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Investigative Ophthalmology & Visual Science, Vol. 32, No. 9, August 1991 Copyright © Associuiion for Research in Vision and Ophthalmology Enumeration of Auforeactive Helper T Lymphocytes in Uveitis E. Mirchel Opremcak, Aimee D. Cowans, Charles G. Orosz, Patrick W. Adams, and Ronald L. Whisler In a one-stage, interleukin-2 (IL-2), limiting-dilution analysis, peripheral blood mononuclear cells from patients with uveitis and normal control subjects were assayed for S-antigen specific, tetanus-specific, and in vivo activated helper T cells. Control subjects consistently demonstrated tetanus-specific responses, but neither in vivo activation nor S-antigen specific helper T cell responses were seen. Patients with active forms of diffuse, posterior, and anterior uveitis were found to have significant frequencies of both in vivo activated and S-antigen specific helper T cells in their peripheral blood. These data show that patients with certain forms of uveitis have a measurable frequency of lymphocytes in the peripheral immunologic compartment capable of secreting IL-2 in response to autologous presentation of ocular autoantigen (S-antigen). Limiting-dilution analysis techniques, generating minimal respondcr cell frequency estimates and distinct IL secretion patterns, may provide an index of disease activity and critical information about the mechanism(s) of ocular inflammation. Invest Ophthalmol Vis Sci 32:2561-2567,1991 Histologic and immunohistochemical analysis of eyes from patients with sympathetic ophthalmia, Vogt-Koyanagi-Harada (VK..H) disease, and birdshot chorioretinitis show T cell infiltration.910 Helper T cell subsets appear to dominate the early phases of the disease, and suppressor T cells are found in the chronic phases.10 In these disorders, in vitro lymphocyte proliferation assays document T cell responsiveness to retinal autoantigens.11"13 From these data, it would appear that cell-mediated immunity and, specifically, autoreactive T cells play an important role in mediating certain forms of uveitis. Most of the information regarding T lymphocyte autoreactivity in uveitis has been qualitative in nature, employing clinical descriptions or immunohistochemistry.910 Other studies used conventional lymphocyte proliferation assays to establish the presence of cell-mediated immune responses to autoantigens (S-antigen or interphotoreceptor retinoid binding protein (IRBP)).11-14 The magnitude of the proliferative response in these assays bears little relationship to the actual numbers of helper T cells required to generate this response.15 Specific cytokine release also is not determined by proliferation assays. Such assays are influenced by multicellular interactions and represent a bulk response.15"17 An accurate method to determine the size and cytokine secretion patterns of the autoreactive T cell pool in patients with uveitis would be clinically important. Antigen-specific forms of immunotherapy and anticytokine drugs could be used based on this information. Disease remission and response to various therapies could be monitored by such a noninvasive, quantitative measure of autoreac- Uveitis is an important cause of visual impairment in the United States.1 In general, the immunologic mechanisms responsible for ocular inflammation are not well denned. Patients with uveitis often are managed empirically because of our lack of adequate information regarding the status and contribution of the underlying immunologic and inflammatory mechanisms. An accurate index of disease activity and more precise knowledge of the predominant pathophysiologic process governing uveitis are required to evaluate, treat, and follow these diseases effectively. Several lines of evidence support a dominant role for autoreactive T lymphocytes in mediating ocular inflammation. Animal models of uveitis showed that immunization with retinal S-antigen and interphotoreceptor retinoid binding protein results in a bilateral chorioretinitis.2"4 Moreover, this inflammation can be induced by the adoptive transfer of in vivo or in vitro sensitized, antigen-specific T cells to naive recipients.5'6 Immunomodulation of T cell function with cyclosporine arrests the inflammation, lending further support to the importance of autoimmune T cells in uveitis.78 From the Ohio State University College of Medicine, Departments of Ophthalmology, Surgery and Medicine, Columbus, Ohio. Supported in part through the Central Ohio Lion's Eye Organization, the Hirsch Funds, and the Kricker Foundation. Submitted for publication: December 12, 1990; accepted April 12, 1991. Reprint requests: E. Mitchel Opremcak, MD, The Ohio State University College of Medicine, Department of Ophthalmology, 456 West Tenth Avenue, Columbus, OH 43210. 