Induction of Experimental Autoimmune Keratitis by Adoptive Transfer of Human Corneal Antigen–Specific T-Cell Line

Abstract

purpose. To establish a permanent human corneal antigen (HuCOAg)-specific T-cell line and to determine whether line cells are capable of inducing inflammatory keratitis by adoptive transfer.

methods. Lymphoid cells harvested from HuCOAg-immunized Lewis rats were expanded to a permanent T-cell line by repetitive cycles of restimulation with HuCOAg and irradiated antigen-presenting cells and propagation in interleukin 2–containing medium. The phenotype and epitope specificity of the line cells were determined. Adoptive transfer was performed after seven cycles by intraperitoneal injection of activated T cells into irradiated recipient rats.

results. A panel of 11 overlapping synthetic HuCOAg peptides to identify T-cell epitopes recognized by the line cells was used. The cells responded selectively to a synthetic peptide containing an immunodominant epitope of HuCOAg (peptides 69–83). Line cells bore the surface phenotype of the T-helper/inducer marker (W 3/25⁺ or CD4⁺). Intraperitoneal inoculation of naive rats with 5 × 10⁷ activated line cells led to maximal clinical signs of stromal keratitis 7 to 9 days after transfer, characterized by corneal haze, conjunctival and episcleral injection, corneal infiltrates, and neovascularization. Histopathologic examination of the tissues revealed numerous lymphocytes and macrophages and some polymorphonuclear leukocytes along with neovascularization. The pathologic lesions were confined to the peripheral corneal stroma. Immunohistochemical studies demonstrated that the overwhelming majority of the inflammatory cells were CD4⁺ T lymphocytes and macrophages; an upregulation of major histocompatibility complex class II antigen expression was also noted.

conclusions. A long-term, rat T-cell line of CD4⁺ phenotype specific for HuCOAg that can induce autoimmune keratitis by adoptive transfer of the line cells to naive syngeneic recipients is described. With the development of this cell line, the mechanisms by which T cells exert their immunopathologic effects in experimental autoimmune keratitis models can be studied.

Sterile corneal ulcerations may occur either as an isolated ocular problem, such as Mooren’s ulcer, or associated with an underlying systemic vasculitic or connective tissue disease such as rheumatoid arthritis or Wegener’s granulomatosis. Regardless of the etiology, these ulcers usually progress relentlessly and may result in loss of vision. Although the exact pathogenesis of noninfectious stromal keratitis remains uncertain, considerable evidence suggests that these disorders may result from an autoimmune response to certain corneal autoantigens. 

We have previously identified a corneal antigen (COAg) and have studied its role in the pathogenesis of Mooren’s ulcer and have demonstrated the occurrence of both cellular and humoral immune responses to COAg in patients with this condition. ^{1 2} We have also cloned both bovine and human COAg (HuCOAg), ^{3 4} and the nucleotide sequence of COAg cDNA is known to be identical with that of neutrophil calgranulin C. Interestingly, the deduced amino acid sequence of HuCOAg cDNA is identical to that of a protein identified on the surface of Onchocerca volvulus extracts from human SC nodules. ⁵ This protein is thought to interact with the nematode surface after release by activated neutrophils and may be involved in attacking microfilaria. ⁵ A subsequent study found that recombinant HuCOAg could immobilize and kill microfilaria and adult worms in an in vitro culture system. ⁶ HuCOAg thus appears to be both a corneal autoantigen and an immune mediator capable of participating in host defense. 

In the present study, we generated a permanent T-cell line specific for the HuCOAg protein to investigate, by adoptive transfer of line cells, the immunopathogenic mechanisms of autoimmune stromal keratitis induced by this stromal antigen. 

