (C) Flow cytometry on cells infiltrated into eyes in a representative experiment. Cells were gated on lymphocyte gate and then live singlet CD45.2+ to analyze frequency of CD4+ and CD8+ T cells, and B cells. (D) Monocytes were gated on CD11b+Ly6C+ and then analyzed for expression of CD11c and MHC II as inflammatory markers. (E) Mean percentages and total cell counts of cells infiltrating the eyes with SEM shown for three experiments (n = 13 mice total per group). *p # 0.05, ****p # 0.0001, Mann–Whitney unpaired test. ns, not significant. The Journal of Immunology 1061 Downloaded from FIGURE 2. Cellular and humoral response toward recoverin autoantigen. Four groups of C57BL/6 mice (n = 5 mice per group) were immunized s.c. in the upper back with an emulsion of CFA with or without mouse recoverin and i.p. injection of PTX as shown in the legend. After 3 wk, mice were euthanized and single-cell suspensions were made from spleens and draining lymph nodes of the individual mice. Legend applies to both panels. (A) Cells (4 3 105 per well) in triplicate wells were stimulated with recoverin (2.5 mg/ml) for 4 d, and supernatants were collected for ELISA of IFN-g, IL-17, or IL-10 cytokines. Mean cytokine concentrations for each mouse were averaged, and error bars show the SE of mean for the five mice in each group. (B) Sera were collected from the immunized mice and analyzed for the presence of recoverin-specific IgG using ELISA (1:400 titer). *p # 0.05, **p # 0.01, ****p # 0.0001, Mann–Whitney unpaired test. ns, not significant. http://www.jimmunol.org/ Immune Epitope Database suggested that aa 66–80 of recoverin, in the retinas within 3 wk (Fig. 6A, 6B), and CD45.2+ immune AQHVFRSFDANSDGT, would have an optimal binding capacity. cells, including CD4+ T cells, MHC II+ B cells, and inflammatory We immunized IL-10 KO mice with recoverin 64–79 sequence, AG- monocytes, had infiltrated into the eyes (Fig. 6C). To ensure re- 16 peptide AYAQHVFRSFDANSDG (Supplemental Fig. 3A) emul- cruitment of the transferred cells into eyes of the recipient mice, sified in M. tuberculosis H37Ra-supplemented CFA followed by CFSE-labeled cells from in vitro recoverin-expanded culture of treatment with PTX. Immunization with AG-16 peptide induced draining lymph nodes of recoverin immunized were adoptively retinal pathology in the eyes (Fig. 5A) that was generally less transferred into naive WT mice. After 7 to 10 d, cells were isolated severe than immunization with the whole protein. There was an from the eyes of the recipient mice and the labeled cells were de- infiltration of CD4+ and CD8+ T cell subsets and inflammatory tected in the eyes (Fig. 6D, Supplemental Fig. 4). Analysis of the by guest on September 28, 2021 monocytes in the eyes of the IL-10 KO mice immunized with AG- labeled cells showed presence of CD4+ and CD8+ subsets of T cells 16 peptide (Fig. 5B). A tetramer of AG-16 peptide bound to I-Ab and CD11b+Ly6C+ cells, whereas CD19+ B cells were not present was used to detect peptide-specific CD4+CD44+ memory T cells among the infiltrated CFSE-labeled donor cells (Fig. 6D). in the eyes, whereas a control tetramer containing CLIP peptide Humoral immune response is not essential for induction of AIR did not bind (Fig. 5C). After in vitro stimulation of cells from draining lymph nodes of AG-16–immunized mice with the peptide Upon immunization with recoverin, there were both cellular and for 72 h, cells were stained with tetramers to confirm the presence of humoral immune responses to this autoantigen. It was not clear if Abs recoverin-specific CD4+ T cells in lymph nodes (Fig. 5D). Lymph have a pivotal role in disease process. To address this issue in a de- node cells displayed a dose-dependent response to recoverin AG-16 finitive way, we immunized mMT mice that are deficient for B cells peptide that included secretion of IFN-g andIL-17(Fig.5E).Anal- with recoverin (Fig. 7). With the absence of B cells in these mice ysis of truncated peptides (Supplemental Fig. 3A) with deletion of (Fig.7A),therewasnoAbformationinresponsetorecoverinim- amino acids at the N terminus resulted in decreased recall responses, munization compared with presence of Ab in the immunized WT indicating the importance of aa 66–68 to the immunogenicity of AG- C57BL/6 mice (Fig. 7B). Despite a lack of humoral response, the 16 peptide (Supplemental Fig. 3B). Cells from the draining lymph immunized mMT mice developed retinopathy in the eye (Fig. 7C, nodes of mice that were immunized with the truncated peptides, VG- 7D). No spontaneous development of retinopathy was detected in the 11 and VS-9, showed measurable but decreasing responsiveness to unimmunized mice lacking B lymphocytes (Fig. 7D). Thus, the role stimulation with recoverin in comparison with mice immunized with of anti-recoverin Abs in pathogenesis is dispensable but humoral AG-16 peptide (Fig. 5F). Intracellular staining for IFN-g of AG-16 immune