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Characterization of Rat CD8⁺ Uveitogenic T Cells Specific for Interphotoreceptor Retinal-Binding Protein 1177–1191¹

Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202

	Abstract

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The uveitogenic T cells that mediate experimental autoimmune uveitis are commonly assumed to be exclusively CD4⁺. In the present study, we showed that, although a panel of long-term cultured rat uveitogenic T cell lines specific for the interphotoreceptor retinal-binding protein peptide, R16, all expressed CD4, 40% of the R16-specific uveitogenic T cells freshly prepared from Ag-immunized rats were CD8⁺TCR⁺, as demonstrated by CFSE staining. We showed that the expansion of these CD8⁺TCR⁺ T cells was Ag-specific and that highly purified CD8⁺ R16-specific T cells were able to induce uveitis on transfusion into naive rats. Moreover, CD8⁺ uveitogenic T cells more readily switched phenotype from, and to, TCR^–CD8^–CD4^– during in vivo or in vitro activation compared with their CD4⁺ counterparts. In a previous study, we showed that highly purified CD8⁺ myelin oligodendrocyte glycoprotein-specific T cells induced more severe autoimmune encephalomyelitis than the corresponding CD4⁺ T cells. In this study, we show that an interphotoreceptor retinal-binding protein peptide consistently activated a high proportion of CD8⁺TCR⁺ T cells, which were uveitogenic in Lewis rats.

	Introduction

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Uveitis is a common cause of human visual disability and blindness. Although a single episode of the disease usually does not cause permanent visual loss, the recurrent nature of uveitis can result in severe clinical complications, such as cystoid macular edema, cataract formation, and glaucoma. Experimental autoimmune uveitis (EAU)³ can be elicited in rodents by immunization with several different Ags, such as surface Ag (S-Ag), interphotoreceptor retinal-binding protein (IRBP) (1, 2), melanin-associated Ag (3, 4), or myelin proteins (5, 6, 7), or by the adoptive transfer of uveitogenic T cells between syngeneic rodents (7, 8, 9, 10). These uveitogenic T cells are assumed to be exclusively CD4⁺ T cells.

In a previous report, we showed that B6 mice immunized with a myelin oligodendrocyte glycoprotein (MOG) synthetic peptide (pMOG35–55) consistently generate CD8⁺TCR⁺ encephalitogenic T cells (11). We also demonstrated that the adoptive transfer of enriched CD8⁺ MOG-specific T cells from MOG-immunized C57BL/6 mice induces more severe clinical and pathologic disease than active immunization with pMOG35–55 (11). To determine whether CD8⁺ autoreactive T cells played a role in autoimmune diseases other than experimental allergic encephalomyelitis (EAE), we have examined whether CD8⁺ autoreactive T cells occur in EAU and play a major role in the pathogenesis. We first used the CSFE-staining technique (12, 13, 14) to study the Ag-specific proliferation and/or expansion of CD8⁺ R16-specific T cells. We then showed that Ag-primed draining lymph node cells were responsive to R16 even after complete depletion of CD4⁺ cells and that, on activation, such T cells were uveitogenic. Finally, we showed that almost 50% of T cell blasts newly activated by immunizing Ag plus APC were TCR^–CD4^–CD8^–, and that most of these became TCR⁺CD8⁺ when cultured in IL-2-containing medium for 3–4 days. Our results provide further evidence that CD8⁺ autoreactive T cells play a major role in the pathogenesis of organ-specific autoimmune diseases (11, 15, 16, 17, 18, 19). Although a large fraction of newly activated R16-specific uveitogenic T cells are TCR^–CD4^–CD8^–, most, if not all, of these "double-negative" T cells subsequently express TCR and CD8, but not CD4, indicating that CD8⁺ uveitogenic T cells down-regulate their surface molecules upon in vivo or in vitro activation. The possibility that the existence of Ag-specific CD8⁺ autoreactive T cells is frequently missed in in vitro studies is discussed.