Materials and Methods

Animals

Inbred female Lewis rats (Charles River, Walkerville, MD) were housed under standard conditions and maintained on laboratory chow and water ad libitum. Rats were used at age 8 to 12 weeks in all experiments. Treatment of animals adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Generation of a HuCOAg-Specific Cell Line

Recombinant HuCOAg protein was prepared as previously described. ⁴ T-cell lines specific for HuCOAg were established from Lewis rats as described by Kojima et al. ⁷ Briefly, we immunized female Lewis rats with 50 μg HuCOAg emulsified in an equal volume of CFA containing 2 mg/ml of Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). Ten days after immunization, draining lymph nodes were aseptically removed, teased, and pressed on wire mesh screen. The cell suspension was washed and cultured (5 × 10⁶/ml) in medium A (RPMI-1640 supplemented with sodium pyruvate, 1 mM; HEPES, 0.01 mM; nonessential amino acids, 0.1 mM; penicillin, 100 U/ml; streptomycin, 100 g/ml; glutamine, 1 mM; 2-mercaptoethanol [2-ME], 5 × 10⁻⁵ M, 1% syngeneic rat serum) and HuCOAg (10μ g/ml) in T-25 flasks for 3 days at 37°C in the presence of 5% CO₂. Blasts we separated on lympholyte (Accurate, Westbury, NY) and expanded in medium B (medium A supplemented with 10% heat-inactivated fetal calf serum [Life Technologies, Gaithersburg, MD] and 15% rat T-cell growth factors [Collaborative Biochemical, Becton Dickinson, Bedford, MA]) in the absence of HuCOAg protein. After incubation for 7 days, cells were washed and re-suspended in medium A (1 × 10⁵ cells/ml) and cultured with irradiated (4000 R) syngeneic thymocytes (1 × 10⁶ cells/ml) for 3 days. The T-cell line was alternately stimulated and expanded until proliferation was restricted to HuCOAg. 

Membrane Phenotype Analysis

We investigated the surface membrane of the HuCOAg-specific, T-cell-line cells by indirect immunofluorescence staining. Monoclonal antibodies (MoAb) directed to the rat surface antigens included pan-T (W3/13), T helper/inducer (W3/25), T suppressor/cytotoxic (OX8), and Ia antigens (OX6; Biosource International, Camarillo, CA). Viable line cells (2 × 10⁵) were washed with PBS containing 0.2% BSA and 10 mM sodium azide and incubated with the primary MoAb for 1 hour on ice. The cells were then washed to remove the primary MoAb and stained with fluorescein-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO) for 1 hour on ice. We determined the surface phenotype using FACScan (Becton Dickinson, Sunnyvale, CA). 

Epitope Specificity of the HuCOAg-Specific T-Cell Line

We used a panel of 11 overlapping synthetic HuCOAg peptides (Quality Controlled Biochemicals, Hopkinton, MA) to identify T-cell epitopes recognized by the HuCOAg-specific T-cell line. Each peptide contains 15 amino acids, covering the entire sequence of HuCOAg, with 7 to 8 amino acids overlapping between adjacent peptides at each end. 

HuCOAg-specific, T-cell line cells (2 × 10⁴ cells/well) were cultured in triplicate with irradiated (4000 R) syngeneic thymocytes (1 × 10⁶ cells/well) as antigen-presenting cells in the presence or absence of the synthetic peptides, HuCOAg, and PPD control protein (10 μg/ml) in 0.2 ml of complete medium in 96-well, round-bottomed microtiter plates. After 56 hours, 1 μCi/well of [³H]thymidine was added, and the cells were harvested 16 hours later on fiberglass filters. We measured incorporation of[ ³H]thymidine with a liquid scintillation counter. The proliferative response was expressed as mean cpm ± SD of the triplicate determination. 

Induction of Experimental Autoimmune Keratitis by Adoptive Transfer

Line cells were activated in culture for 3 days with HuCOAg (10μ g/ml) and irradiated syngeneic thymocytes before the adoptive transfer. All recipient animals were irradiated at 650 R before the transfer. 

We performed the adoptive transfer by intraperitoneal injection of the activated cells at variable concentrations (5 × 10⁶ to 50 × 10⁶), washed the cells three times with RPMI-1640, and suspended them in 1.0 ml RPMI-1640. Control rats were injected with Con A–activated lymphocytes harvested from the spleen and lymph nodes of naive animals. We monitored the animals daily for clinical signs and severity of corneal lesions. 