2561 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017 2562 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Augusr 1991 tivity. Limiting-dilution analysis provides this information by obtaining minimal frequency estimates for a specific T cell population. This system also avoids the "bulk" response problem inherent in conventional lymphocyte proliferation data.15"19 By quantifying autoreactive T cells and identifying their antigen specificity, lymphokine secretion patterns, activation status, and unique immunologic compartment differences, limiting-dilution analysis also may provide fundamental information about the immunopathogenesis of uveitis. We describe a method to enumerate autoreactive helper T cells in the peripheral blood of patients with uveitis. We used limiting-dilution analysis techniques to compare minimal frequency estimates of T cells in the peripheral blood of patients with active uveitis that were capable of secreting interleukin-2 (IL-2) in response to the autologous presentation of tetanus and S-antigen with those of normal control subjects. Materials and Methods Culture Media Culture media for the limiting dilution analysis consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 1.6 mM L-glutamine, 0.27 mM folic acid, 0.27 mM L-asparagine, 0.55 mM L-arginine, 10 mM N-2-hydroxyethylpiperazine-N-2ethane sulfonic acid buffer, 1.0 mM sodium pyruvate (Gibco, Grand Island, NY); 100 units of penicillin and streptomycin, and 25 /xg of amphotericin B (Whittaker, Walkersville MD); 5 X 10~5 M beta-mercaptoethanol (Sigma, St. Louis, MO); and 10% heatinactivated human AB serum (Whittaker). Mononuclear Cell Preparation Peripheral blood mononuclear cells (PMBC) were isolated from heparinized whole blood obtained from patients with uveitis or normal human volunteers after informed consent. Cells were obtained using standard Ficoll-Paque density centrifugation (Pharmacia, Piscataway, NJ) at 400 X g.20 Monocyte preparations were obtained by subsequent Percoll gradientdensity sedimentation (Sigma).21 T helper cells were defined operationally as lymphocytes capable of secreting IL-2. In vivo activated T cells are lymphocytes spontaneously secreting IL-2 in the absence of specific antigen. Helper T cells secreting IL-2 in response to the autologous presentation of antigen are regarded as antigen-specific T lymphocytes. Indicator Cell Line CTLL-20 cells, a T cell growth factor-dependent murine cell line, were maintained in complete DMEM supplemented with 10% concanavalin A stimulated rat splenocyte supernatant and 10% heat-inac- Vol. 02 tivated fetal bovine serum.15 The CTLL-20 cells were washed three times and resuspended in the culture media at a concentration of 4 X 104 cells/ml for a final concentration of 1 X 103 cells/microwell for the IL-2 limiting-dilution analysis. CTLL-20 cells proliferate in response to murine IL-2, murine IL-4, and human IL-2 but not human IL-4.22 Thus, CTLL-20 cell proliferation is an indirect measure of IL-2 production. Test Antigens Bovine retinal S-antigen was provided generously by Dr. Robert Nussenblatt (National Eye Institute, Bethesda, MD) and was used at a final concentration of 20 Mg/ml in the IL-2 limiting-dilution analysis. Tetanus toxoid (Wyeth-Ayerst, Marietta, PA) was used at a final dilution of 1:999 of stock. Tetanus toxoid was selected as the control antigen because both tetanus and the experimental antigen (S-antigen) are soluble peptides. In addition, most subjects can be expected to be immunized against tetanus and should have effector cells specific for this antigen. Limiting-Dilution Assay To determine the frequency of antigen-specific, IL2-secreting helper T cells in the peripheral blood, a limiting-dilution assay was developed as described previously.15 Briefly, the appropriate serial dilutions of the effector cell were plated into 96-well, V-bottom microtiter plates (INC/Flow, Los Angeles, CA) resulting in 20 replicate wells each with 20,000, 10,000, 5000, 2500, 1250, or 625 lymphocytes per well. Irradiated (7500 rad) autologous monocytes (5000 cells per well) were