|Molecular Vision 2006;
Received 9 February 2005 | Accepted 28 February 2006 | Published 10 August 2006
Effects of cytotoxic T lymphocyte antigen 4 (CTLA4) signaling and locally applied steroid on retinal dysfunction by recoverin, cancer-associated retinopathy antigen
Akiko Maeda, Tadao Maeda, Yan Liang,
Melda Yenerel, David A. Saperstein
Department of Ophthalmology, University of Washington, Seattle, WA
Correspondence to: David A. Saperstein, MD, University of Washington, Department of Ophthalmology, Box 356485, Seattle, WA, 98195-6485; Phone: (206) 221-6425; FAX: (206) 221-6784; email: firstname.lastname@example.org
Purpose: To assess the cytotoxic T lymphocyte antigen 4 (CTLA4) pathway in the recoverin peptide (R64; AYAQHVFRSF) mouse model of cancer-associated retinopathy (CAR) and to assess the protective effects of subconjunctival triamcinalone injections in this model.
Methods: To study the role of the CTLA4 pathway on the R64-induced mouse model of CAR, BALB/c mice were immunized with R64. The mice were further intraperitoneally treated with anti-CTLA4 antibody to get stronger immunoreaction. The development of CAR was evaluated by electroretinogram (ERG) examinations 21 days after treatment. A cytotoxicity assay was employed to detect induction of R64-specific cytotoxic T lymphocytes (CTLs). Immunoblotting to assess the development of anti-recoverin antibody and a T cell proliferation assay to determine the activity of lymphocytes against R64 were examined in two experimental groups, anti-CTLA4 antibody treated and untreated mice.To study the protective effect of subconjunctival triamcinalone in this model, mice immunized with R64 peptide and anti-CTLA4 antibody were either treated with 50 mg/kg/body weight of triamcinalone or phosphate buffered saline (PBS). These mice were assayed using ERG and histological examination 35 days after the first R64 immunization.
Results: When mice were challenged with R64 peptide and anti-CTLA4 antibody, R64 peptide-specific CTLs were induced and decreased b-wave amplitudes were observed in ERG. Conversely, no CAR symptoms were detected in mice not treated with anti-CTLA4 antibody. Anti-CTLA4 antibody treatment did not give any significant differences in T cell proliferation and humoral reaction against recoverin. Subconjunctival triamcinalone treated mice show a trend toward improved survival of outer nuclear layer cell bodies, but did not show significant improvement of ERG amplitudes compared to the untreated mice.
Conclusions: Inhibition of the CTLA4 pathway is essential for the development of recoverin-induced murine CAR, suggesting that strengthening negative T cell signaling through CTLA4 may lessen the retinal degenerations in CAR-affected subjects. The positive effects of attenuation of the CTLA4 pathway must be weighed against a potential negative effect on survival since this pathway may also provide natural immunotherapy against the underlying malignancy. Subconjunctival injections of triamcinalone may have beneficial effects on the integrity of the outer nuclear layer (ONL) of the retina in the CAR model, although there was no significant effect on the ERG recordings.
Cancer-associated retinopathy (CAR) is a paraneoplastic syndrome usually associated with small cell carcinoma of the lung [1,2]. This nonmalignant complication has been shown to be secondary an autoimmune reaction to anomalous expression of recoverin in cancerous cells [3,4]. Recoverin is a retina-specific Ca2+ binding protein that is expressed in photoreceptors and retinal bipolar cells . The discovery of anti-recoverin antibodies in CAR patients sera led to the hypothesis that the retinal degeneration that occurs in CAR syndrome is attributed to retinal damage caused by these recoverin-specific antibodies that react to normal retinal tissue [6,7]. While this hypothesis is widely accepted, the method upon which the antibodies reach the retina remains controversial.
