The Uveitogenic Potential of Retinal S-Antigen in Horses

Abstract

purpose. To investigate the uveitogenic potential of retinal S-antigen (S-Ag) in horses.

methods. Horses were immunized subcutaneously with S-Ag or BSA as control antigen, emulsified in complete Freund’s adjuvant. Simultaneously, Bordetella pertussis was given intravenously. Antigen specific T- and B-cell responses were analyzed in a 3-day interval. Disease development was judged clinically and histopathologically. Two identical booster immunizations were given every 4 weeks to test induction of recurrences.

results. T- and B-cell responses specific for S-Ag were observed in all immunized horses but were absent in control animals. However, uveitis developed in only one of five animals. Reimmunization with S-Ag did not lead to a uveitic relapse in this horse. All other horses of the S-Ag– and BSA-treated groups neither showed any signs of uveitis, nor had inflammatory infiltrates of the inner eye.

conclusions. In contrast to interphotoreceptor retinoid-binding protein (IRBP), S-Ag is a weak autoantigen in horses. Even though S-Ag immunization leads to the activation of autoreactive T- and B-cells, infiltration of the inner eye and induction of uveitis are controlled in most horses.

Equine recurrent uveitis (ERU) is the only spontaneous animal model for autoimmune uveitis. ¹ Recent research has shown that this disease, as its human homologue, is mediated by autoaggressive CD4⁺ T cells reacting to retinal antigens. ² The immune reactions of ERU-diseased horses indicated a subsequent verification of these findings by immunization of eye-healthy horses with IRBP leading to an ERU-like disease. ³ Interphotoreceptor retinoid-binding protein (IRBP)–induced equine uveitis can be reliably reinduced several times by further immunizations with IRBP. Therefore, the equine models of spontaneous and induced uveitis are most valuable in the investigation of the pathophysiology of recurrences and the testing of new therapeutic concepts for the control of a relapsing autoimmune disease in the future. Relapses are a key feature of several autoimmune diseases, including multiple sclerosis. Epitope spreading to pathogenic epitopes is one of the mechanisms thought to contribute to relapses in autoimmune diseases such as experimental allergic encephalitis (EAE), a model for multiple sclerosis. ⁴  

Intra- and intermolecular epitope spreading occurs with subsequent immunizations in the equine model of induced uveitis. ³ Intermolecular spreading was observed to occur with S-antigen (S-Ag), a well-characterized retinal autoantigen in the Lewis rat. ⁵ Immune reactions to S-Ag were also detected in patients with uveitis. ⁶ Furthermore, intramolecular spreading to specific immunodominant epitopes of S-Ag was observed in patients with certain entities of uveitis. ⁷ Although spreading in patients at early disease stages was restricted to particular determinants, chronic stages were characterized by spreading over many determinants. ⁷  

The widely used rodent model for autoimmune uveitis is S-Ag–induced experimental uveitis (EAU) in the Lewis rat. ⁸ The disease onset is acute and self-limiting, leading to a considerable damage of the retina. ⁹ The disadvantage of this model is the monophased nature of the induced uveitis, which limits the insights in potential causes for recurrences. Studies are needed to help to elucidate the mechanisms of disease recurrences and to develop therapies that prevent recurrent inflammations. ⁹ The goal of our study was to investigate the uveitogenic potential of S-Ag in horses to evaluate the relevance of epitope spreading to S-Ag and to compare the uveitogenic potential of this antigen with the one previously described for IRBP. 

Materials and Methods

Antigens

Bovine S-Ag was isolated from retinas, as described previously. ¹⁰ S-Ag– and IRBP-derived peptides were purchased from Biotrend (Cologne, Germany). The following peptides were used in in vitro proliferation assays: PDSAg (bovine S-Ag; aa 342-355 ¹¹ ), peptide M (bovine S-Ag; aa 303-320 ¹² ), S-Ag 286 (bovine S-Ag aa 286-297 ¹³ ), S-Ag 281 (bovine S-Ag; aa 281-296 ¹⁴ ), R 14 (bovine IRBP; aa 1169-1191 ¹⁵ ), PI 536 (bovine IRBP; aa 536-549 ¹⁶ ), R4T (bovine IRBP; aa 1163-1176 ¹⁴ ), R4/R14 (human IRBP; aa 1169-1181, ¹⁴ PI731 (human IRBP; aa 731-745 ¹⁴ ) and PI1137 (human IRBP; aa 1137-1153 ¹⁴ ). PPD (purified protein derivative of tuberculin; Sigma-Aldrich, Deisenhofen, Germany) was used as a positive control. 