added to the wells as autologous, antigen-presenting cells for the protein limiting-dilution analysis. The lymphocyte cultures are incubated for 18 hr at 37°C in 10% CO2 with the test and control antigens, allowing antigen-specific T cell activation to occur. The plates are irradiated with 2500 rad to prevent mononuclear cell proliferation and overlaid with 1 X 103 CTLL-20 cells/well. After an 8-hr incubation at 37°C, the plates were pulsed with 3H-thymidine (New England Nuclear, Wilmington, DE). The plates then were incubated for 12 hr, harvested, and counted for thymidine uptake by scintillation on a beta counter. Important controls for the limiting-dilution analysis include CTLL-20 proliferative responses to: (1) control 1—0% IL-2 alone (CTLL-20 negative control), (2) control 2—10% IL-2 alone (CTLL-20 positive control), (3) control 3—monocytes (5000 cells/ well) and antigen without effector lymphocytes (stimulator control and negative point for statistics), and (4) control 4—monocytes (5000/well) and lymphocytes (20,000 cells/well) without tetanus (spontaneous, antigen-nonspecific, IL-2 release in vivo activation). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017 No. 9 2563 AUTOREACTIVE HELPER T LYMPHOCYTES IN UVEITIS / Opremcok er ol Experimental wells contained limiting dilutions of irradiated, effector PBMC, irradiated autologous monocytes (5000 cells/well), CTLL-20 cells (1000 cells/well), and test antigen. Critical to the limiting-dilution analysis is the establishment of an optimal limiting-dilution analysis environment and a single dilutional factor. Neither the concentration of antigen nor the number of antigen-presenting cells can be a limiting factor to fulfill the requirements of the limiting-dilution analysis. We found that a concentration of 20 Mg/ml for S-antigen, 1:999 dilution for tetanus antigen, and 5000 autologous monocytes per well provided nonlimiting antigen stimuli in the limiting-dilution analysis microculture. Under such conditions, the responder cell number was the only limiting variable in the experimental system. As a result of spontaneous activation in the patient population, a complete IL-2 limiting-dilution analysis was done without test antigen. This generates a frequency estimate for in vivo activated T helper cells. tion for the 20 replicate wells was plotted against effector cell dilutions. Figure 1A shows an antitetanus response using limiting-dilution analysis. Under the experimental conditions, the percent negative wells for IL-2 secretion was directly proportional to the effector cell concentration. At cell concentrations above 20,000 cells per well, it was common to note a dropoff in both the magnitude of proliferative response and the number of positive wells. This may represent competition for IL-2 by other cells in the microwell environment or the presence of suppressor cell activities at the higher cell concentrations and was comparable to the conditions present in standard lymphocyte proliferation assays. Figure 1B illustrates the absence of a T helper response to S-antigen and represents the normal control population's lack of response to self antigens. Normal Control Tetanus LDA 4000- Limiting-Dilution Analysis and Statistics : 30003 H Thymidine Incorporation (CPM'S) t i ; s 2000- ; n ; 1000- j: 0# Cells/Well % Neg Wells : : ! • j !! :: ' '• 1: : )• •! j 20000 10000 5000 40 30 60 : : i- :•• ::• The limiting-dilution analysis generates a minimal frequency estimate for a discrete subset of effector cells in a given population. This frequency is calculated by analysis of the Poisson distribution between the number of responder cells added to the limitingdilution microwells and the percentage of replicate microwells that do not produce detectable IL-2.1516 Limiting-dilution microcultures are considered positive for IL-2 production if thymidine incorporation by CTLL-20 cells is greater than the thymidine incorporation (mean plus three standard deviations) observed in 20 replicate stimulator control microwells. Frequency calculations were determined by computer using chi-square minimization analysis as described previously.16 This analysis yields minimal frequency estimates (1/f), the 95% confidence limits of the frequency estimate, and a chi-square estimate of probability (P). In this system, significance is indicated by P > 0.05. Minimal estimates were reported because of the possibility of multiple effector cells occurring in one microwell and the uncertainty regarding the absolute number of T helper cells required to produce detectable IL-2. 