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(Investigative Ophthalmology and Visual Science. 2000;41:3480-3484.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Prospective Determination of T-Cell Responses to S-Antigen in Behçet’s Disease Patients and Controls

Marc D. de Smet1,2 and Molly Dayan1

1 From the Laboratory of Immunology, Clinical Immunology Section, National Eye Institute, Bethesda, Maryland; and the 2 Department of Ophthalmology, Academic Medical Center, University of Amsterdam, The Netherlands.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To prospectively determine, using two different assays, the lymphocyte proliferative response to a retinal autoantigen (S-antigen) in patients with Behçet’s disease who are under treatment for ocular inflammation.

METHODS. Patients were evaluated at each visit for signs of ocular inflammation. Peripheral blood leukocytes were harvested and cultured in the presence of bovine S-antigen in a standard culture assay, as well as by limiting dilution using multiple short-term T-cell lines.

RESULTS. Five patients were observed for 2 to 10 months. During follow-up, three patients had episodes of ocular inflammation. No consistent change in proliferative response was observed in standard proliferation assays. However, an increase in established T-cell lines was correlated to the presence of ocular inflammation in all three patients. Ocular activity was associated with an increase of 9- to 30-fold in the frequency of short-term T-cell lines. This increase returned to baseline within 1 to 3 months.

CONCLUSIONS. An increase in S-antigen–responsive lymphocytes is found in the peripheral blood of patients with Behçet’s disease during episodes of ocular inflammation. This increase cannot be measured using standard proliferation assays but requires the use of techniques exploiting the principles of limiting dilution analysis.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Behçet’s disease is characterized by recurrent episodes of severe intraocular inflammation. Several immunologic abnormalities have been reported in this disease that suggest that autoimmunity may play a role in its pathogenesis.1 2 3 Proliferation assays using peripheral blood lymphocytes have commonly been used to detect cellular immunity to retinal antigens. A heightened response to S-antigen (S-Ag) has been observed in a variety of patients with uveitis, including those with Behçet’s disease.4 5 6 7 8 9 However, this assay remains qualitative in nature. Although capable of providing evidence of a response, lymphocyte proliferation assays are unable to provide information on the severity of ocular inflammation and elicit variable proliferative responses over time.10 11 12

Circumventing the qualitative nature of the lymphocyte proliferation assay, limiting dilution assays (LDA),13 allow determination of the number of cells that respond to a specific antigen or antigens (precursor frequency).14 15 LDAs have been used in surveillance of the level of response to the soluble tetanus toxoid antigen over time.16 Using a cell culture–based assay that incorporates elements of a limiting dilution technique, we showed that it is possible to estimate the T-cell precursor frequency to bovine S-Ag in patients with diverse forms of uveitis.17 Using a similar approach, others determined the precursor frequency of T cells responding to fragments of myelin basic protein (MBP) in patients with multiple sclerosis (MS) and after spinal cord injury.18 19 20 Adjusting the culture conditions allowed the demonstration of in vivo clonal expansion of MBP-reactive T cells in both the blood and the cerebrospinal fluid (CSF) of patients with MS, suggesting active trafficking of these lymphocytes across the blood–brain barrier.18 20 A similar study in Behçet’s disease would imply the isolation of lymphocytes from the vitreous of a patient with active inflammation, a procedure that is rarely required and is not without risk. However, it is possible to study the influence of ocular inflammation on lymphocytes within the peripheral blood.

Using our modified LDA, we decided to explore the change in S-Ag responsiveness over time in peripheral blood lymphocytes of patients with Behçet’s disease. We were particularly interested in determining whether fluctuations in responsiveness could be detected after episodes of intraocular inflammation. Bovine S-Ag was used as the stimulating antigen. Because standard lymphocyte proliferation assays have not been carefully studied in this setting, we also wanted to characterize the response of this simpler culture method to ocular inflammation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Patient Selection and Assessment of Disease
Five patients with Behçet disease, seen in the uveitis clinic of the National Eye Institute, were selected for long-term monitoring of their cellular immune responses. All patients met the diagnostic criteria established by the International Behçet’s Disease Study Group.21 All patients signed an informed consent approved by the Investigational Review Board of the National Eye Institute. Patients were tested for immune response at each regularly scheduled patient visit for a minimum of three visits and were monitored until the patient was discharged from the eye clinic or chose to forego further testing. Before drawing blood, patients had a complete ocular examination that included measurement of visual acuity and examination of the anterior and posterior segments for evidence of intraocular inflammation. To determine baseline responses, four normal individuals were also tested. An acute inflammatory episode was deemed present when one of the following events was noted on clinical examination: an increase in vitreous cells associated with a vision decrease of more than two lines on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart, or the appearance of a focus of chorioretinitis, vascular sheathing, or intraretinal hemorrhage. The study abided by the guidelines set forth in the Declaration of Helsinki as well as the US Code of Federal Regulations pertaining to research on human subjects (45 CFR 46).