Animals

Eight horses of a standard outbred background (German warmblood) were purchased on the slaughter livestock market. The horses were serologically typed for their MHC (ELA) haplotype. ¹⁷ The haplotypes were comparable to the haplotypes of animals used in the IRBP study. ³  

Five horses were used for the uveitis induction group and three age- and sex-matched horses served as the control. The five experimental horses had to be killed for reasons unrelated to this study (horses 1, 4, and 5 had chronic degenerative joint disease and horses 2 and 3 displayed aggressive and dangerous behavior). Previous episodes of uveitis were excluded by clinical examination and the history given by the owner. The horses had not received any medication during the past 6 months, as EU legislation does not allow steroids or non–steroidal anti-inflammatory drugs in this period in horses that are used in meat production. The horses were accompanied by a document that showed every medication received throughout their life. All horses used in this study had routine hematologic and clinical biochemistry checks at day 0 of each immunization. The after parameters were analyzed: red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), hemoglobin, percentage differential white count, aspartate aminotransferase (AST), alkaline phosphatase (AP), glutamate dehydrogenase (GLDH), γ-glutamyl transpeptidase (γ-GT), bilirubin, lactate dehydrogenase (LDH), creatine kinase (CK), cholesterol, triglycerides, total protein, albumin, urea, creatinine, glucose, calcium, and phosphate. None of these parameters indicated an ongoing systemic disease or immunosuppression. The preimmune sera did not react to S-Ag or IRBP, but were positive to tetanus toxoid, a vaccination antigen. 

The experiments were approved by the Review Board of the Government (Regierung von Oberbayern, AZ 211-2531-31/99). All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Uveitis Induction

Five horses were immunized by subcutaneous injection in the neck of 200 μg bovine S-Ag dissolved in 200 μL RPMI and emulsified with 200 μL complete Freund’s adjuvant (CFA; Sigma-Aldrich). At the same time point, each horse received an intravenous injection of 10¹⁰ killed Bordetella pertussis bacteria. The B. pertussis bacteria were dissolved in a 2-mL injection solution. Two identical booster immunizations were given in 28-day intervals for potential disease reinduction. Control horses received identical injections, with S-Ag replaced by an irrelevant protein (BSA). 

Clinical Disease Evaluation and Scoring

Horses were monitored daily for clinical signs of disease. Uveitis was scored according to a protocol established previously for scoring experimental uveitis in horses. ¹⁴ Briefly, we graded nine clinical parameters of uveitis on a scale from 0 (no disease) to 4 (severe disease). The clinical parameters were conjunctivitis, keratitis, corneal edema, anterior chamber inflammation, miosis, vitreal inflammation, anterior synechia, opacification of the lens, and retinal inflammation and detachment. The evaluation was performed by a clinician in a blinded fashion. 

T-Cell Proliferation Assay

Peripheral blood lymphocytes (PBLs) were obtained from fresh heparinized blood samples by single-density gradient centrifugation (Ficoll; Amersham, Braunschweig, Germany) at days 0, 7, 14, 21, and 28 after each immunization. Cells were cultured in triplicates (5 × 10⁵ per well) in flat-bottomed microtiter plates (Nunc, Wiesbaden, Germany) for 5 days in the presence of antigens or peptides. Triplicates were stimulated with 5 μg/mL whole S-Ag or S-Ag– or IRBP-derived peptides. Subsequently, cells were labeled with 2 μCi [³H] thymidine (Amersham) per well for 18 hours and harvested on day 6. [³H] thymidine incorporation was measured by β-scintillation counting. The results are presented as the stimulation index (SI; mean cpm of triplicates with antigen/mean counts per minute of triplicates without antigen). An SI ≥ 2 was considered a positive reaction. 