Histologic Assessment of Stromal Keratitis

Nine days after induction of disease, we killed the animals for immunohistologic evaluation. The enucleated eyes were immersed for 1.5 hours at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) and 5% sucrose. The specimens were then rinsed twice in cool (4°C) 0.1 M PB with 20% sucrose and incubated overnight in the same solution. The specimens were placed in plastic biopsy molds containing a 2:1 mixture of 0.1 M PB with 20% sucrose and OCT. The plastic molds were frozen using a mixture of dry ice and methylbutane, and the blocks were stored at −70°C until sectioning. 

Serial frozen sections (8 μm), cut across the center of the cornea along the optic axis, were placed on gelatinized slides, and the slides were kept at room temperature for 1 hour before immunohistochemical staining. After brief fixation in methanol and quenching of endogenous peroxidase with 3% H₂O₂, the sections were washed and stained by an enhanced four-step immunoperoxidase method (Vectastatin Elite ABC; Vector Laboratories, Burlingame, CA). We used mouse anti-rat antibodies at a concentration of 1 to 2 μg/ml as primary antibodies (Table 1) . After overnight incubation in a moist chamber at 4°C, the primary antibodies were washed from the cells. We then applied the secondary antibody, biotin-labeled horse anti-mouse IgG depleted of antibody cross-reactivity with rat IgG. After a 30-minute incubation, the cells were washed, and avidin-biotin-peroxidase complex 1:100 was added for 45 minutes. The slides were again washed and developed in the substrate of 3-amino-9-ethyl-carbazole (EAC) solution. We used frozen sections of rat spleen as positive controls. 

We quantified the infiltration of immunopositive cells into the tissues as follows: −, absent; ±, very few positive cells; +, some positive cells; + +, many positive cells; and + + +, very many positive cells. Positively stained cells were scored in three different areas: limbus, peripheral cornea, and central cornea. 

Results

Epitope Specificity of the HuCOAg-Specific T-Cell Line

A T-cell line specific for HuCOAg protein was selected from lymph node cells of Lewis rats immunized 10 days earlier with recombinant HuCOAg in CFA. Initially, lymph node cells responded to both HuCOAg and PPD. After three cycles of stimulation with HuCOAg and propagation with IL-2, the cell line lost responsiveness to PPD and responded exclusively to stimulation with the HuCOAg protein. The cell line was propagated and selected for four more rounds before transfer to naive recipients. 

To evaluate the epitope specificity of the line cells, we tested proliferation responses with 11 synthetic peptides covering the entire sequence of HuCOAg. Cells responded only to peptides 69 to 83, located in the C-terminal sequence of the HuCOAg protein (Fig. 1) . Other peptides failed to produce a proliferative response in this T-cell line. 

Surface Phenotype of the HuCOAg-Specific T-Cell Line

The surface membrane phenotype of the cell line was determined with a panel of specific monoclonal antibodies. The great majority of the line cells were strongly stained with antibodies W3/13 and W3/25, which are specific for T cells and for the helper/inducer T-cell subset, respectively (Table 2) . Minimal or no staining occurred with antibodies OX8 and OX6, which define suppressor/cytotoxic T cells and Ia antigens in the rat. 

Clinical Features of Keratitis

To determine the ability of the HuCOAg-specific T-cell line to induce clinical signs of autoimmune stromal keratitis (corneal infiltration and neovascularization) in recipient rats, we injected graded doses of cells into recipients intraperitoneally. We found a relationship between the cell dose and the severity of clinical signs in the recipient animals. The smallest number of cells required to produce clinical disease was 5 × 10⁶. When 5 × 10⁷ activated line cells were injected, approximately 80% of the recipients developed severe clinical signs of stromal keratitis. The first clinical signs were noticeable on days 4 to 5 after the transfer. 