2500 70 : 1250 85 625 85 Normal Control S-Ag LDA 5000- B 40003 H Thymidine Incorporation 3000(CPM'S) 2000- 1000- Results Normal Control Population To establish normal values for the frequency of tetanus-specific, S-antigen-specific, and in vivo activated helper T cells in peripheral blood, healthy subjects without a history of uveitis were assayed. Figure 1 is a representative IL-2 limiting-dilution analysis for a normal subject. In this example, thymidine incorpora- :::._ ::::. :::!• :::: 0. # Cells/Well % Neg Wells 20000 10000 5000 2500 1250 625 99.99 99.99 99.99 99.99 99.99 99.99 Fig. 1. Representative IL-2 limiting dilution analysis from a normal control subject. Thymidine incorporation from 20 replicate microwells is plotted against the effector cell dilution. (A) illustrates a strong tetanus response. (B) shows the absence of S-antigcnspecific helper T cells in this healthy subject. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017 2564 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Augusr 1991 Table 1 summarizes these data for the normal control population. In vivo activated helper T cells, spontaneously secreting IL-2 were infrequent (one of eight). Only one subject (12), recovering from an upper respiratory illness, was found to show evidence for this activity with a minimal frequency estimate of 7 T cells in 106 peripheral blood lymphocytes (PBL). Most subjects had a significant number of tetanusspecific responder cells with frequency estimates ranging from 25-200 T cells/106 PBL. One subject (3) did not have a detectable tetanus response before immunization. Six weeks after a booster shot, the level became greater than 3000 helper T cells/106 PBL. As reported by others, limiting-dilution analysis was found to be more sensitive than lymphocyte proliferation assays in detecting immune responses in normal subjects.2223 Several subjects responded to tetanus antigen in limiting-dilution assay but did not respond in a parallel lymphocyte proliferation assay. Only one normal subject (12) responded to S-antigen with a frequency of 8 autoreactive T cells/106 PBL. A small percentage of normal subjects have been reported by others to have proliferative responses to this ocular autoantigen.1314 Unlike standard lymphocyte proliferation assays that detect multicellular interactions, the limiting-dilution assay is designed to recognize and quantify a discrete T cell function, eg, specific cytokine release (IL-2) after autologous presentation of precise antigen. This technique appears to be more sensitive than proliferation assays.17"19 This may be due to the elimination of other influencing factors present in bulk culture assays such as competition for cytokines and suppressor cell activity. It also appears to be very sensitive; all healthy control subjects were documented to have a significant frequency of helper T cells commitTable 1. Limiting dilution analysis—normal subjects Subject Spontaneous Tetanus #1 NA NA NA 125 0 25 0 71 34 48 63 55 111 83 67 200 20 77 0 0 #2 #3 #4 0 #5 #6 0 #7 0 NA NA 0 NA 7 0 0 #8 #9 #10 #11 #12 #13 #14 0 S-antigen 0 NA NA 0 0 0 0 0 8 0 0 The frequencies of telanus. S-antigcn. and spontaneously activated, 1L-2secreting T cells arc given as the number of cells/106 PBL. All values are significant (P> 0.05) unless noted (NS). Frequencies reported as zero represent undctcctable levels of responder cells (less than 3 cells/106 PBL) (NA = not assayed). Vol. 32 ted to tetanus control antigen. Tetanus responses provide an important control for basic T cell function in the subject and patient population and for this assay. Our system also appears to be very specific; only one (1 of 12) normal subject (12) had evidence for autoreactivity. Uveitis Patient Population Patients with uveitis were diagnosed by standard criteria of history, extensive review of systems, physical examination, ocular examination, and supplemental laboratory testing. Most patients had been receiving nonspecific antiinflammatory regimens. All patients had active disease and were assayed within several weeks of establishing a diagnosis. Figure 2 shows a representative tetanus and S-antigen limiting-dilution analysis for a patient (22) with VK.H disease. In this example, the S-antigen-specific response was observed with a frequency of 42 helper T cells/106 PBMC. Table 