Recoverin-specific cytotoxic T lymphocytes (CTLs) that react with recoverin derived peptides R49(QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) that correspond to human recoverin amino acid sequence 49-57, 49-58, and 64-73 have also been identified in the peripheral blood of CAR patients . These CTLs may confer an improved prognosis concerning the underlying malignancy and may be related to the retinal degeneration mechanism. CAR patients are clinically characterized by the presence of photopsia, progressive visual loss, attenuated retinal arterioles and attenuation of the rod ERG recordings .
Adamus et al.  successfully induced CAR-like retinal dysfunction by injection of anti-recoverin antibody into the Lewis rat; Ohguro el al. [7,11] demonstrated potential treatments with systemic steroids, immunosuppressive agents, and Ca2+ blockers with an intravitreous antibody injected model. The effects of the immunosuppressive therapy in these experiments are limited by the nature of the CAR model. Since the Ohguro model is created by direct injection of anti-recoverin antibody in the vitreous, the cell-mediated portion of the immune response may not be elicited; therefore, the therapeutic effect of immunosuppression on this pathway cannot be ascertained. Recently a murine CAR model created by immunization with a R64 recoverin derived peptide and anti-CTLA4 antibody was developed . This model has the advantage over the antibody-generated model, as it more closely resembles the actual human disease by allowing both cell-mediated and antibody-mediated pathways to exert their respective forces on the disease process.
CTL-associated Ag 4 (CTLA4; CD152), a homolog of CD28 that binds with higher affinity to B7 molecules expressed in activated T cells, functions as a negative regulator of immune responses . CTLA4 ligation results in inhibition of IL-2 production and recent data indicate that blockage of negative T cell signaling through CTLA4 leads to continuous T cell activation, and plays important roles to induce experimental autoimmune diseases and tumor regression by antigen-specific CTLs in vivo [14,15]. Modulating CTLA4 signaling is expected to be powerful tool both for treating patients with autoimmune diseases with CTLA4Ig  and providing experimental model with anti-CTLA4 Ab .
Systemic and local therapies with steroids have been the first line treatments for inflammatory diseases of the retina. More recently, much focus has been on local steroid treatments because of the potential advantage of less systemic side effects and increased drug levels to the effected tissue. In the case of CAR, local ocular therapy may have an additional advantage since systemic immunotherapy may attenuate the body's natural immunotherapeutic effects against the underlying malignancy.In this study the goals were to: 1. Assess the role of the CTLA4 pathway in a murine CAR model generated by immunization with a recoverin peptide (R64) by employing competitive inhibition of the CTLA4 pathway with an anti-CTLA4 antibody, and 2. Treat the same mice with locally (subconjunctival) delivered steroid therapy in attempt to attenuate the retinopathy.
All experiments with mice employed procedures approved by the University of Washington Animal Care Committee and conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia. Female BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice used in these experiments were eight weeks old. Four experimental groups of mice were generated by immunization to assess the role of the CTLA4 pathway in the development of CAR in the mouse. In group 1, R64 peptide immunized mice were treated with an intraperitoneal challenge of anti-CTLA4 antibody (+R64/+CTLA4 antibody). In group 2, R64 peptide immunized mice were treated with an intraperitoneal challenge of PBS (+R64/-CTLA4 antibody). In group 3, mice were treated with anti-CTLA4 antibody without immunization with R64 (-R64/+CTLA antibody). In group 4, mice were immunized (-R64/-CTLA4 antibody). For a single replicate of the experiment, six mice were used for each of groups 1 and 2 and three mice were used for each of groups 3 and 4 (for a total of 18 mice for a single set). Three sets of the experiments were carried out. For the steroid experiments, five mice were treated with or without steroid (for a total of ten mice).
Antigens and anti-CTLA4 antibody treatment
R64 (AYAQHVFRSF) peptide that corresponds to human recoverin 64-73 (Genbank accession number P35243) was synthesized using a solid-phase peptide synthesizer PSSM-8 (Shimadze Co., Kyoto, Japan). The peptide was purified using C18 reverse-phase high-performance liquid chromatography (HPLC, Millipore/Waters, Milford, MA).