The first signs of disease were episcleral and conjunctival hyperemia and peripheral corneal haze, followed by corneal edema and in some cases episcleral hemorrhages. Peripheral neovascularization of the cornea developed 5 to 7 days after transfer. Maximal clinical signs occurred 7 to 9 days after inoculation, followed by a rapid clinical recovery. Control rats, irradiated with transferred Con A–activated T cells, had some transient corneal edema (and hyphema in one eye only), but no neovascularization or corneal inflammation developed. 

Histopathology

We performed histopathologic and immunohistochemical analyses of the enucleated globes on day 9 after the adoptive transfer. Light microscopic examination of the hematoxylin-eosin–stained sections showed an inflammatory infiltrate composed of numerous lymphocytes and macrophages and some polymorphonuclear leukocytes, mainly in the limbus and peripheral cornea, associated with intense neovascularization (Fig. 2) . Immunohistochemical studies showed that the overwhelming majority of the inflammatory cells were T lymphocytes and macrophages (Fig. 3) , which were most prominent in the limbus and peripheral corneal stroma (Table 3) . Examination of other structures, including retina, lens, and sclera, failed to reveal any abnormalities. No W3/25, W3/13, or CD8 cells were evident in the control rate eyes, but some OX6- and ED1- and numerous ED2-positive cells were observed only in the limbal area. 

Discussion

We describe here the development of a long-term rat T-cell line of CD4⁺ phenotype specific for HuCOAg that is capable of inducing autoimmune keratitis by adoptive transfer of line cells to naive syngeneic recipients. 

Several autoreactive T-cell lines capable of adoptively transferring disease have been developed for experimental autoimmune encephalomyelitis (EAE), ⁸ experimental autoimmune thyroiditis (EAT), ⁹ experimental autoimmune arthritis (EAA), ¹⁰ experimental autoimmune uveoretinitis (EAU), ¹¹ and experimental autoimmune keratitis (EAK). ¹² These lines generally produce their immunopathogenicity through a delayed-type hypersensitivity (DTH)-like mechanism elicited by the specific antigen. However, some of the EAE lines generate an antigen-specific cytotoxic response against syngeneic targets, and only those lines with cytolytic capacity can clinically induce EAE. ^{13 14} The EAU lines have not yet been shown to cause specific cytolysis in vitro. 

The HuCOAg-specific T cells developed in this study appear to exert their immunopathogenic effects through a DTH-like reaction. Presumably, HuCOAg-specific T cells enter the cornea after intraperitoneal injection, with MHC class II–positive cells presenting HuCOAg to the CD4⁺ T cells. The release of lymphokines would amplify the inflammatory reaction by recruiting additional antigen-nonspecific cells. Previous adoptive transfer experiments have indeed demonstrated that only small numbers of infiltrating cells are specifically sensitized against the initiating antigen ^{15 16} and that many of the inflammatory cells are nonspecific mononuclear cells recruited by inflammatory cytokines released by the sensitized lymphocytes. ¹⁷ Thus, it is likely that tissue damage is mediated through the activities of these antigen-nonspecific inflammatory cells, mainly macrophages. The immunohistologic analysis in this study confirmed that macrophages and T lymphocytes are the major constituents of the inflammatory infiltrate in our EAK model. 

Several studies have sought to determine the role of humoral and cellular autoimmunity in the pathogenesis of sterile corneal melting. The presence of corneal epithelial antibodies in patients with sterile corneal ulcerations has been demonstrated, suggesting a role for humoral immunity in the pathogenesis. ^{18 19} However, other studies have shown that the presence of corneal epithelial autoantibodies was not restricted to patients with corneal inflammation; patients with uveitis also have cornea-specific antibodies. ²⁰ In a later study, a soluble 54-kDa corneal epithelial antigen, BCP 54, the major protein in corneal epithelium, was isolated from the serum of a patient with corneal ulceration and presence of autoantibodies to this antigen in patients with corneal ulcerative diseases, corneal transplants, and uveitis as well as in a small percentage of the normal population was demonstrated. ²¹ BCP 54 was identified as a class 3 aldehyde dehydrogenase (3-ALDH). ²²  