response might affect the severity of clinical scores peptide-stimulated cells from AG-16 peptide–immunized mice (Fig. 7D), as pathology in the eyes of mMT mice were milder than + demonstrated expansion of CD4 TH1 cells (Fig. 5G). WT mice, (exact p value = 0.034) and progression toward disease occurred at slower pace. Adoptive transfer of recoverin-specific T cells induces retinopathy Discussion To further test the hypothesis that cellular immunity was primarily In this study, we have established a novel mouse model for in- responsible in inducing AIR, adoptive transfer studies were per- duction of retinopathy in response to immunization with recoverin, formed. Lymph node cells were isolated from recoverin-immunized a protein normally expressed by photoreceptor cells. A high level of IL-10 KO mice and restimulated in vitro with 5 mg/ml recoverin for immune cell infiltration into the eyes was observed corresponding 4 d. At the end of the culture, 10 million cells were adoptively to development of inflammatory lesions on the retinas, and in transferred into unimmunized WT C57BL/6 mice, followed by severe cases, the presence of cells invading the vitreous humor. injections of PTX on days 0 and 2. There were pathological changes Systemic immune responses to recoverin protein immunization 1062 RECOVERIN-INDUCED RETINAL AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Accelerated AIR toward recoverin in the absence IL-10 cytokine. WT and IL-10 KO mice were immunized at 8–10 wk of age s.c. in the upper back with recoverin emulsified in CFA followed by i.p. injection of PTX. IL-10 KO mice in control groups were left unimmunized (A–C) or received CFA without recoverin (D). After 3 wk, each eye was clinically scored and mice were euthanized, and single-cell suspensions were made from spleen and draining lymph nodes. (A)In- cidence of retinopathy (cumulative clinical score of .2 per mouse) in WT and IL-10 KO mice immunized with recoverin (solid lines). Clinical scores of age-matched, unimmunized IL-10 KO, and WT mice (12–16 wk) are shown as the controls (dashed lines). (B) Pathological changes visualized by fundus examination and optical coherence tomography in IL-10KO mice 3 wk after immunization (score 2) and an age-matched (12 wk old) unimmunized control mouse (score 0.5). (C)NaiveIL-10 KO mice at different ages and 3 wk after immunization were examined for retinal changes by fundus examination and optical coherence tomography to determine clinical scores of the retinopathy. (D) Lymph node cells (4 3 105 per well) from the four in vivo conditions were cultured with or without recoverin (2.5 mg/ml) for 4 d, then supernatants were analyzed for IFN-g and IL-17 by ELISA. Significance is shown for the comparison of recoverin-stimulated cells from the two recoverin- immunized groups. (E) Lymph node cells from recoverin-immunized IL-10KO mice (4 3 105 per well) were either stained immediately without stimulation after isolation (ex vivo) or cultured with recoverin (2.5 mg/ml) for 4 d, then treated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 h, and GolgiPlug was added at the last 2 h. Cells were surface stained with CD3, CD4, CD8, and intracellularly with IFN-g and IL-17. (F) Cumulative intracellular cytokine data after culture of lymph node cells from recoverin-immunized IL-10KO mice (n =5).**p # 0.01, ****p # 0.0001, Mann–Whitney unpaired test. ns, not significant. The Journal of Immunology 1063 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Accelerated inflammatory cell infiltration in the eyes of IL-10 KO mice after induction of AIR with recoverin. WT and IL-10 KO mice on C57BL/6 background were immunized s.c. in the upper back with recoverin emulsified in CFA, followed by i.p. injection of PTX. Age-matched (8 wk old) WT and IL-10 KO mice in a control groups were left unimmunized. After 3 wk, mice were euthanized and the eyes were harvested and pooled in each condition for isolation of the infiltrating cells. (A) Flow cytometry on isolated eye cells from recoverin immunized or unimmunzed IL-10 KO mice to identify CD4+ T cells, CD8+ T cells, and inflammatory monocytes. Representative figures of at least three experiments with reproducible results for five to six mice per group in each experiment. (B) Averages of frequency and absolute cell counts for three experiments (n . 