Carboxyfluorescein Labeling of Lymphocytes

To characterize the type of responding T cells, we labeled the centrifugation-separated lymphocytes with an intracellular fluorescent dye, 5(6)-carboxyfluorescein diacetate succinimidyl-ester (CFSE). Lymphocytes (5 × 10⁶ were incubated in 20 μM CFSE for 30 minutes at room temperature after which dye uptake was evaluated by flow cytometry (BD Biosciences, Heidelberg, Germany). Labeled cells were then stimulated with 5 μg/mL S-Ag for 5 days in 24-well flat-bottomed plates. Control cells were left without antigen as an unstimulated negative control or stimulated with 5 μg/mL PPD. Cells were harvested after 5 days, and cell division was analyzed with flow cytometry (FACScan; BD Biosciences), using the equine-specific mAbs MCA1078 for anti-CD4 staining and MCA1080 for anti-CD8 (Serotec, Eching, Germany). 

Detection of S-Ag–Specific Autoantibodies

Sera of experimental and control horses were tested for the presence of autoantibodies against S-Ag by enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Maxisorp; Nunc) were coated with 2 μg S-Ag per milliliter coating buffer (pH 9.2) overnight. Before each incubation, the plates were washed three times. Blocking was performed for 1 hour with 1% BSA in PBS-0.05% Tween 20 (PBS-T; Sigma-Aldrich). Samples were diluted (1:1600) in PBS-T and incubated for 1 hour. The secondary antibody (anti-horse IgG peroxidase [POD]; Sigma) was added at a 1:10,000 dilution in PBS-T for 1 hour. For immunoglobulin isotype determination, mouse anti-equine IgA; IgM; IgGa, IgG-b, IgG-c; and IgG(T) antibodies (Biozol, Eching, Germany) were used, followed by an anti-mouse κ-light chain POD antibody (Biozol). The assay was developed with tetramethylbenzidine (TMB; Sigma-Aldrich). The reaction was stopped with 1M sulfuric acid. The absorbance was measured at 450 nm using an ELISA reader (Easy Reader; SLT, Salzburg, Austria). 

Histopathological Examination

Eyes were fixed in Bouin’s solution (Sigma-Aldrich). For adequate and fast fixation the eyes were dissected into four pieces as described. ¹⁸ Eyes were sectioned in four equivalent pieces by cutting in the meridional plane crossing the optical disc and in a second vertical/rectangular meridional plane. After dehydration, the sections were embedded in paraffin. Histopathological changes were evaluated on sections stained with hematoxylin and eosin. 

Immunohistochemistry

The antibody CD3-12 (kindly provided by Elisabeth Kremmer, GSF Grosshadern, Germany) was used for the staining of T cells. Specific staining of equine T cells in paraffin-embedded sections has been demonstrated. ¹⁹ Sections were stained by the avidin-biotin complex–alkaline phosphatase (ABC-AP) method (Vectastain ABC-AP Kit; Linaris, Wertingen, Germany). Binding was then visualized (Vector Red Substrate Kit; Linaris). In double-labeling procedures with anti-CD3 and -S-Ag antibodies, the peroxidase system (Vectastain ABC; Linaris) was used for the second labeling and visualized with brown 3′3′diaminobenzidine (DAB; Sigma-Aldrich). 