	Materials and Methods

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Animals

Pathogen-free female Lewis rats (5–6 wk old) were purchased from Harlan Sprague-Dawley (Indianapolis, IN), and were housed and maintained at the animal facilities of the University of Louisville (Louisville, KY).

All animal studies conformed to the Association for Research in Vision and Ophthalmology statement on the use of animals in Ophthalmic and Vision Research. Institutional approval was obtained and institutional guidelines regarding animal experimentation were followed. IRBP peptide R16 (residues 1177–1191 of bovine IRBP; ADGSSWEGVGVVPDV), IRBP81–90 (SWEGVGVVPD), and IRBP82–90 (WEGVGVVPD) were synthesized by Sigma-Aldrich (St. Louis, MO).

Animal model of EAU

Induction of uveitis in Lewis rats by immunization with peptide R16 has been previously described (20, 21). Briefly, the rats were immunized s.c. with 200 µl of an emulsion containing 30 µg of R16 and 500 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in CFA (Sigma-Aldrich), distributed over six spots on the tail base and flank.

Induction of uveitis by adoptive transfer of R16-specific T cells into naive Lewis rats was performed as previously described (21, 22, 23), and the animals were examined daily for clinical signs of uveitis by slit-lamp biomicroscopy. Intensity of uveitis was scored blind on an arbitrary scale of 0–4 as previously described (21). Inflammation in the eye was confirmed by histopathology. Many inflammatory cells were seen in both the anterior and posterior chamber, together with a disorganized retinal architecture, retinal detachment, and photoreceptor cell damage.

R16-specific T cell lines

R16-specific T cell lines were isolated from R16-immunized Lewis rats (11, 24, 25, 26). T cells were isolated 10 days after immunization from lymph node cells or spleen cells by passage through a nylon wool column, then 1 x 10⁷ cells were stimulated with 20 µg/ml R16 in 2 ml of complete RPMI 1640 medium in a six-well plate (Costar, Cambridge, MA) in the presence of 2 x 10⁷ irradiated syngeneic spleen cells as APCs. After 2 days, the activated lymphoblasts were isolated by gradient centrifugation on Lymphoprep (Robbins Scientific, Mountain View, CA) and cultured in RPMI 1640 medium supplemented with 15% IL-2-containing medium (supernatant from Con A-stimulated rat spleen cells). These T cell lines were maintained by periodic (approximately every 10 days) restimulation for 48 h with Ag in the presence of irradiated syngeneic APCs. The lines were used after 3–4 stimulation/resting cycles.

Proliferation assay

R16-specific T cells (3 x 10⁴ cells/well) in a total volume of 200 µl were cultured at 37°C for 48 h in 96-well microtiter plates in medium with or without R16 in the presence of irradiated syngeneic spleen APCs (2 x 10⁵), and [³H]thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard Instrument, Meriden, CT). The proliferative response was expressed as the mean cpm ± SD of triplicate determinations.

Purification of CD8⁺ T cells using a StemSep column (StemCell Technologies, Vancouver, Canada)

CD8⁺-enriched T cells were isolated from freshly obtained draining lymph nodes and spleen using the negative selection StemSep kit (StemCell Technologies). The rat lymph node and spleen cells were first incubated with bispecific Ab complexes, each of which binds both to a cell S-Ag on rat hemopoietic cells (OX43, CD45RA, CD161, SIRP, or CD4) and to dextran beads. The cells were then treated with StemSep Magnetic Colloid (StemCell Technologies) for 15 min at 4°C, loaded into a magnetic column, and washed with 15 ml of medium according to the manufacturer’s protocol. The flow-through fraction containing CD8⁺ enriched cells was collected. The purity of the isolated cell fraction was determined by flow cytometric analysis using FITC-conjugated anti-TCR and PE-conjugated Abs directed against CD8 or CD4.

Purification of CD8 cells by complement-mediated killing of CD4⁺ cells

T cells were incubated with an optimal amount of anti-rat CD4 (OX-35) Ab (BD Pharmingen, San Diego, CA) for 1 h at 37°C, then the cells with bound Ab were lysed using fresh rabbit serum (complement). Depletion of CD4⁺ T cells was confirmed by FACS analysis.