For mice immunization, 50 μg of peptides in emulsion with complete Freund adjuvant (CFA) containing Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI; 1:1, v/v) were injected subcutaneously three times every week at days 0, 6, and 20.
Anti-CTLA4 antibody (CTLA4 antibody) clone UC10-4F10-11 that was established by immunization of mouse CTLA4-mouse IgG2a fusion protein  (BD Biosciences Pharmingen, San Diego, CA) was given intraperitoneally 0, 3, and 6 days after first peptide immunization at the amount of 100 μg/mouse.
The cytotoxic activity of stimulated T cells was measured using a conventional 51Cr release assay . The target cells were labeled with 100 mCi of Na51CrO4 for 2 h at 37 °C, harvested, washed, and resuspended. Peptide-pulsed target cells were prepared by incubating the target MethA cells, a fibrosarcoma cell line derived from BALB/c mice obtained from American Type Culture Collection (Manassas, VT), at 5x105 cells/ml with 50 μg/ml of the peptide for 18 h at 37 °C, and then labeling them with 51Cr. The effector cells were placed in each well of a v-bottom microtiter plates. The labeled target cells were then added to the wells at a concentration of 5x103 cells/well in a total volume of 0.2 ml. After 4 h incubation, the release of 51Cr was measured in 0.1 ml of supernatant using a gamma counter. Measurements were carried out in triplicate, and standard deviations (SD) were calculated. The percentage of specific cytotoxicity was determined from the fraction of specific 51Cr release using the following expression: ([experimental release - spontaneous release]/[maximum release - spontaneous release]). The spontaneous release was determined by incubating the target cells alone, in the absence of effector cells, and the maximum release was obtained by incubating the target cells with 2% Nonidet-P40.
T cell proliferation assay
Draining lymph node cells pooled from three mice were passed over nylon wool columns to enrich T cells. These cells (4x105/well) were cultured with 30 Gy-irradiated syngeneic spleen cells as antigen presenting cells with 5 μg/ml of peptide in a 96 well flat-bottom microtiter plate for 72 h. Cells were pulsed with 18.5 kBq [3H]-thymidine for 8 h and [3H]-thymidine incorporation was measured. Measurements were carried out in triplicate. Means and standard deviations (SD) were calculated.
On days 7 and 21 after peptide immunization, a long lasting corticosteroid (triamcinolone acetonide; Kenalog-40®, Bristol-Myers Squibb Company, Princeton, NJ) was injected in the periocular space (subconjunctival) to anesthetized mice (intraperitoneal injection with 15 μl/g/body weight of 6 mg/ml of ketamine and 0.44 mg/ml of xylazine diluted in PBS) at dose of 50 mg/kg/body weight.
Mice were dark-adapted overnight before the experiments and anesthetized by intraperitoneal injection with 15 μl/g body weight of 6 mg/ml of ketamine and 0.44 mg/ml of xylazine diluted in PBS. The pupils were dilated with 1% tropicamide. A contact lens electrode was placed on the eye with a drop of methylcellulose and a ground electrode placed on the ear. Electroretinograms (ERG) were recorded and analyzed with the electrophysiologic system UTAS E-3000 (LKC Technologies, Inc., Gaithersburg, MD). The mice were placed in a Ganzfield chamber and single-flash ERG recordings were obtained from both eyes. Flash stimuli had a range of intensities (-3.7 to -1.4 log cd.s.m-2), and white light flash duration was adjusted according to intensity (from 20 μs to 1 ms).