Results

Uveitogenicity of S-Ag in Horses

To understand better the role of S-Ag in the pathophysiology of ERU, we first intended to induce the disease by S-Ag immunization, as previously described for IRBP. ³  

The clinical scores of all horses are shown in Figure 1 . Only one of five horses (horse 1) injected with B. pertussis and S-Ag emulsified in CFA showed bilateral clinical signs of uveitis after the second immunization. First signs of uveitis were conjunctivitis, keratitis, a mild corneal edema, a marked miosis and a change of vitreous color to yellow on day 42 (Fig. 1) . A hypopyon in the anterior chamber was found on day 43. On day 50, the clinical signs progressed to chemosis, vascularization of the cornea, hyphema, and a complete miosis. Clinical signs subsided on day 56 and stayed constant up to the end of the experiment on day 70, due to a mild miosis, anterior synechia formation, and opacification of the lenses. The turbidity of the vitreous was unchanged. The third immunization on day 56 did not lead to additional inflammatory signs in horse 1. Four horses remained clinically unaffected by all three identical immunizations with retinal S-Ag. The severity of induced uveitis in horse 1 peaked in both eyes at day 49 and led to blindness. The remaining horses (2–5) of the S-Ag induction group showed no signs of inflammation or vision impairment throughout the whole experiment (Fig. 1) . Horses of the control group (BSA) did not show any signs of uveitis either. A reimmunization of horse 1 did not result in reinduction of uveitis. 

Induction of S-Ag–Specific T-Cell Response

The failure to induce disease in most of the horses raised the question of whether S-Ag immunization was insufficient to activate autoreactive cells. To address this, we first analyzed the T-cell response. In vitro proliferation assays of PBLs demonstrated a proliferative response to whole S-Ag in all S-Ag–immunized horses (Fig. 2) . Seven days after the first immunization, lymphocytes of horse 1 showed the strongest response to stimulation with whole S-Ag. After the second immunization the PBLs of horse 2 reacted strongest to stimulation with S-Ag, although no clinical signs were observed. Horse 4 also showed a marked in vitro reaction to primary and secondary immunization. In comparison to the four clinically unaffected animals, the lymphocytes of horse 1 proliferated from day 7 after the second immunization (day 35) at each time point analyzed. The reactions of horses 2 to 5 declined to negative levels within the following 2 weeks (Fig. 2) . CFSE experiments revealed a CD4⁺ phenotype of proliferating cells in all five horses after in vitro stimulation with S-Ag (Fig. 3) . Control horses did not show any positive response. 

Because all S-Ag–immunized horses had a significant T-cell response to the autoantigen, we further investigated epitope specificities. 

Epitope-Specificity of the T-Cell Reaction

The observation that all S-Ag–immunized horses showed a significant T-cell response to the autoantigen led us to compare the epitope specificities as a potential explanation for the highly divergent clinical outcomes. In vitro restimulation assays showed that after the first S-Ag immunization, all horses reacted to PDSAg, the major uveitogenic epitope of S-Ag in the rat model of experimental autoimmune uveitis. ¹¹ In addition, no differences were detectable in the recognition pattern of other well-known uveitogenic epitopes of S-Ag between horse 1, in which uveitis developed, and nondiseased horses 2 to 5. As shown in Table 1 , peptides S-Ag 281 and peptide M elicited an immune response in the PBLs in one horse and S-Ag 286 in the PBLs of two horses at one time point. Epitope spreading toward IRBP-derived peptides was not detected in any horse (data not shown). 

Autoantibody Production in S-Ag–Immunized Horses

Because differences in T-cell responses were not observed, we analyzed the B-cell response. The formation of autoantibodies against S-Ag was measured by indirect ELISA using monoclonal antibodies to horse IgG isotypes. All horses were negative for S-Ag–specific autoantibodies before immunization. First, anti-S-Ag titers were measurable at day 14 after the first immunization in the sera of all S-Ag–immunized horses compared with the sera of the BSA-immunized control group. The second immunization led to a further increase in anti-S-Ag titers, peaking at day 42 and staying constant until the end of the experiment. IgGa and IgGb autoantibodies developed in horses 1, 3, and 5, whereas IgGb autoantibodies developed preferentially in horses 2 and 4 (serum dilution 1:1600; Fig. 4 ). S-Ag recognition by autoreactive antibodies could also be demonstrated in situ. All sera of S-Ag–immunized horses clearly stained S-Ag at the photoreceptor outer segments in paraffin–embedded sections (Fig. 5B) . Staining was absent with preimmunization sera of the respective horses (Fig. 5A) and horses of the BSA control group. 