It is known that a single injection of a soluble antigen in rabbit cornea causes a severe delayed type of inflammatory response called Wessely’s phenomenon, which is mainly mediated by complement (Wessely K, Über anphylaktische Erscheinungen an der Hornhault, Munch med Wschr., 1911). In the rat model of this reaction there appeared striking differences clinically and histopathologically compared with rabbits. The corneal inflammation was markedly less, and no plasma cells were observed in the corneal sections. ²³ Furthermore, experiments with intracorneal antigen challenge in complement-depleted rats suggested that the antigen-induced keratitis model in rats is primarily cell mediated. ²⁴ In their most recent study, Verhagen et al. ¹² reported an experimental autoimmune keratitis model in rats induced by adoptive transfer of BCP 54–sensitized T cells. Using MoAb staining, they found numerous T cells and macrophages and an aberrant expression of MHC class II antigens, suggesting a cellular immune response. With this data, we intended to produce a rat model of Mooren’s ulcer by transferring the HuCOAg-sensitized syngeneic T cells. However, we achieved the clinical signs of a peripheral stromal inflammation rather than frank ulceration. Although the histopathologic findings were similar to those in Mooren’s ulcer, with heavy lymphocyte infiltration in the corneal stroma sparing the endothelium and absence of vasculitis, no plasma cells in the vicinity of the stromal infiltrate were visualized. The results of our experiments also confirmed that a cellular immune response, likely a DTH type, is the immunopathologic mechanism of autoimmune stromal keratitis in rats. Plasma cells do not play a role in rat model in contrast to human disease. Whether this phenomenon explains the difference in clinical severity is unknown. 

The T-cell line developed in this study is specific for a novel human corneal autoantigen, HuCOAg, which was characterized earlier ^{4 5} and found to be identical with calgranulin C. The line to this antigen shares several fundamental characteristics with a previously described autoagressive T-cell line to the S100 protein, S100β. ⁷ S100β (calgranulin B) is similar to HuCOAg (calgranulin C), which belongs to the S100 superfamily of Ca²⁺-binding proteins. S100β is expressed by astrocytes in the central nervous system (CNS), by Schwann cells in the peripheral nervous system, and by Müller cells in the retina. ²⁵ Adoptive transfer of S100β-specific T-cell-line cells into naive recipients induces intense inflammation throughout the CNS, uvea, and retina. ⁷ HuCOAg-specific T-cell transfer induces intense inflammation, which is confined to the corneal stroma. The tissue specificity of disease induction by these two lines is a consequence simply of the distribution of tissue-specific autoantigen (S100β in the retina, HuCOAg in the corneal stroma). 

S100β antigen shares a 42% overall sequence homology with HuCOAg, but there are striking differences between the amino acid sequences of these two proteins at the C-terminal ends. ^{12 13} Interestingly, the pathogenic epitopes of these proteins are located at the C-terminal ends (peptides 76–91 for S100β and peptides 69–83 for HuCOAg) and completely differ in amino acid sequence: FVSMVTTACHEFFEHE (S100β, calgranulin B) and DEQEFISLVAIALKA (HuCOAg, calgranulin C). 

The S100β-specific T-cell lines do not cause in vitro specific cytolysis of syngeneic astrocytes pulsed with S100β. ⁷ These line cells secrete high levels of IFN-α into the culture medium, indicating that the line cells belong to the T-helper lymphocyte type 1 (Th1) subset of CD4⁺ T cells. Because Th1 cells are known to mediate a DTH response, ^{26 27} experimental autoimmune panencephalitis and uveoretinitis mediated by S100β-specific T-cell line are likely to result from Th1 cytokine-mediated DTH reactions. We adopted this method of selection and propagation to establish our HuCOAg-specific T-cell line, and the autoimmune stromal keratitis in our animal model likely represents a DTH response with subsets of CD4⁺ T cells generating inflammatory and regulatory cytokines that recruit and direct antigen-nonspecific cells to exert tissue damage. However, both CD4⁺ lymphocytes and cytotoxic lymphocytes were present in the active disease. Repeated antigen selection of bulk T-cell lines, as in our study, may lead to a limited representation of the most active lines. All other T-cell specificities in the immunized lymph node population were lost during the in vitro selection procedure. 