Serum titer of anti-recoverin antibody
Serum titer of anti-recoverin antibody was analyzed by immunoblot and ELISA. Briefly, recombinant mouse recoverin (1 μg) was analyzed by SDS-PAGE using a 12.5% polyacrylamide gel . The protein electrotransfer onto Immobilon-P (Millipore, Bedford, MA) was carried out employing a Hoeffer mini-gel system (Amersham Pharmacia Biotech. Piscataway, NJ). For immunoblotting, the membrane was blocked with 3% BSA in PBS and incubated for 3 h with 400 times diluted sera from treated mice. A secondary antibody conjugated with alkaline phosphatase (Promega, Madison, WI) was used at a dilution of 1:5000. Antibody binding was detected by incubation with NBT/BCIP (Promega). For ELISA, 0.3 μg of recombinant mouse recoverin were adsorbed on the well surface of a 96-multiwell plate, and blocked with 1% milk in PBS. Serum samples from mice were diluted with PBS and added to each well. After 1 h incubation, unbound serum was washed away and horseradish peroxidaseconjugated anti-mouse IgG was added. The excess of conjugated IgG was washed away, and the substrate solution was added to each well and the absorbance at 405 nm was measured.
On day 35 after first immunization, eyes were enucleated and fixed for 1 h in 4% phosphate buffered glutaraldehyde and transferred into 10% phosphate buffered formaldehyde until processing. Tissues were stained with hematoxylin-eosin technique.
The number of nucleus of the outer nuclear layer (ONL) in 10 μm2 area was counted to assess the treatment's effect against retinal cell apoptosis, and compared between triamcinalone treated and not treated groups. Statistical analysis was carried out using the one way ANOVA test.
Effect of anti-CTLA4 antibody treatment
Recently, anti-tumor epitopes were identified on recoverin in CAR patients' peripheral blood that correspond to R49, R49.2, and R64 . R64 peptide also produced anti-tumor activities in BALB/c mice in vivo under usage of anti-CTLA4 antibody, which facilitates the induction of the antigen-specific T cell response in vivo, and blockage of CTLA4 signal was essential. Therefore, we tested whether anti-CTLA4 antibody treatment is required to induce retinal dysfunction .
Four experimental groups of mice were generated by immunization to assess the role of the CTLA4 pathway in the development of CAR in the mouse. Group 1; R64 peptide immunized mice were treated with an intraperitoneal challenge of anti-CTLA4 antibody (+R64/+CTLA4 antibody). Group 2; R64 peptide immunized mice were treated with an intraperitoneal challenge of PBS (+R64/-CTLA4 antibody) Group 3; Mice were treated with anti-CTLA4 antibody without immunization with R64 (-R64/+CTLA antibody). Group 4; One was not treated with either immunization (-R64/-CTLA4 antibody). All CTLA4 antibody injections were given on days 0, 3, and 6 post-first immunization, and ERGs were recorded after 21 days.
As shown in Figure 1, mice immunized with R64 peptide alone (+R64/-CTLA4 antibody) showed no significant changes in the ERG amplitudes as compared to either of the control arms (-R64/+CTLA4 antibody and -R64/-CTLA4 antibody), suggesting no retinal degeneration was present. In contrast, ERG amplitudes of +R64/+CTLA4 antibody treated mice were approximately 200 μV lower between -3.7 and -1.4 log cd s m-2 stimulus intensity than those of control mice, suggesting retinal degeneration.
Next, to investigate whether R64-specific immunoreactivity that induces retinal dysfunction is affected by the treatment with anti-CTLA4 antibody, T cell responces and antibody production were examined. Because R64 reactive CD8+ T cells are known to exist in CAR patients and model animals, CTLs activity of treated mice were evaluated by cytotoxic analysis. Cytotoxicity testing revealed that no specific killing activity was detected from mice without anti-CTLA4 antibody treatment, whereas peptide-specific cell lysis was observed in +R64/+CTLA4 antibody mice in an effector cell ratio dependent manner (Figure 2A). These R64-specific CTLs did not show any killing activity on control recoverin peptide pulsed cells (data not shown).
To assess the reaction of immunocytes against R64 peptide, the T cell proliferation assay was performed with +R64/+CTLA4 antibody or +R64/-CTLA4 antibody and -R64/±CTLA4 antibody mice. The T cell proliferation assay showed similar immunoreactivity in both groups treated with R64 peptide (+R64/+CTLA4 antibody and +R64/-CTLA4 antibody). The mice not immunized with R64 peptide (-R64/+CTLA4 antibody and -R64/-CTLA4 antibody) showed no specific T cell activity. (Figure 2B).