T-Cells in the Inner Eyes of Nonresponding Horses

Histopathological evaluation was performed at the end of the experiment on day 70. It revealed that the retinal architecture of horse 1 was completely destroyed (Fig. 5C) . The retina was ablated, and a massive infiltration by lymphocytes was detectable surrounding the remaining neuronal cells. Only small remnants of the retinal pigment epithelium (RPE) were found throughout the eye (Fig. 5C) . The vitreous was infiltrated by inflammatory cells, which also accumulated beside remaining photoreceptor cells. The infiltrating cells were predominantly CD3⁺ (Fig. 5D) . Double staining with anti-S-Ag and anti-CD3 antibody demonstrated the accumulation of T cells at the same locations where S-Ag was detectable (Fig. 5E) . The retinas of the nonuveitic eyes (horses 2–5) were devoid of inflammatory cells. Nevertheless, small numbers of CD3⁺ cells where detectable in the choroid beneath the RPE in all eyes of S-Ag–immunized horses, whereas these cells were absent in the control horses. Horse 3 showed development of three lymphoid follicles in the bulb conjunctiva, consisting mainly of T cells (Fig. 5F) . In the eyes of the control horses no cellular infiltrates were detectable. 

Discussion

Horses represent a unique model for the investigation of autoimmune uveitis. Although previous studies underscored the important role of IRBP in pathogenesis in this species, the relevance of S-Ag, the major autoantigen in the Lewis rat model, is still unclear. Our recent observation of intermolecular epitope spreading to S-Ag in horses with an IRBP-induced uveitis indicated that S-Ag could be uveitogenic itself or could be involved in the induction of recurrent episodes of the diseases. To investigate further the role of S-Ag, we used exactly the same approach as successfully applied for IRBP. Our results show that the susceptibility of horses to S-Ag–induced uveitis was low, with only one animal of five having intraocular inflammation. The diseased animal had no immune response to IRBP or S-Ag before S-Ag immunization, indicating that there was no previous ocular autoimmunity. This result is in striking contrast to that in the IRBP trials in which uveitis developed in seven of seven horses. ³ Furthermore, our attempt to reinduce uveitis in the only affected animal by repeated application of S-Ag was unsuccessful. This finding is also contrary to IRBP-triggered horse experimental uveitis, where the induction of relapses was possible in all tested horses ³ and therefore represents an important feature of this disease model. 

The failure to induce uveitis by S-Ag application in most of the horses supports the findings of an earlier study in which S-Ag failed to induce uveitis in ponies. ²⁰ Our decision to repeat this experiment was based on several assumptions. First, the strain dependence of susceptibility in rodents is well documented. MHC and non-MHC genes play important roles in these models. ²¹ Treatment with B. pertussis or purified pertussis toxin (PTX) concurrent with the immunization overcomes some non-MHC–related mechanisms of resistance. ²¹ In contrast to the immunization protocol applied in our successful IRBP induction model, Hines et al. ²⁰ did not use B. pertussis in ponies. We therefore tried to improve the chance of disease induction by concomitant application of S-Ag and B. pertussis. Analysis of the data provided by Hines et al. ²³ and ourselves strongly supports the conclusion that S-Ag is a weak autoantigen in horses and may play a minor role in the pathogenesis of uveitis. 