As our results show, immunodominant T-cell specificities that arise during selection of T-cell lines with the whole HuCOAg molecule define the pathogenic epitope on HuCOAg. Because T cells mediate their effects via cytokines, which cytokines these cells produce at the site of inflammation is highly relevant. Work is now under way to examine the Th1 and Th2 cytokines in the cornea during experimental autoimmune keratitis induced by adoptive transfer of the HuCOAg-specific T-cell line. Understanding T-cell mechanisms of inducing keratitis may be important in developing immunotherapeutic strategies for treating human autoimmune corneal disease. 

 Supported by National Eye Institute Grant EY11096.

 Submitted for publication April 18, 2000; revised August 8, 2000; accepted September 8, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Antibodies Used in this Experiment

Clone	Source	CD Specificity
W3/13	Serotec; MAS010c	CD43+ T lymphocytes, plasma cells, polymorphonuclear cells
W3/25	BioSource International; ARS0401	CD4+ T-helper lymphocytes, peripheral T cells, macrophages
OX8	Sera-Lab; MAS041c	CD8+ T-cytotoxic/suppressor cells/subset of natural killer cells
ED1	Serotec; MCA341	Macrophages, monocytes, and dendritic cells (cytoplasmic antigen)
ED2	Serotec; MCA1333	Resident macrophages (cell surface marker)
MRC OX6	Serotec; MCA46R	MHC class II (rat I-A)

Figure 1.

 

Epitope specificity of the HuCOAg-specific T-cell line. Cultures were established with 2 × 10⁶ line cells and 1 × 10⁶ irradiated (4000 R) thymocytes as antigen-presenting cells (APC) in each well. HuCOAg was added at 10 μg/ml. The cells were incubated for 72 hours, and [³H]thymidine was added 16 hours before harvest. The counts of [³H]thymidine incorporation are expressed as cpm ± SD minus the cpm of background wells (1268 ± 20 cpm, line cells and APC without antigen or peptide).

Table 2.

 

Surface Phenotype of HuCOAg-Specific T-Cell-Line Cells

Monoclonal Antibody	Specificity	% Positive Cells*
;l>W3/13	Pan-T	90
W3/25	T helper/inducer	83
OX8	T suppressor/cytotoxic	2
OX6	Ia antigens	0

*  The background (percentage of cells positive for second antibody alone) was 1–4% and has been subtracted.

Figure 2.

 

Histopathology of the stromal keratitis induced by HuCOAg-specific T-cell line cells. This H&E-stained corneal section was obtained from a representative recipient 9 days after adoptive transfer of 5 × 10⁷ line cells. Inflammatory cell infiltration and neovascularization are restricted to the stroma. Original magnification, ×200.

Figure 3.

 

Immunohistochemistry of peripheral corneal sections with EAK. The positive cells stain brown with amino ethyl carbazole. Original magnification, ×64. (A) W3/13. PMC, plasma cells, T cells. (B) W3/25. T-helper lymphocytes. (C) OX8. T-suppressor lymphocytes. (D) ED1. Macrophages, monocytes, and dendritic cells. (E) ED2. Resident macrophages. (F) OX6. MHC class II antigen.

Table 3.

 

Immunohistologic Analysis of Inflammatory Cells

	W3/13 PMC, Plasma Cells, T Cells	W3/25 T-Helper Lymphocytes	OX8;0>T-Suppressor Lymphocytes	ED1;0>Macrophages, Monocytes, and Dendritic Cells	ED2;0>Resident Macrophages	OX6;0>MHC Class II
Limbus	++	+	+	++	+++	++
Peripheral cornea	+++	++	+	+++	+	+++
Central cornea	+	∓	−	+	−	++