Lymphocyte Isolation and Culture
Peripheral blood samples were collected at several time points, as indicated in Table 1 . For each time point, 60 ml of peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were separated on a gradient (Isolymph; Gallard–Schlesinger, Carle Place, NY). One fraction was used for both culture assays as indicated below and was processed immediately. The remaining PBMCs were frozen to be used for antigen presentation. For the standard proliferation assay, cells were cultured in flat-bottomed 96-well microplates, at 2 x 105 cells per well, in 0.2 ml RPMI 1640 medium with HEPES (Cellgro, Herndon, VA), supplemented with 10% heat-inactivated human AB serum (Biocell Laboratories, Carson, CA). Six replicate cultures were stimulated with bovine S-Ag at 20 or 100 µg/ml. Phytohemagglutinin (PHA) at 1 µg/ml was used as a control of adequate proliferation. Cells were cultured for 5 days, pulsed with [3H]-thymidine (3H-TdR; NEN, Boston, MA; 2 Ci/mmol, 0.5 µCi/10 µl per well), during the last 18 hours of culture. Bovine S-Ag was prepared according to the method of Dorey et al.22 The results are expressed as the stimulation index (SI = mean counts per minute in cultures with stimulant/mean counts per minute in control cultures without stimulant).


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  		Table 1. Patients’ Characteristics at Baseline

 
Estimation of the Proportion of Circulating T Cells Reactive to S-Ag
In a previous study, we determined the optimal cell density to detect S-Ag–reactive T cells.17 This was found to be 2 x 105 cells/well, similar to studies in patients with MS.23 At this concentration, polymerase chain reaction (PCR) analysis demonstrated monoclonality of cell lines.23 Cultures for all subjects were performed in exactly the same manner.

PBMCs were incubated at 37°C, with bovine S-Ag 100 µg/ml for 1 hour at a cell density of 1 x 107/ml. These were then washed twice and resuspended in RPMI 1640 at a concentration of 1 x 106/ml and plated at 200 µl per well into 480 wells on five round-bottomed 96-well microtiter plates (Costar, Cambridge, MA). Beginning on the third day and every 3 days thereafter, half of the medium was replaced with fresh medium containing 5% T-cell stimulant (Collaborative Research, Bedford, MA) and 2 U/ml of human recombinant interleukin (rIL)-4 (Genzyme, Boston, MA).

On day 12 to 14, each well was analyzed for its reactivity to bovine S-Ag. An aliquot from each culture well was split into four aliquots (10,000 cells per aliquot), and placed into fresh, round-bottomed 96-well plates. Two wells were cultured with 105 PBMCs pulsed for 4 hours with S-Ag before irradiation (3000 rads). The two other wells received unpulsed irradiated PBMCs. Wells were considered responsive to bovine S-Ag if the SI was above 3.0 and if the coefficient of variation for each duplicate culture was less than 30%. Results are expressed as the percentage of positively responding wells.

Statistical Analysis
Statistical analysis was performed by computer (Prism; GraphPad Software, San Diego, CA) without assuming a gaussian distribution using the Mann–Whitney test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Five patients with Behçet’s disease were included in this study. Four women and one man were observed for a period of 2 to 10 months (mean, 4.5 months) with three to six sets of analyses performed per patient. The average patient’s age was 29 years (range, 23–37 years). Behçet’s disease had been diagnosed for an average of 3.4 years (range, 1–8 years) at the time of study enrollment. Disease in all patients had been controlled for a minimum of 3 months, with a combination of low-dose prednisone (2.5–30 mg/d) and cyclosporine (0.5–5 mg/kg per day). During the follow-up period, three patients had an ocular flare-up. When a flare-up was diagnosed, the cyclosporine dose was doubled and then tapered over 1 to 3 months, based on response to therapy. The prednisone dose was similarly increased. Baseline data on patients and control subjects are summarized in Table 1 .