To further examine whether anti-CTLA4 antibody regulates anti-recoverin antibody production that is thought to be correlated with retinal dysfunction, immunoblotting and ELISA using recombinant mouse recoverin were performed to measure these levels in the serum. Immunoblot assays revealed the serum titer of anti-recoverin antibody was similar between anti-CTLA4 antibody treated and untreated mice (Figure 2C). ELISA results showed 0.95±0.24 or 0.98±0.14 (absorbance, mAU) at 400 times dilution of their sera, and anti-recoverin antibody titer was also comparable between steroid treated and untreated mice. Control serum from mice that were not treated with R64 recoverin peptide showed 0.047±0.008 (absorbance, mAU) in the ELISA.
Histology and steroid treatment
The administration of subconjunctival triamcinalone had no statistically significant difference between the control and steroid treated group on the ERG amplitudes at day 35 after the first peptide immunization (Figure 3). Anti-recoverin antibody titer was detected equally in the both steroid treated and untreated +R64/+CTLA4 antibody immunized mice (Figure 4A). In addition, ELISA results indicated 1.04±0.18 or 0.97±0.24 (absorbance) at 400 times dilution of their serum, whereas recoverin untreated control mouse sera showed 0.053±0.005 (absorbance). Both immunoblot and ELISA data suggested that anti-recoverin antibody titer of steroid treated or untreated mice was similarly high level, and steroid treatment did not reduce anti-recoverin antibody production. Histopathologic examination of the +R64/+CTLA4 antibody immunized mice showed no evidence of cellular infiltrate in the neural retina. There was some evidence of decreased cell number in the ONL in these mice as compared to the unaffected mice. Treating the +R64/+CTLA4 antibody mice with subconjunctival injections of triamcinalone did not appear to prevent the ERG loss seen in these mice (Figure 3). However, histopathologic examination of the steroid-treated mouse retinas revealed a trend toward the prevention of ONL thinning and the number of nucleus in ONL was preserved significantly (Table 1; p<0.05, one way ANOVA) as compared to the untreated CAR mice (Figure 4B).
Albert et al.  first reported the possibility of neuron-specific CTL activity in paraneoplastic syndrome and showed neural cell lysis by cdr2-specific CTLs. Subsequently, neuron-specific CTLs were detected in paraneoplastic syndrome patients; these CTLs are proposed to contribute to the antigen-specific organ damage and rejection of cancer cells expressing the same antigen [8,20]. CAR patients possess recoverin-specific CTLs in their peripheral blood, and recoverin-specific CTLs can recognize aberrantly-expressing recoverin in cancer cells . In CAR, retinal degeneration is known to be induced by anti-recoverin antibody, but the roles of recoverin-specific CTLs on retinal degeneration are still unclear. Immunoreaction against recoverin is one of the key mechanisms that underlie this immune-mediated retinal degeneration. In this study, T cell proliferation and antibody production against recoverin were almost comparable between +R64/+CTLA4 antibody treated mice and +R64/-CTLA4 antibody treated mice, whereas cytotoxic activity was observed only in +R64/+CTLA4 antibody treated mice. This fact indicates that retinal dysfunction induced by recoverin subcutaneous immunization requires the recoverin-specific CTLs existence together with production of anti-recoverin antibody.
CTLA4 is a suppressive signal of T cell activation, and Leach et al.  suggested that in mice the anti-CTLA4 antibody treatment is essential to elicit antigen-specific CTLs in vivo. The mechanism of inhibition of T cell activation is a result of enhanced signals through B7/CD28 pathways. Linsley et al.  bound an extracelluar domain of mouse CTLA4 to a constant region of human IgG1, forming a fusion protein CTLA4Ig, which had high affinity with B molecules of CD28. Treatment with CTLA4Ig recombinant fusion protein can lead to a satisfactory immune result in heart, kidney, and islet transplantations [21-23]. In addition, therapeutic effects of CTLA4Ig were reported in CD4+ T cell mediated autoimmune diseases, such as systemic lupus erythematosus (SLE), experimemtal autoimmune uveoretinitis, experimental encepharomyelitis, etc [24-26].