10 mice per group) of the immune cell infiltration into the eyes of IL-10 KO mice after 3 wk of immunization with recoverin in comparison with the eyes of WT mice after 3 wk of recoverin immunization. *p # 0.05, Mann–Whitney unpaired test. ns, not significant included activation of IFN-g, IL-17, and IL-10–producing T cells, as All three cohorts of human subjects had measurable cellular and hu- well as anti-recoverin IgG production. The model was dependent in moral immune responses toward recoverin, but PBLs from the recently part on the addition of two doses of PTX injections, a method that is diagnosed AIR patients had an increased ratio of IFN-g to IL-10 in frequently used in models of autoimmunity to overcome barriers response to recoverin in comparison with both control groups (13). to lymphocyte extravasation. In the current study, injection of PTX More often, PTX has been used to enhance lymphocyte egress resulted in reduced production of IL-10 from splenocyte cultures from blood vessels, for example by breaking the blood-brain barrier restimulated with recoverin in vitro and a minor increase in circu- in the experimental autoimmune encephalomyelitis model. A major lating anti-recoverin IgG Abs. mechanism of action of PTX has been reported to be induced The evidence of a shift in the relative amounts of recoverin-induced production of IL-1b from CD11b+Ly6C+ and CD11b+Ly6G+ my- IFN-g and IL-10 production was very intriguing in light of data that eloid cells to promote Th1 and Th17 cells and suppress IL-10 we have recently gathered from comparing patients with active AIR to production (15). Other studies have also shown that PTX treat- patients with noninflammatory retinal dystrophy or healthy controls. ment can lower IL-10 production (16) and decrease regulatory 1064 RECOVERIN-INDUCED RETINAL AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Recoverin antigenic peptide induces AIR. IL-10 KO mice were immunized s.c. in the upper back with a recoverin antigenic peptide AG-16 (100 mg) emulsified in CFA followed by i.p. injection of 200 ng PTX on days 0 and 2. Control mice were not immunized. (A) After 4 wk, mice were examined for retinal changes by fundus examination. (B and C) At week 4, mice were euthanized and the eyes were harvested and pooled for each condition and treated with collagenase to isolate cells. (B) T cells and inflammatory monocytes infiltrated into the eyes of the immunized mice averaged for three experiments (n . 10 mice per group). (C) Tetramer-specific CD4+ T cells among CD45.2+ infiltrated cells in the eyes of the immunized mice. Cells were stained with CD3, CD4, CD44, and AG-16/I-Ab or control tetramer and gated on CD3+CD4+ T cells. (D) AG-16/I-Ab tetramer or control tetramer staining of cells from draining lymph nodes of mice immunized with AG-16. Bar graph shows the mean 6 SEM for six individual mice in two experiments. (E) Cells from draining lymph nodes of AG-16 immunized mice (4 3 105 per well) were cultured with serial dilutions of recoverin (Figure legend continues) The Journal of Immunology 1065

T cells (17). The data from human AIR patients and the success of immunization. In the same line, Abs against recoverin have been the AIR model once PTX was included in the protocol prompted us reported in most patients with uveitis, and their frequency reaches to further investigate the role of IL-10 in protecting mice from 100% among patients with birdshot chorioretinopathy (24). Most developing AIR. remarkably, other anti-retinal Abs did not show the same pattern in Immunizations of IL-10–deficient mice with recoverin resulted in patients with uveitis (24). Lewis rats were highly susceptible to accelerated retinal pathology, a modest increase in anti-recoverin retinopathy, as immunization with recoverin led to appearance of IgG, and significantly increased IFN-g and IL-17 responses toward clinical features of severe uveoretinitis (25, 26). Thus, these path- recoverin earlier after immunization. IL-10–deficient mice were ological conditions might not be considered as two separate disease also susceptible to immunization with an antigenic peptide of entities but rather as different stages of progression of one disease recoverin and retinal pathology developed when recoverin- with varying antigenic targets. It is plausible that there is a spectrum stimulated lymphocytes from IL-10 deficient mice were trans- of clinical features based on genetic composition and environmental ferred into unimmunized WT C57BL/6 recipients. This is in factors that control the level of susceptibility and severity of disease agreement with reports of an accelerated disease for mice deficient in different individuals. for IL-10 in other autoimmune diseases (18). Mice deficient for The factors that predispose patients to lose tolerance to recoverin and IL-10 are susceptible to colitis (19) that stems from excessive other retinal Ags are poorly understood. One possibility is that damage proinflammatory Th1 and Th17 T cell responses (20–22). These to photoreceptor cells, which naturally occurs due to damage from light mice develop spontaneous inflammation at environmental surfaces exposure, can lead to increased availability of retinal Ags. People with such as gut, skin, and lungs (23). Germ-free IL-10–deficient mice enhanced retinal degeneration driven by mutations affecting photore- do not develop colitis, which points to the importance of gut ceptor viability may also be at increased risk, especially if clearance Downloaded from microbiota in the disease process. mechanisms or immune regulatory mechanisms are impaired. In an In contrast to the current dogma of a major role for anti-recoverin RCS rat model of retinal degeneration, Abs and T cell responses to autoantibodies in the pathogenesis of AIR, our findings in humans and retinal Ags arose spontaneously in unimmunized rats, possibly as a now in mice collectively suggest that T cell responses are a primary result of necrosis of photoreceptor cells (27). Once immune tolerance cause of disease. In this respect, our AIR model has similar features to of T cells to recoverin, IRBP, or any other initiating autoantigen in the