CFSE staining

T cells from the draining LN and spleen from immunized rats were prepared by passage through nylon wool column and stained with the vital dye, CFSE (Molecular Probes, Eugene, OR) as previously described (12). Briefly, the cells were washed and resuspended at 50 x 10⁶ cells/ml in serum-free RPMI 1640 medium, then incubated at 37°C for 10 min with gentle shaking with a final concentration of 10 µM CFSE, washed twice with, and resuspended in, complete culture medium with 10% FCS, stimulated with peptides and irradiated APCs, and analyzed by flow cytometry.

Immunofluorescence flow cytometry

Aliquots of 2 x 10⁵ cells were double-stained with combinations of FITC- or PE-conjugated mAbs against rat TCR (R73), CD4, or CD8. All Abs were purchased from BD Biosciences (La Jolla, CA). Data collection and analysis were performed on a FACSCalibur flow cytometer using CellQuest software (San Jose, CA).

Pathological examination

For histology, whole eyes were collected, immersed for 1 h in 4% phosphate-buffered glutaraldehyde, then transferred to 10% phosphate-buffered formaldehyde until processed. The fixed and dehydrated tissue was embedded in methacrylate, and 5-µm sections cut through the pupillary-optic nerve plane and stained with H&E. The presence or absence of disease was evaluated blind by examining six sections cut at different levels for each eye. Severity of EAU was scored on a scale from 0 (no disease) to 4 (maximum disease) in half-point increments, as described previously (21).

Statistical analysis

The data are expressed as the mean ± SD. Each experiment was repeated at least three times. Student’s t test was used to analyze the results.

	Results

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The majority of long-term rat R16-specific uveitogenic T cell lines express CD4

We have previously generated a panel of R16-specific uveitogenic T cell lines (23) derived either from the draining lymph nodes or spleen of R16-immunized rats at 10–14 days after immunization by repeated Ag selection in vitro and restimulation every 7–10 days using R16 and syngeneic irradiated APC (25, 27, 28). Six of these lines were tested for CD4 and CD8 expression, with similar results. Fig. 1, A and B, shows a typical FACS-staining pattern for one of the lines, showing that the cells were mainly CD4⁺TCR⁺, while Fig. 1C shows that the responses of these lines were specific for the immunizing peptide and dose dependent.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Long-term cultured rat R16-specific uveitogenic T cell lines are CD4+TCR+. A and B, The rat R16-specific T cell line IRBP21 was stained with the indicated mAb (OX-35, anti-CD4; R73, anti-pan- rat T cells) and FITC-conjugated rat anti-mouse IgG; the control group was stained with FITC-conjugated rat anti-mouse IgG only. The results are representative of those obtained for six of six different lines. C, The specificity of the proliferative response of T cell lines IRBP21 and IRBP5LN to R16.

We have previously reported that CD8⁺ autoreactive T cells play a major role in the pathogenesis of MOG-induced autoimmune encephalomyelitis (11, 29). However, due to the fact that CD8⁺ T cells are frequently overgrown by CD4⁺ T cells in vitro, to examine the activation and proliferation of CD8⁺ autoreactive T cells, it is usually necessary to first deplete CD4⁺ autoreactive T cells from the mixed T cell population using short-term, rather than long-term, cultures (11). However, the existence of CD8⁺ autoreactive T cells in a mixture of T cells can be demonstrated successfully using the CSFE-labeling technique, in which CSFE labels the cellular proteins and the intensity of its fluorescence decreases with each cell division; T cell expansion can then be readily identified by the bands of green CFSE fluorescence (each representing one division), seen when the cells are sorted for CD4 and CD8 phenotypes by staining with PE-labeled anti-CD4 or anti-CD8 Abs. Splenic R16-primed T cells from Lewis rats 10 days after immunization with R16/CFA were prepared by passage through a nylon wool column. The T cells were then labeled with 10 µM CFSE before in vitro stimulation with R16 or other truncated peptides and APCs, and the activated T cell blasts were separated 48 h later by Ficoll gradient centrifugation, and cultured in IL-2-containing medium for various times before FACS analysis, after staining with PE-conjugated anti-rat CD4 or anti-rat CD8 Abs.