As the selected pony strain used by Hines et al. ²⁰ is rarely affected by spontaneous ERU, one explanation for the failure of uveitis induction could be a resistant genetic background. As in the IRBP-experiments, in the present study we used warmblood horses from an outbred background, verified by serologic equine MHC typing. ¹⁷ It is notable that most of these horses showed the same resistant phenotype as ponies, despite the fact that the MHC haplotypes were comparable to those horses in which uveitis was readily inducible by IRBP. However, this phenotype was not exclusive, since one horse had severe uveitis with complete destruction of the retina and a massive infiltration of CD3⁺ T lymphocytes. MHC-dependent resistance of rodent strains to the development of uveitis is based on nonrecognition of pathogenic epitopes. ²¹ To evaluate further whether the resistance was based on a general unresponsiveness to the retinal autoantigen, we compared T- and B-cell responses of the resistant animals and the susceptible horse. We observed a similar pattern of autoimmune responses in these animals. In vitro, all S-Ag–immunized horses showed a proliferative response to S-Ag and PDS-Ag, the major pathogenic epitope in Lewis rats. Furthermore, the isotype pattern of S-Ag–specific immunoglobulins revealed no differences. Thus, application of the retinal autoantigen S-Ag in combination with the strong immunomodulators CFA and B. pertussis was not sufficient to induce uveitis, despite the induction of a strong T- and B-cell response. 

An important finding was that CD3⁺ cells were present in the choroids of all S-Ag–immunized horses, but only in horse 1 had the cells managed to pass the blood–retinal barrier formed by the RPE. These findings point to a crucial difference in the target organ instead of a difference in the immune response of responder and nonresponders. So far, we have not determined whether the affected horse had a defective blood–retinal barrier or whether its S-Ag–specific T cells differed from those of the nonaffected animals with respect to activation markers or expression of cytokine or chemokine/chemokine receptors. Both hypotheses are supported by published data in the rodent uveitis models. Highly proliferating and thus activated autoantigen-specific T cells are not necessarily highly pathogenic. ¹³ Taking this into account, it is impossible at this stage to conclude whether the proliferating T cells in horse 1 were effector cells that induce uveitis. 

Differences in the target organ have been discussed as an important mechanism by groups studying the genetic control of EAU in rodents. One supposed mechanism is the involvement of genetic factors that influence the local vasoactive amine release causing changes in blood vessel permeability of the target organ. ²² However, the equine retina is largely devoid of blood vessels, ¹⁸ inflammatory cells enter the retina through the RPE in spontaneous as well as in IRBP-induced uveitis. ^{3 14} In horses, the sensitivity of RPE cells to vasoactive amines may be more critical for maintaining the blood–retinal barrier. Despite intensive ophthalmic examination of the eyes before immunization, we cannot exclude that a previous event could have disturbed the blood–retinal barrier in the eyes of the affected horse, thus facilitating the invasion of S-Ag–specific T cells. The RPE of horse 1 was completely destroyed, which may suggest that cells had invaded the eye through the retinal pigment layer, but overall destructive alterations in the eyes of this horse made it impossible to draw conclusions about the initiating events or sites of invasion. 

A key feature of IRBP-induced uveitis is the reinducibility of acute inflammation. In horse 1, in which uveitis developed from S-Ag immunization, we were not able to induce any relapses. A possible explanation for this difference may be that the pathogenic S-Ag–specific T-cell response of this horse was followed by the induction of regulatory cells, which prevented further relapses, as described in a rat model of EAE. ²³ Caspi et al. ²⁴ reported that, in EAU, rats of the susceptible Lewis strain can become resistant to disease induction by injection of CD8⁺ T-suppressor cells that release a soluble factor that suppresses CD4⁺ effector cells. We cannot exclude that such a mechanism is also responsible for the failure of disease reinduction in our horse; however, we did not observe an active suppression of effector cells, preventing uveitis relapses in IRBP-induced uveitis in the horse. IRBP has a uveitogenic potential in Lewis rats ²⁵ and is the only retinal autoantigen capable of inducing uveitis in susceptible mouse strains. ²⁵ In contrast to experimental autoimmune uveitis in the rat, murine EAU can relapse. ²⁶ In comparison to our model established in the horse, relapses in the murine model are autonomous, and the frequency and time points of the relapses cannot be determined in advance. 

After gaining considerable insight into the effector mechanisms in experimental uveitis, the future direction is to understand recurrences and events at the very onset of the disease. ⁹ S-Ag–induced uveitis in the horse and other species has not been suitable for studies of recurrences. In contrast, IRBP-induced equine experimental uveitis and spontaneous equine recurrent uveitis are of great relevance in seeking to answer these questions. The horse, as does many other large animal models, represents both a valuable model and an important clinical target on its own. ²⁷  

 Supported by Deutsche Forschungsgemeinschaft (DFG) Grants DE 719/1-5 and SFB 571.