experimental autoimmune encephalomyelitis and uveitis models in retina is broken, the autoimmune response may spread to other retinal http://www.jimmunol.org/ terms of T cells and inflammatory monocyte-derived dendritic cells proteins and, as described above, manifest as uveitis if there is a delay present in lesions of the target organ. That AIR was inducible using an in diagnosis or a major impairment in immune regulation. antigenic peptide of recoverin and occurred in naive hosts following Recoverin is thought to be functional solely in the retina in a normal adoptive transfer primarily of T cells suggests that humoral responses setting. However, it is one of a small set of retinal Ags that is expressed may be less important to pathogenesis than previously suspected. In in the thymus (28). It has been shown that lowered expression of fact, mMT mice that lack mature B cells and the ability to make anti- retinal Ags in the thymus correlated with more immunogenicity of recoverin IgG Abs still had AIR following recoverin immunization, retinal proteins (28). This suggests that recoverin may normally be although the disease appeared to be milder. The model described in involved in driving immune tolerance of the retina. We found that this article is expected to provide a better framework for studying the both C57BL/6 and BALB/c mice were susceptible to experimental by guest on September 28, 2021 pathogenesis and treatment of AIR as a T cell–mediated disease. AIR induction despite the fact that recoverin mRNA is present in the The major distinctions that have been made immunologically be- thymus in these strains (28). However, those WT mice do not develop tween AIR and autoimmune uveitis are that the immune responses are pathology in the eye until 6 to 8 wk after immunization despite the targeted against different retinal Ags, pathological changes in AIR are presence of a proinflammatory T cell response in draining lymph restricted to the retinal tissue, and the effector mechanisms of human nodes and high titers of Ab toward recoverin as early as 3 wk after AIR have previously been attributed solely to the anti-retinal Abs, immunization. In the absence of immunoregulatory cytokine, IL-10, including anti-recoverin Abs. Unlike AIR, autoimmune uveitis has the augmented Th1 and Th17 T cell responses in IL-10 KO mice that been extensively studied as a T cell–mediated disease using the ex- developed just within a short time after immunization led perimental autoimmune uveitis model, which is based on immuni- to accelerated retinal pathology. This indicates that tolerogenic zation with the photoreceptor protein, IRBP (14). By definition, mechanisms mediated by IL-10 are delaying onset at early stages of uveitis requires the influx of lymphoid cells into the vitreous humor. In disease and limiting disease severity. our recoverin-immunization model, there was a spectrum of changes Recoverin is one of the many neoantigens that is frequently in the retina with WT mice in earlier stages of disease (3–5 wk), expressed by malignant cells (29). Tumor-bearing mice show ab- demonstrating a milder form that resembled the clinical features ob- normal ERG and other features of retinopathy (30). It has been shown served in AIR patients. Progression of the disease from 6 to 8 wk that immunization of mice with recoverin or its peptides can lead to postimmunization was accompaniedbymoresevereinflammationin tumor regression with side effects that included changes in ERG and the retina and often the presence of lymphocyte aggregates in the induction of retinopathy (1, 31, 32). Our data on the exacerbated vitreous, more like autoimmune uveitis. In the absence of IL-10, immune response to recoverin and clinical severity in IL-10 KO mice disease progressed more rapidly, and most mice had evidence of are also in line with another study in mice that demonstrated that cellular infiltration of the vitreous humor. neutralization of CTLA4 signaling leads to a more pronounced im- Our data therefore indicate that both AIR and uveitis could be mune response to recoverin that resulted in prevention of tumor induced at different time points in the same mouse after recoverin progression of a cancer that expressed recoverin (31).