2 summarizes the data from 29 patients with active uveitis. Helper T cell frequencies for tetanus-committed cells in these patients were comparable to normal controls and ranged from 5-500 helper T cells/106 PBL. One patient (12) did not have a detectable tetanus response. In contrast to the normal population, patients with active uveitis had both spontaneously activated and S-antigen-committed helper T cells in the peripheral blood (Table 2). Thirteen of our 29 patients (45%) had evidence of spontaneous T cell activation with frequencies ranging from 3-50 activated T cells/106 PBL. Spontaneously activated T cells were found in some of the patients in each of the forms of uveitis including idiopathic, presumed infectious (toxoplasmosis and Epstein-Barr virus), Behcet's disease, subretinal fibrosis and uveitis syndrome (SFU), sympathetic ophthalmia, birdshot choroidopathy, and VKH disease. Eighteen of the 29 patients with active uveitis (62%) had a distinct subpopulation of T cells capable of secreting IL-2 in response to the autologous presentation of S-antigen. Frequency estimates in these patients for S-antigen-committed helper T cells ranged from 4-43 T cells/106 PBL. The diffuse uveitis group (eight of nine) had the highest percent (89%) of S-antigen-positive patients and the highest average number of responder cells (18 T cells/106 PBL). Of the patients with posterior forms of uveitis, 58% had evidence for autoreactive T cells specific for S-antigen. As a group, however, the posterior forms of uveitis had a significantly lower average number of responder cells (3 T cells/106 PBL) than the diffuse group (P < 0.01). None of the four patients with intermediate uveitis could be stimulated to produce IL-2 when challenged with S-antigen. The patients with recurrent forms (three of four) of severe, anterior uveitis (histocompat- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017 7000 A Uveitis Patient Tetanus LDA 6000 5000 3 H Thymldlne Incorporation 4000 (CPM'S) 30002000•»!!. 1000- # Cells/Well % Neg Wells 3 H Thymldlne Incorporation (CPM'S) 20000 .01 10000 25 5000 25 X + 3 SD 2500 60 Uveitis Patient S-Ag LDA Patient Spontaneous Tetanus 17 0 0 0 83 NS 142 S-Ag Anterior Uveitis 4000 #1 B27 #2 B27 #3 B27 #4 B27 3000- 2000- 33 11 43 0 142 10 0 0 5 0 142 NS 0 0 0 0 0 21 12 13 3 0 7 13 3 NS 0 0 37 71 166 111 5 0 NS 500 ••X + 3 SD 1000# Cells/Well % Neg Wells with uveitis may be clinically useful. The size of this population could be an index of disease activity and may parallel response to therapy. We examined the frequency estimates for an autoreactive helper T cell pool in patients with select forms of uveitis by limiting-dilution analysis. This technique is the only quantitative assay capable of generating a minimal frequency estimate for a specific responder cell in a given population.15"19 Unlike standard lymphocyte proliferation assays that detect multicellular interactions, this system can be designed to recognize a single lymphocyte function.18-24 Limiting-dilution analysis techniques also offer more sensitivity than proliferation assays. This is due primarily to the elimination of other influencing factors present in bulk culture such as competition for cytoTable 2. Enumeration of autoreactive, cytokinesecreting (IL-2) T cells in patients with uveitis—S-Ag 5000 B 2565 AUTOREACTIVE HELPER T LYMPHOCYTES IN UVEITIS / Opremcok er ol No. 9 Intermediate Uveitis 20000 10000 5000 2500 1250 625 35 75 75 99.99 99.99 99.99 Fig. 2. Representative IL-2 limiting dilution analysis from a patient with uveitis (VKH syndrome). This shows both a positive tetanus (A) and S-antigen response (B). ibility antigen H LA-B27 associated) also had a population of S-antigen-reactive T cells ranging from 0-33 cells per million PBL (average, 10 cells/106 PBL). Discussion Only limited information is available regarding the underlying immunologic milieu of patients with uveitis. Our understanding of the mechanism of uveitis would be enhanced by an appropriate tool to monitor autoreactivity. Most investigators used conventional lymphocyte proliferation assays to establish the presence of a population of T cells responsive to specific ocular autoantigens.9"14 There is little correlation, however, between the magnitude of proliferation and the number of helper T cells required to generate this response.1724 Such quantitative information is prerequisite to our understanding of the dynamic relationship between