Standard Proliferation Assays
A comparison of the proliferative response between control subjects and patients was performed at study entry. Data on control subjects are provided in Table 2 . There was no statistically significant difference between the two groups when tested at the 20-µg/ml concentration of bovine S-Ag (mean SI: 2.3 control subjects, 2.5 patients). Similarly there was no statistically significant difference in S-Ag response at 100 µg/ml (mean SI: 2.2 control subjects, 4.3 patients), although a significant coefficient of variation was present between the two groups. Reducing the difference in this variation by logarithmic transformation did not change the level of significance.


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  		Table 2. Proliferative Responses in Control Subjects to Bovine S-Ag

 
During the follow-up period, stimulation indices varied significantly. An increased SI was correlated with a flare-up in only one of three cases of acute inflammation. In two of the three patients who had a flare-up of uveitis, a significant SI was noted at some time before the flare-up. Significance was defined as an SI above the mean SI of control subjects + 2 SD.5 24 In the remaining patient, the SI remained below this level of significance. Table 3 summarizes the proliferation assay data in patients.


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  		Table 3. Standard Lymphocyte Proliferation Responses to Bovine S-Ag in Patients with Behçet’s Disease

 
Estimation of the Proportion of Circulating S-Ag–Reactive T Cells
In all patients and control subjects, it was possible to establish short-term T-cell lines. Between 80% and 95% of cultures had a coefficient of variation of less than 30% and thus could be analyzed according to the antigen response. One assay elicited a much lower response. In patient 1, assay 3, only 62% of cultures met the inclusion criteria. At study entry, there was no statistically significant difference in the number of established lines between the two groups. Of the five patients tested, an increase over the baseline number of generated T-cell lines was noted in three cases. These were temporally related to the onset of an episode of intraocular inflammation. The number of established T-cell lines returned to baseline within 1 to 3 months after resolution of the inflammatory episode. Of note, in one patient, the increase in generated T-cell lines was temporally associated with a decrease in response observed with the lymphocyte proliferation assay (patient 5). Results are summarized in Table 4 .


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  		Table 4. Short-term T-cell Line Assay Responses to Bovine S-Ag in Patients with Behçet’s Disease

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Patients with Behçet’s disease are known to be sensitized to retinal autoantigens, S-Ag, and interphotoreceptor retinol-binding protein (IRBP).5 9 They are also known to be sensitized to 65-kDa heat shock proteins25 and possibly to other antigens. Although the exact trigger for the onset of disease is yet to be identified, once sensitization to retinal autoantigens has occurred, these T cells are recruited to the site of inflammation. Adoptively transferred S-Ag–sensitized T cells can be found in rat retinas within the first 24 hours of transfer.26 So far, it has not been possible to demonstrate a role for S-Ag–sensitized cells in human patients. Demonstration in humans would require isolation of T cells from an inflamed eye and delineation of their proliferative response to retinal antigens. This approach was successfully used to delineate the specificity of T cells toward herpes simplex virus in the vitreous of patients with acute retinal necrosis but is impractical in patients with Behçet’s disease, because they rarely require an operative procedure during an inflammatory episode.27

Alternatively, an attempt can be made to demonstrate the presence of an expanded pool of responsive T cells in the peripheral blood after an intraocular inflammation episode. In patients with MS, activated MBP-reactive T cells were shown to undergo in vivo clonal expansion in both the blood and the CSF, indicating the presence of an active traffic of MBP-reactive T cells across the blood–brain barrier.20 Assuming that a similar process is present in patients with ocular inflammation, we decided to observe a group of patients with Behçet’s disease prospectively by using two assays to measure T-cell proliferation to S-Ag.

Standard proliferative assays have been used by numerous groups to demonstrate an association with human disease.9 12 24 An often-quoted cutoff for immunologic significance is an SI of 2.0. Using this cutoff value, our present group of patients all showed a positive immune proliferative response during the follow-up period, whereas only three patients had intraocular inflammation. Using a more restrictive cutoff of significance (mean of control subjects + 2 SDs) allowed us to identify two of three patients with ocular inflammation. The heightened response was measured either before or at the time of ocular inflammation. This second approach more appropriately selected patients at risk but missed one individual. In addition, once inflammation developed, subsequent measurements were significantly reduced in intensity. Thus, standard proliferation assays can help predict patients at risk of development of uveitis but cannot be used for surveillance of the immune response during the active phase of inflammation.