Preliminary kinetic studies showed that FACS analysis of the cells at 96–120 h after activation was most suitable for examining the Ag-driven expansion of rat T cell subsets, as T cells that are proliferating vigorously pass through 5–6 divisions in this time, while nonexpanding cells remain quiescent. Fig. 2 shows the results of one representative assay at 96 h after stimulation. As shown, a significant portion (40%) of the R16-specific T cells expressed CD8 and had passed through five divisions. This CD8⁺ T cell response was Ag-specific, as stimulation using a truncated weakly antigenic IRBP peptide (IRBP82–90) did not induce significant T cell proliferation and expansion.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Detection of CD8+ R16-specific T cells using CFSE staining. A, Nylon wool-enriched T cells, prepared from R16-immunized Lewis rats 10 days after immunization, were stained with CFSE, then stimulated with R16 and APCs, and the activated T cell blasts separated on a Ficoll gradient and recultured in IL-2-containing medium for 2–3 days, as described in Materials and Methods, then stained with either PE-anti-rat CD4 or PE-anti-rat CD8, and examined by FACS. B, Specificity of the T cell proliferative response to R16 and the poorly antigenic R16-derived truncated peptide (IRBP82–90). Nylon wool-enriched T cells (3 x 104 cells/well) from R16-immunized rats were cultured at 37°C for 48 h in 96-well microtiter plates with medium, R16, or truncated peptide and irradiated APCs (2 x 105), and [3H]thymidine incorporation during the last 8 h was assessed. The proliferative response is expressed as the mean cpm ± SD of triplicate determinations.

We wished to determine whether CD4-depleted R16-specific T cells were uveitogenic. Due to the fact that none of the anti-rat CD4 Abs available now has in vivo-depleting effect (OX-35 can fix complement in vitro but not deplete CD4⁺ T cells in vivo; W3/25 has neither complement-fixing effect in vitro nor CD4-depleting effect in vivo), the depletion study has been limited in vitro. To examine this, activated R16-stimulated T cell blasts from R16-immunized rats were depleted of CD4⁺ T cells using anti-rat CD4 (OX-35) Ab and complement, as described in Materials and Methods, and CD4 depletion was confirmed by FACS analysis (Fig. 3, A–D). The specific response to the peptide R16 of the CD4-depleted T cells was determined by proliferation assay. As shown in Fig. 3E, these cells proliferated significantly in response to the R16 peptide. When 5 x 10⁶ CD4-depleted T cell blasts were injected into naive rats, all the recipient rats (n = 4) developed EAU within 4–5 days (Fig. 3F). The clinical intensity of the disease and the pathological pattern induced by CD4-depleted and CD4-nondepleted R16-specific T cells were indistinguishable (Fig. 3, G and H).

View larger version (91K): [in this window] [in a new window]   FIGURE 3. Uveitogenic activity of CD4-depleted R16-specific T cells. A–D, Activated T cell blasts from R16-immunized rats were depleted of CD4+ T cells using anti-rat CD4 Ab (OX-35) and complement, as described in Materials and Methods. CD4+ and CD8+ T cells before (A and B) and after (C and D) complement treatment were analyzed by flow cytometry for expression of CD4/CD8 and TCR. E, Proliferative response of CD4-depleted T cells to R16. CD4-depleted T cells were cultured at 37°C for 48 h in 96-well microtiter plates with medium or R16 and irradiated APCs. [3H]Thymidine was added during the last 8 h. The proliferative response is expressed as the mean cpm ± SD of triplicate determinations. F, Disease course and severity in four rats receiving 5 x 106 CD4-depleted T cell blasts evaluated by slit-lamp biomicroscopy. G and H, Histopathology of EAU in rats receiving either untreated R16-specific T cells (G) or CD4-depleted R16-specific T cells (H) (H&E, original magnification, x40). The arrows indicate inflammatory cell infiltration, new vessel information, serous retinal detachment, and photoreceptor cell damage (grade 2).