the peripheral immunologic compartment and the eye. Knowing and following the size of such an autoreactive T cell pool in a group of patients #5 Sarcoid #6 Sarcoid #7 Sarcoid #8 Pars planitis 0 29 142 Posterior Uveitis #9 Birdshot #10Birdshot #11 Birdshot # 12 Behcet's #13SFU #14MCP #15 Idio #16EBV #17Toxo #18Toxo #l9Histo #20 TB 58 NS 66 0 71 4 7 0 0 0 8 16 NS 4 0 5 9 0 Diffuse Uveitis #21 VKH #22 VKH #23 VKH #24 VKH #25 VKH #26 SO #27 SO #28 SO #29 SO + 0 0 0 50 3 12 0 37 111 125 0 100 333 77 142 773 23 42 5 13 0 20 7 16 11 The frequency of tetanus. S-antigcn. and spontaneously activated. IL-2-secrciing T cells is given as cclls/106 PBL. All values are significant (/>> 0.05) unless noted (NS). Frequencies reported as zero represent undetectable levels of responder cells (less than 3 cells/106 PBL). Frequency per 1 X I0 6 PBMC. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017 2566 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1991 kines and suppressor cell activity.17"19'24 By using these techniques, healthy control subjects had a significant frequency of helper T cells capable of secreting IL-2 in response to soluble tetanus antigen without significant in vivo activated or S-antigen-specific effector cells (Table 1). Only one randomly selected subject had evidence of both spontaneous and S-antigen-specific responder cells. Patients with uveitis had comparable tetanus-specific helper T cells frequencies. It would appear that healthy subjects and patients with uveitis have precursor T helper cells in the peripheral blood capable of an anamnestic response to this exogenous antigen when appropriately presented. Ocular inflammation does not appear to affect this unrelated and antigen-specific helper T cell function. Unlike control subjects, 45% of uveitis patients (13 of 29) had spontaneously activated T cells in their peripheral blood. Activated T cell frequencies ranged from 3-50 cells/106 PBMC. Spontaneously activated T cells were found in some of the patients in each of the forms of uveitis including idiopathic, presumed infectious (toxoplasmosis and Epstein-Barr virus), Behcet's disease, SFU, sympathetic ophthalmia, birdshot choroidopathy, and VKH. The frequency of in vivo activated T cells in any autoimmune disease was not previously reported to our knowledge. The presence of activated T cells in the peripheral blood of patients with uveitis is not, however, unexpected and may be secondary to the lack of lymphatic drainage in the eye to regional lymph nodes. The splenocameral route for lymphocyte trafficking and immune processing has been well established for several antigen systems.25"27 Inflammatory cells and antigens have direct access to the central immunologic compartment by aqueous and postcapillary venules. Therefore, activated helper T cells leaving the eye would not encounter regional lymphoid tissue and instead would circulate and be processed in the spleen. Although we did not ascertain the precise antigen specificity of these cells, this activity was not noted in the normal population. It can be postulated that these activated cells are involved in mediating ocular inflammation through interaction with specific antigens found in the eye. Such antigens may include toxoplasmic antigens, viral antigens, or unique ocular autoantigens. Eighteen of the 29 patients with active uveitis had a distinct subpopulation of T cells capable of secreting IL-2 in response to the autologous presentation of Santigen (4-43 cells/106 PBL). Eighty-nine percent of the patients with diffuse uveitis were reactive and had the highest average number of responder cells (18 T cells/106 PBL). All four patients with sympathetic ophthalmia and 80% of patients with VK.H (four of five) had finite frequencies of these cells. Of the patients with posterior forms of uveitis, 58% had evi- Vol. 02 dence for autoreactive T cells specific for S-antigen; 67% of patients with birdshot retinochoroiditis were positive. Patients with diffuse uveitis had a significantly higher (P < 0.01) number of responder cells compared with posterior forms of uveitis (3 T cells/ 106PBL, P < 0.01). Patients with intermediate uveitis could not be stimulated to produce IL-2 when challenged with S-antigen. It would appear that this form of uveitis is not mediated by IL-2-secreting, S-antigen-reactive T cells. Immunotherapy directed against S-antigen may not be valuable in this disease. Unexpectedly, patients with