peptide AG-16 for 4 d, then supernatants were analyzed for IFN-g and IL-17 by ELISA. (F) Mice were immunized s.c. with AG-16, VS-11, and VS-9 peptides in CFA, and then at 3 wk, draining lymph node cells were harvested and cultured with 5 mg/ml of whole recoverin protein for 4 d, at which time the supernatants were analyzed for IFN-g by ELISA. (G) Splenocytes from unimmunized or AG-16 immunized IL-10 KO mice were cultured for 4 d with AG-16 peptide and then treated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 h, with GolgiPlug added for the last 2 h. Cells were surface stained with Abs against CD3 and CD4 and then intracellularly for IFN-g. Cells are gated on CD3+ T cells. *p # 0.05, **p # 0.01. Mann–Whitney unpaired test. ns, not significant. 1066 RECOVERIN-INDUCED RETINAL AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Adoptive transfer of recoverin-stimulated cells induces AIR in the recipient mice. Donor IL-10 KO mice on the C57BL/6 background (n =5per group in three experiments) were immunized s.c. in the upper back with recoverin (200 mg)emulsifiedinCFA,followedbyi.p.injectionof200ngPTXondays 0 and 2. After 3 to 6 wk, mice were euthanized and single-cell suspensions from draining lymph nodes were pooled and stimulated with recoverin (2.5 mg/ml) for 4 d. Cells harvested from culture were transferred (10 million cells per mouse) i.v. into naive, WT C57BL/6 mice. For the control group, naive WT mice (n =10 total) without cell transfer were used. (A) After 3 wk, the recipient WT mice were examined for pathological changes in fundus imaging. (B) Summary of clinical scores for naive mice or those receiving recoverin-stimulated cells. (C) Recipient mice were euthanized and eyes were pooled, minced, and digested with collagenase (1 mg/ml) at 37˚C for 30 min. Cells were passed through 40-mm cell strainer and then stained for flow cytometry. Cells were gated on CD45.2+ to analyze frequency and total counts of the infiltrated hematopoietic cells in eyes of recipient mice. CD11b+Ly6C+ monocytes were further analyzed for expression of MHC II I-Ab and CD11c. Bar graphs show mean 6 SEM data for three experiments (total n . 9 mice per group). (D) Adoptive transfer of CFSE-labeled recoverin-stimulated cells was performed. Cells isolated from the eyes were gated on singlet live CD45+ cells to identify the CFSE-labeled cells infiltrating the eyes. Representative figures from two experiments with three mice per group in (D). *p # 0.05, ***p # 0.001, Mann–Whitney unpaired test. The Journal of Immunology 1067

FIGURE 7. Induction of AIR in B cell–defi- cient mice. WT and mMT mice (n = 8 per group in two experiments) on C57BL/6 background were immunized s.c. in the upper back with recoverin emulsified in CFA followed by i.p. in- jection of PTX. Age-matched (8 wk old) WT and mMT mice (n = 8 per group) in control groups were left unimmunized. (A) Confirmation of ab- sence of B cells in the immunized mMT mice. (B) Detection of anti-recoverin IgG in sera from WT and mMT mice in a representative experiment (n = 4 mice per group) after immunization. p , 0.001 (C) Representative fundus images of eyes preimmunization and 7 wk post- immunization. Clinical scores for these repre- sentative eyes are four for WT and one for mMT. (D) Summary of clinical scores for unimmunized and recoverin-immunized WT and mMT mice (n = 8 mice per group) from two experiments Downloaded from harvested 8 wk postimmunization. *p # 0.05, ***p # 0.001, ****p # 0.0001, Mann–Whitney unpaired test. http://www.jimmunol.org/ Recoverin expression has been found in many types of solid tumors Disclosures and is especially common in melanoma and breast and lung cancer, and The authors have no financial conflicts of interest. abnormal ERG have been reported in cancer patients (30, 33, 34). Malignant melanoma is derived from neuro-ectodermal melanocytes, which share stem cell lineage with retinal cells, making it not so sur- References 1. Lu, Y., S. He, L. Jia, N. W. Khan, and J. R. Heckenlively. 2010. Two mouse models prising to see expression of recoverin and other retinal Ags by skin for recoverin-associated autoimmune retinopathy. Mol. Vis. 16: 1936–1948. melanoma cells (35). More than 85% of non–small cell lung cancer 2. Heckenlively, J. R., and H. A. Ferreyra. 