To ensure that treatment with OX-35 Ab and complement in vitro indeed completely removed CD4⁺ T cells from R16-stimulated T cells, we phenotyped the R16-specific T cells. Before in vitro R16 restimulation, the CD4⁺:CD8⁺ ratio in T cells from R16-immunized rats was 2:1 (Fig. 4A). After 2 days of stimulation with R16, CD4⁺TCR⁺ and CD8⁺TCR⁺ cells accounted for, respectively, 22% and 19% of the total activated T cell blasts (Fig. 4B), while a large population of the newly activated T cell blasts (>50–60%) expressed neither CD4/CD8 nor TCR (double negative). After treatment of these cells with OX-35 Ab and complement, the proportions of both the CD8⁺TCR⁺ and double-negative cells increased; specifically, all the TCR⁺ cells were stained with CD8 (Fig. 4C). Interestingly, after 3 days culture in IL-2-containing medium, most, if not all, of these double-negative T cells became TCR⁺CD8⁺ (Fig. 4D).

View larger version (31K): [in this window] [in a new window]   FIGURE 4. R16-activated T cells contain a high portion of TCR–CD4–CD8– cells, which, on culture in IL-2-containing medium, re-express CD8 and TCR. TCR/CD4 or TCR/CD8 subsets from R16-immunized rats were stained with combinations of labeled mAbs and analyzed by flow cytometry. A, Nylon wool-enriched splenic T cells; B, cells after 48-h stimulation with R16 plus APC; C, depletion of CD4+ T cells using anti-rat CD4 Ab (OX-35) plus complement; D, cells as in C after a further 72-h culture in IL-2-containing medium.

	Discussion

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In a previous study, we showed that the synthetic peptide, pMOG35–55, has the unexpected ability to induce CD8⁺ encephalitogenic T cells (11), although a high proportion of CD8⁺ encephalitogenic T cells could only be better demonstrated using a specific experimental protocol, in which CD4⁺ T cells were removed before Ag stimulation. We also showed that the CD8⁺ encephalitogenic T cells survive better than the corresponding CD4⁺ cells in vivo, even though they are easily overgrown by their CD4⁺ T cell counterparts in vitro. Interestingly, adoptive transfer of MOG-specific CD8⁺ T cells causes a much more severe and progressive form of EAE than the transfer of MOG-specific CD4⁺ T cells (11). We hypothesized that currently used tissue culture protocols may favor the growth of CD4⁺ T cells over that of CD8⁺ T cells in vitro, with the result that long-term cultured autoreactive T cell lines are not suitable for revealing the activity of autoreactive CD8⁺ T cells. Such a hypothesis has been supported by a recent study showing that CD8 T cells specific for -galactosidase induced ocular inflammation in transgenic mice expressing -galactosidase (35). To further determine whether autoreactive CD8⁺ T cells play a major role in other autoimmune diseases, we set out to identify and characterize such T cells in EAU.

Based on our previous observations that autoreactive CD8⁺ T cells are most easily found in primary cultures before becoming overgrown by their CD4⁺ counterparts, we examined freshly prepared R16-specific T cells. We first used CFSE staining to demonstrate the presence of CD8⁺ R16-specific T cells. CSFE prelabeling of in vivo-primed T cells derived from Lewis rats immunized with an uveitogenic peptide does not affect their Ag-specific response to the immunizing peptide (data not shown). Both CD4⁺ and CD8⁺ T cells proliferated in response to the specific Ag and not to a truncated weakly antigenic derivative, demonstrating that CFSE staining is a sensitive assay system for the detection of an Ag-specific CD8⁺ T cell response.