recurrent forms of severe, anterior uveitis (HLAB27 associated) also had a population of S-antigenreactive T cells ranging from 0-33 cells/106 PBL. This observation was not reported previously, to our knowledge, and may be a manifestation of the increased sensitivity in the limiting-dilution assay environment. Although all patients had active clinical disease at the time of the assay, the stage of the disease was less well defined. Recently, investigators emphasized the importance of the stage of the disease to help ascertain initiating and mediating events from unrelated secondary phenomena.28 Vrabec et al28 showed the development of S-antigen reactivity in lymphocyte proliferation assays after laser photocoagulation in patients with diabetic retinopathy. In their studies, proliferative responses against S-antigen developed in the absence of clinical uveitis and illustrate the importance of identifying pathogenic T cells mechanisms such as lymphocyte activation and cytokine secretion. Antigen epitope influence also may play an important role in inducing clinical disease. We used bovine S-antigen; limiting-dilution assay responses may be different against other S-antigen peptide moieties. Of the S-antigen-responsive patients, 50% lacked evidence for in vivo activation. These responder cells therefore represent circulating S-antigen-committed, precursor helper T cells and were not found in the normal population. The other half of the S-antigenpositive patients, however, had evidence of spontaneous activation. These cells may represent circulating S-antigen-committed, activated T helper cells (nonprecursor) suggesting in vivo contact with this autoantigen. Given that the normal control population had neither spontaneous nor S-antigen-responder cells, the presence of both subpopulations of T helper cells would support their role in mediating uveitis. The experiments as designed, however, cannot determine the antigen specificity of the spontaneously activated T cells. Therefore, an alternative explanation would be that the activated T cells were directed against another antigen(s). The cytokine IL-2 is involved with lymphocyte communication, specifically T helper cell function. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017 No. 9 AUTOP£ACTIVE HELPER T LYMPHOCYTES IN UVEITIS / Opremcok er ol An IL-2 assay was chosen to determine the role of this pivotal compound in patients with uveitis. Limitingdilution analysis techniques can be designed to determine minimal frequency estimates for other exogenous or autologous antigens and used to detect other lymphokines or lymphocyte functions. End-organ responses can be studied to investigate lymphocyte trafficking patterns and distinct immunologic compartment differences in ocular inflammatory disorders. With further experience, frequency estimates may be used to follow the clinical course and responses to therapy and provide a tool to understand the predominant pathophysiologic mechanism(s) governing these diseases better.29 13. 14. 15. 16. 17. Key words: uveitis, limiting-di. .tion analysis, helper T lymphocytes, inlerleukin-2, auloimmune References 1. The 1983 Report of the National Advisory Eye Council, Vision Research: A National Plan: Summary Panel Reports on Retinal and Choroidal Diseases. 1983, pp. 23. 2. Wackcr WB, Donosos LA, Kalsow CM, Yankeelov JA, and Organisciak DT: Experimental allergic uveitis: Isolation, characterization and localization of a soluble uveitis-pathogenic antigen from bovine retina. J Immunol 1 19:1949, 1977. 3. Nussenblatt RB. Kuwabara T, de Monasterio FM. and Wacker WB: S-antigen uveitis in primates: A new model for human disease. Arch Ophthalmol 99:1090. 1981. 4. Gcry 1, Wiggcrt B, Redmond FM, Kuwabara T, Crawford MA, Vistica BP. and Chandcr GJ: Uvcoretinitis and pinealitis induced by immunization with interphotoreccptor retinoidbinding protein. Invest Ophthalmol Vis Sci 27:1296, 1986. 5. Caspi RR, Roberge FG, McAllister CA, El-Siaed M, Kuwabara T, Gcry I, Hanna E, and Nussenblatt RB: T cell lines mediation experimental autoimmune uveorctinitis (EAU) in the rat. J Immunol 136:928, 1986. 6. Mochizuki M. Kuwabara T, McAllister C, Nussenblatt RB, and Gery I: Adoptive transfer of experimental autoimmune uveorctinitis in rats. Invest Ophthalmol Vis Sci 26:1, 1985. 7. Nussenblatt RB, Rodrigues MM. Wacker WB, Cevario SJ, Salinas-Carmona MC, and Gcry I: Cyclosporin A inhibition of experimental autoimmune uveitis in lewis rats. J Clin Invest 67:1228, 1981. 