 Submitted for publication November 11, 2003; revised December 19, 2003; accepted February 23, 2004.

 Disclosure: C.A. Deeg, None; S. Reese, None; H. Gerhards, None; G. Wildner, None; B. Kaspers, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Clinical score of the eyes of S-Ag–immunized horses. Uveitis developed only in horse 1. The episode started on day 42, which was day 14 after the second immunization. The severity of the inflammation increased until day 49. The third immunization (day 56) did not induce another episode. Horses 2, 3, 4, and 5 did not show any clinical signs of inflammation during the experiment.

Figure 2.

 

T-cell response after in vitro stimulation with S-Ag. Assays were performed at days 0, 7, 14, 21, and 28 after each immunization. Arrows first and subsequent immunizations. Results are expressed as the SI. Each symbol represents the same horse as shown in Figure 1 .

Figure 3.

 

CFSE labeling of dividing cells. Cells were stimulated for 5 days with 5 μg/mL S-Ag (left) or 5 μg/mL PPD (middle) or were left unstimulated as the negative control (right). Staining with equine-specific anti-CD4 and -CD8 antibodies demonstrated a CD4 phenotype of dividing T cells (left). Results are of one representative experiment (horse 5).

Table 1.

 

S-Ag Epitope Detection

Horse	S-Ag	PDSAg	SAg 281	SAg 286	Peptide M
1	129.4	6.3	1.0	1.0	2.2
2	25.9	65.4	1.0	1.0	1.0
3	3.0	2.4	3.9	2.4	1.0
4	3.4	3.8	1.0	2.1	1.0
5	12.7	3.9	1.0	1.0	1.0

 The results are given as the maximum SI achieved in the individual horse. All S-Ag–immunized horses reacted toward the major pathogenic epitope of S-Ag, PDSAg. T cells of horse 2 proliferated strongly to PDSAg, whereas horse 1 had the highest stimulation index to whole S-Ag. Peptide M, SAg 286 and SAg 281 elicited immune responses at one time point in different horses.

Figure 4.

 

Autoantibody production in response to S-Ag. Results are expressed as optical densities. Each bar represents the production of the indicated antibody isotype toward S-Ag by horses 1 to 5. Control horses were negative for S-Ag autoantibodies (data not shown). S-Ag–immunized horses were negative for S-Ag autoantibodies before immunization (data not shown).

Figure 5.

 

Histology of eyes from S-Ag–immunized horses. (A) Normal equine retinas stained with preimmune serum of horse 2. (B) Normal retina stained with anti-S-Ag serum from horse 2. Photoreceptor outer segments were clearly labeled (red, *). (C) Complete destruction of retinal architecture. Infiltration of CD3⁺ T cells (brown) around retinal neuronal cells in the right eye of horse 1. Leftover RPE and neovascularization were visible in the retina (*). (D) Retinal infiltration by T cells (CD3⁺, brown, arrows). Destruction of photoreceptor outer segments with some remaining cells from the inner or outer nuclear layer and formation of epiretinal gliosis (EG). NL, nuclear layer. (E) Severely destroyed retinal fold in horse 1. Infiltration of CD3⁺ T cells (brown, arrows) in the nuclear layer (NL) of the remaining photoreceptor cells (visualized by red staining for S-Ag) and in the neuronal cell layer at the borderline to a severe epiretinal gliosis (EG). (F) Subconjunctival lymphoid follicle of horse 5 (CD3⁺ cells, red). (*) Sclera. Bars (A, C) 25 μm; (B) 20 μm; (D) 15 μm; (E) 40 μm; (F) 120 μm.

The authors thank Thomas Göbel and Stephan Thurau for critical discussions, Barbara Amann and Sylvia Mitterer for excellent technical assistance, and Sonja Kothlow for help with the CFSE assay.