To examine the response of CD8⁺ T cells from Ag-primed rats on stimulation with the immunizing Ag, we first prepared highly purified CD8⁺, CD4⁺, and TCR^–CD8^–CD4^– T cells using two different or a combination of the following methods, affinity column purification, and complement-mediated depletion, and thentested the Ag-specific response of the separated T cell subpopulations. The affinity-purified CD8⁺ T cells did not show any appreciable response to the immunizing peptide (data not shown), but the CD4-depleted cells (double-negative and CD8⁺ cells) responded well. In addition, we found that, unlike T cells prepared from naive rats, T cells from immunized rats, both before and after in vitro stimulation, contained large numbers of double-negative cells, as shown by FACS analysis. These observations prompted us to determine whether the double-negative cells contained activated CD4⁺ or CD8⁺ T cells in which the expression of surface CD4 or CD8 was down-regulated. Two alternative approaches should allow us to identify down-regulated CD8⁺ T cells in the double-negative cells. First, we analyze CD4 and CD8 expression at the mRNA levels; and second, we can analyze the CD4 and CD8 expression using FACS analysis of cultured double-negative cells. Therefore, we did kinetic studies on Ficoll-separated T cell blasts exposed to immunizing peptide and APC for 48 h. Surprisingly, we found that >50% of the Ficoll-separated T cells expressed neither CD4 nor CD8 at 48 h after activation, but that, when cultured in IL-2-containing medium for 3–4 days, most, if not all, of the double-negative cells became TCR⁺CD8⁺. This implies that the double-negative T cells within the newly activated T cell blasts were predominantly derived from activated CD8⁺ T cells with down-regulated TCR and CD8 expression.

The fact that the affinity-purified CD8⁺ T cells isolated from immunized rats did not significantly respond to immunizing peptide could be due to: 1) the column-purified CD8⁺ T cells being largely Ag nonspecific, whereas the R16-specific T cells had down-regulated their CD8 expression and thus were not present in the affinity-separated cells; or 2) the highly purified CD8⁺ T cells being unable to in mount an Ag-specific response in vitro in the absence of CD4 help. We have also performed assays testing whether CD8⁺ pathogenic T cells infiltrate the inflamed eye. However, the overwhelming majority of the T cell infiltrating the inflammation is recruited by bystander response and it is thus far not possible to distinguish between the CD4 and CD8 cells that are bystander cells vs the Ag-specific cells (36, 37).

It is unclear why all the TCR^–CD8^–CD4^– cells re-expressed CD8 and TCR, but not CD4. One possibility is that CD8 is more easily down-regulated than CD4; another is that both CD4⁺ and CD8⁺ T cells undergo down-regulation, but either CD8⁺ T cells are more able to recover from down-regulation or the majority of T cells with down-regulated CD4 undergo apoptosis. Whatever the mechanism, we have previously reported that both CD4⁺ and CD8⁺ autoreactive T cells contribute to the pathogenesis of MOG-induced EAE (11, 29). In the current study, we show that uveitogenic T cells separated from R16-immunized rats remain highly uveitogenic after complete depletion of CD4⁺ T cells and that uveitogenic CD8⁺ T cells easily lose surface expression of CD8 and TCR and become double-negative when newly activated, but re-express TCR and CD8 when cultured in IL-2-containing medium.

	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 work was supported in part by National Eye Institute Grants NEI EY12974 (to H.S.) and NEI-EY014366 (to D.S.), RG3413A4 (National Multiple Sclerosis Society), the Commonwealth of Kentucky Research Challenge Trust Fund, and Research to Prevent Blindness, Inc. (New York City, NY). H.S. is a recipient of a career development award from Research to Prevent Blindness.

² Address correspondence and reprint requests to Dr. Deming Sun, Kentucky Lions Eye Center, Department of Ophthalmology and Vision Sciences, University of Louisville. 301 East Muhammad Ali Boulevard, Louisville, KY 40202. E-mail address: d0sun001{at}louisville.edu

³ Abbreviations used in this paper: EAU, Experimental autoimmune uveitis; EAE, experimental allergic encephalomyelitis; IRBP, interphotoreceptor retinal-binding protein; MOG, myelin oligodendrocyte glycoprotein; S-Ag, surface Ag.

Received for publication March 17, 2004. Accepted for publication June 2, 2004.

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