8. Nussenblatt RB, Rodrigues MM, Salinas-Carmona MC, Gery I. Cevario S, and Wacker WB: Modulation of experimental autoimmune uveitis with cyclosporin A. Arch Ophthalmol 100:1146. 1982. 9. Kaplan HJ. Waldrcp JC. Nicholson KA. and Gordon D: Immunologic analysis of intraocular mononuclcar cell infiltrates in uveitis. Arch Ophthalmol 102:572, 1984. 10. Jakobicc FA, Marboe CC, Knowlcs DM, Iwamoto T, Harrison W, Chang S. and Coleman DJ: Human sympathetic ophthalmia: An analysis of the inflammatory infiltrate by hybridomamonoclonal antibodies, immunochemistry, and correlative electron microscopy. Ophthalmology 90:76. 1983. 11. Nussenblatt RB, Mital KK, Ryan S. Green WR, and Maumenee E: Birdshot retinochoroidopathy associated with HLAA29 antigen and immune responsiveness to retinal S-antigcn. Am J Ophthalmol 94:147, 1982. 12. Doekcs G, vandcrGaag R, Rothova A, vanKooyk Y, Broersma L. Zaal MJ. Dijkman G. Fortuin ME, Baarsma GS, and Kijl- 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 2567 stra : Humoral and cellular immune responsiveness to human S-antigen in uveitis. Curr Eye Res 6:909, 1987. Nussenblatt RB, Gcry 1, Ballintinc EJ, and Wacker WB: Cellular immune responsiveness of uveitis patients to retinal S-antigen. Am J Ophthalmol 89:173, 1980. Hirose S, McCallislcr CG, Mittal KK, Vistica P, Shinohara T, and Gery I: A cell line and clones of lymphocytes from a healthy donor with specificity to S-antigen. Invest Ophthalmol Vis Sci 29:1636, 1988. Orosz CG. Adams PW, and Ferguson RM: Frequency of human alloantigcn-rcactivc T lymphocytes: II. Method for limiting dilution analysis of alloantigcn-reactivc helper T cells in human peripheral blood. Transplantation 43:718, 1987. Taswell C: Limiting dilution assays for the determination of immunocompetcnt cell frequencies. J Immunol 126:1614, 1981. Miller RA and Stutman O: Enumeration of IL-2 secreting helper T cells by limiting dilution analysis and demonstration of unexpectedly high levels of IL-2 production per responding cell. J Immunol 128:2256, 1982. Rozans MK, Smith BR, Burakoff SJ, and Miller RA: Long lasting deficit of functional T cell precursors in human bone marrow transplant recipients revealed by limiting dilution methods. J Immunol 136:4040, 1986. Clerici M, Stocks Nl, Zajac RA, Boswell RN, Bernstein DC, Mann DL, Shearer GM, and Bcrzofsky JA: Interlcukin-2 production used to detect antigenic peptide recognition by Thclpcr lymphocytes from asymptomatic HI V-scropositivc individuals. Nature 339:383, 1989. Boyum A: Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 21:77, 1968. Ulmer A and Flad HD: Discontinuous density gradient separation of human mononuclcar leukocytes using Pcrcoll as a gradient medium. J Immunol Methods 30:1, 1979. Mosmann T. Yokato T, Katsclein R, Zurawski S, Arai N, and Takabe Y: Species specificity of T cell stimulating activities of IL-2 and BSF-1 (IL-4): Comparison of normal and recombinant human IL-2 and BSF-1 (IL-4). J Immunol 138:1813. 1987. Huang YP, Perrin LH, Micscher PA, and Zublcr RH: Correlation of T and B cell activities in vitro and scrum IL-2 levels in systemic lupus erythematosus. J Immunol 41:827, 1988. Clouse KA, Adams PW. and Orosz CG: Enumeration of viralantigen reactive helper T lymphocytes in human peripheral blood by limiting dilution for analysis of viral antigen-reactive T cell pools in virus-scropositivc and virus-scronegative individuals. J Clin Microbiol 27:2316, 1989. Kaplan HJ and Strcilein JW: Immune response to immunization via the anterior chamber of the eye. J Immunol 120:689, 1978. Strcilein JW and Neidcrkorn JY: Characterization of the suppressor cell(s) response for anterior chamber associated immune deviation (ACAID) induced in BALB/c mice by P815 cells. J Immunol 134:1381, 1985. Wetzig RP. Foster CS, and Green MI: Ocular immune response: I. Priming of A/J mice in the anterior chamber with azobenzencarsonate-deriviiizcd cells induces sccond-orderlike suppressor T cells. J Immunol 128:1753, 1982. Vrabec TR? Rebcr RN, Magargal LE, and Donoso LA: S-antigen: Identification of human T-ccll lymphocyte proliferation sites. Arch Ophthalmol 108:1470, 1990. Nussenblatt RB, Mittal KK, Fuhrman S, Sharma SD, and Palestine AG: Lymphocyte proliferalive responses of patients with ocular toxoplasmosis to parasite and retinal antigens. Am J Ophthalmol 107:632, 1989. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933160/ on 06/17/2017