2008. Autoimmune retinopathy: a review tissues expressed recoverin (36). Immune reactivity of PBMCs to and summary. Semin. Immunopathol. 30: 127–134. 3. Grange, L., M. Dalal, R. B. Nussenblatt, and H. N. Sen. 2014. Autoimmune by guest on September 28, 2021 recoverin peptides is also reported for patients with CAR having certain retinopathy. Am. J. Ophthalmol. 157: 266–272.e1. HLA alleles (37). Given the expression of recoverin as a neoantigen in 4. Heckenlively, J. R., A. A. Fawzi, J. Oversier, B. L. Jordan, and N. Aptsiauri. many cancers, and our data showing the importance of immune reg- 2000. Autoimmune retinopathy: patients with antirecoverin immunoreactivity and panretinal degeneration. Arch. Ophthalmol. 118: 1525–1533. ulation to preventing AIR, there is cause for concern that increasing use 5. Adamus, G. 2017. 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Supplementary Figure 1. Clinical scoring of autoimmune retinopathy and immune response to recoverin. A: C57BL/6 mice were immunized s.c. in the upper back with an emulsion of complete Freund’s adjuvant with or without mouse recoverin and intraperitoneal injection of pertussis toxin (PTX). Mice were examined for retinal changes in fundus of the eyes at indicated time points. A representative clinical score assigned for each individual mouse is shown on the lower panels. Low score (0.5-1) indicates presence of few degenerative spots. Higher scores indicate high numbers of spots that coalesce to form degenerative patches in fundus. Also, mice with high scores often had aggregated cells that infiltrated into vitreous as assessed by OCT (data not shown). B: Wild type mice were immunized s.c. in the upper back with recoverin emulsified in CFA followed by intraperitoneal injection of pertussis toxin (PTX). After 3 weeks, mice were euthanized and single cell suspensions were made from draining lymph nodes. Cells from recoverin immunized mice (4 x 105/well) were cultured with recoverin (2.5 μg/ml) for 4 days. Cells were then treated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 hours and GolgiPlug was added at the last 2 hrs. Cells were surface stained with CD3, CD4, CD8, and intracellularly with IFN-γ and IL-17. Quadrant positions were based on staining with isotype control antibodies.

Supplementary Figure 2. Funduscopy and OCT on eyes from naïve and immunized IL-10 KO mice A: Fundus images shown at different ages from very young to over 6 month old naïve IL-10 KO mice. B: Fundus images show pathological changes after immunization with recoverin at the indicated time points for IL-10 KO mice immunized at 7-10 weeks of age. C: Infiltration of cells into vitreous humor in the eyes of 4 different IL-10 KO mice after 4 weeks of immunization with recoverin.

Supplementary Figure 3. T cell response toward shorter fragments of recoverin antigenic peptide. IL-10 KO mice on C57BL/6 background were immunized s.c. in the upper back with recoverin antigenic peptide AG-16 (100 μg) emulsified in CFA followed by intraperitoneal injection of 200 ng pertussis toxin (PTX) on days 0 and 2. A: Truncated peptides of AG-16 were produced for analysis of minimal epitopes necessary for T cell response. The predicted anchor residues of peptides for binding to MHC class II are shown in red. B: Cells from draining lymph nodes (4 x 105/well) were cultured with recoverin peptides AG- 16, AG-14, VG-11, VS-9 for 4 days then supernatants were analyzed for IFN-γ and IL-17 by ELISA. Representative figures of 3 experiments with reproducible results for 3 mice per group in each experiment.

Supplementary Figure 4. Adoptive transfer of CFSE labeled T cells into recipient mice. Two groups of WT mice were immunized s.c. in the upper back with either recoverin or OVA emulsified in CFA followed by intraperitoneal injection of 200 ng PTX on days 0 and 2. At 6 weeks, mice were sacrificed and cells from draining lymph nodes of recoverin and OVA immunized mice were pooled for each group and cultured with recoverin (5 μg/ml) or OVA (5 μg/ml), respectively, for three days. Cells were labeled with CFSE and adoptively transferred into two groups of naïve WT mice. Cells isolated from the eyes were gated on singlet live CD45+ cells to show recruitment of the CFSE labeled cells into the eyes.