These observations indicate that blocking CTLA4 signaling leads to activation of T cell response. Therefore, blocking of CTLA4 activity might be important to observe CTL activities in vivo as previously proposed . Our study showed that CTLA4 modulation was required to obtain CTL activity and retinal dysfunction. However, immunocyte invasion into the retina was not observed in the anti-CTLA4 antibody treated or untreated mice. Therefore, we conclude that the attenuation of a- and b-waves in ERG which were detected in the mice treated with recoverin peptide and anti-CTLA4 antibody, was not induced by direct infiltration of activated immunocytes. This silent immunoreaction to retina is similar to rejection of allogeneic retinal tissue implanted in the subretinal space . At the early stage of CAR, no retinal degeneration is observed, and only ERG abnormality is recorded . These phenomena might result from the existence of immune privilege in the subretinal space. Histopathologic studies of later stage disease revealed loss of photoreceptor cells in CAR patients and significant thinning of outer nuclear layer, inner nuclear layer, and outer segments similar to the rat model . However, inflammatory changes such as destruction of the morphology and lymphocyte infiltrations were not reported. In the mouse model presented here, no significant inflammatory changes were detected in the retina. Additionally, there was only minimal anatomical destruction compared to human CAR or the anti-recoverin antibody injection model, which showed that when anti-recoverin antibody was injected into the vitreous, a nonrecordable ERG and retinal nuclear cell apoptosis were detected . This suggests that anatomical retinal degeneration starts with thinning of the ONL in CAR. In our study, the +R64/+CTLA4 antibody treated group had fewer cells in the ONL compared with the group injected with only +R64/-CTLA4 antibody (data not shown). Thus, this model may mimic the early stage of degeneration.
Local steroid treatment is a common therapy for chronic or severe noninfectious inflammatory eye diseases . Systemic immunosuppressive therapy can have many adverse side effects. Particularly in the CAR patient, one of these effects could result in lowering the body's ability to provide autoimmune therapy to treat the underlying malignancy. In this study, local steroid treatment did not have a protective effect on the ERG amplitudes similar to the previous systemic steroid treatment done by Ohguro et al. . This is likely because the local therapy does not reduce the formation of anti-recoverin antibody. Despite the lack of apparent effect on the ERG, the local steroid treatment may have a positive effect on ONL cell survival that was not detected in the previous study examining systemic steroid medication . Although these data do not support the use of local steroid to treat CAR, the trend toward ONL survival warrants further study of the effects of local immunosuppresion in CAR.
In summary, to induce CAR-like symptoms in vivo, not only exposure to recoverin antigen, but also further immunomodulation was required. This may explain why, although aberrant recoverin expression in cancer cells occurs frequently, CAR remains a very rare paraneoplastic syndrome . This suggests that complicated impairments of immunosignaling are likely occurring in paraneoplastic syndromes like CAR. To discover effective treatments for CAR patients may require equally sophisticated therapies. These may include local and systemic immunosupression and specific immunomodulating agents that target CTLA4-Ig and anti-apoptotic molecules.
We would like to thank Dr. K. Palczewski for his great help during the course of this study. This research was supported in part by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists, the Naito Foundation, The Foundation Fighting Blindness Career Development Award. Health Service grants EY01730 and EY08061 from the National Eye Institute, National Institutes of Health, Bethesda, MD, an unrestricted Grant from Research to Prevent Blindness, Inc. (RPB), New York, NY to the Department of Ophthalmology at the University of Washington, grants from the Ruth and Milton Steinbach Fund and the E. K. Bishop Foundation.
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