In another study, a patient with an unspecified retinal scar, present for more than 30 years, was repeatedly tested over an 18-month period against S-Ag.28 A large variation in lymphocyte responsiveness was attributed to the microenvironment present in the culture well. Cytokines produced by one or more cell populations present in the microwell prevented the expression of the S-Ag phenotype.29

Limiting dilution assays have traditionally been used to quantify cells with observable functional qualities. Appropriate dilution of the seeding cell population, allows the interpretation of data according to "single-hit kinetics" (0-term) of the Poisson distribution. Use of unfractionated cell samples dispersed into limiting dilution cultures yields nonlinear titration curves due to opposing forces generated by suppressor and effector cells.29 30 31 To overcome the suppressor effects requires prolonged cultivation and the supply of needed growth factors such as T-cell growth factor.29 Studies performed in other laboratories have shown that this approach generates CD4 single-cell lineages in the majority of wells that often are responsive to a single peptide determinant.18 20 32 Given the number of wells showing a positive response in these studies, single-hit kinetics apply. Thus, the number of positively responding wells is reflective of the circulating pool of responsive T cells. Of the five patients who were observed, three had a transient ocular inflammatory response. In each of these cases, there was an increase in the number of identifiable T-cell lines concurrent with the ocular inflammation. The expansion was between 9- and 30-fold, and this increase rapidly returned back to baseline between 1 and 3 months.

Assessing limiting dilution using this approach makes certain assumptions. The cell populations within the culture well behave according to single-hit kinetics. Memory T cells, which were probably responsible for the activity observed in these experiments, require multiple hits for activation.33 However, in the presence of IL-2, a major component of the T-cell stimulant, it is converted back to single-hit behavior. Thus, addition of the T-cell stimulant favors cells that are capable of growing in its presence. The adequacy of the technique can be checked by setting up cultures at different dilutions. Increases in the number of cells should lead to a linear increase in the number of positive wells. Deviations from linearity, in particular leveling off in the curve, indicates the presence of suppressive elements in the culture well.19 This phenomenon has been observed in patients with MS. We do not know to what extent this effect was present in the assays performed in our patients, because the chosen cell dilution was based on previous work with patients with uveitis, in whom disease was under control at the time of analysis. It is possible that the observed reduction in the proportion of responding wells was partially due to enhanced suppression provided by other cells present in the culture well. Performing titration experiments on a broad range of concentrations that extend beyond the zone of linearity should help to answer this question.29

In experimental models, both CD8 responses to a viral challenge and CD4 responses to antigen challenge are characterized by three distinct phases.34 35 Initial activation and expansion of the lymphocyte pool lasts for approximately 7 days. It is characterized for a novel agent by a 100- to 5000-fold increase in the number of specific lymphocytes.34 36 37 38 In the case of mice exposed to lymphocytic choriomeningitis virus (LCMV), this expansion was calculated to represent 15 divisions or 1 division every 13 hours for 8 days.37 Rapid expansion is followed by a period of cell death, lasting anywhere from 8 to 30 days and is realized primarily through apoptosis.34 35 36 After 30 days, a stable pool of memory T cells is generated that represents approximately 5% of the initial response. Re-exposure to an antigen or virus leads to a rapid expansion from this pool of memory T cells. Although this response occurs more rapidly than the initial response, the measured lymphocyte expansion in peripheral blood or lymph nodes amounts only to a 5- to 100-fold expansion over baseline.35 36 Our data on patients with Behçet’s disease closely parallels these experimental findings. Patients were seen within a few days of recurrence and were noted to have a 9- to 30-fold increase in responsive lymphocytes. As seen in experimental models, this increase rapidly returned to the pre–flare-up level.

This is the first time that a correlation has been observed between peripheral antigen responsiveness and inflammatory disease in humans. Adjusting the culture conditions to favor effector T-cell proliferation has allowed us to unmask this responsiveness. These findings suggest a role for S-Ag in the autoimmune uveitis associated with Behçet’s disease, but it does not imply causality. As previously observed, patients with established disease respond to a number of autoantigens.5 It is likely that some if not all these antigens contribute or help to perpetuate the inflammatory response.

In conclusion, adjustment in culture conditions that promote clonal expansion of reactive T cells has allowed us to demonstrate a variation in the number of responsive lymphocytes in the peripheral blood of patients with Behcet’s disease with active intraocular inflammation. Though technically more demanding, this approach can yield considerably more information on the immune status of patients with uveitis than is currently provided by standard lymphocyte culture methods. It is a valuable tool for quantifying cells at a functional level.


   Footnotes
 
Supported in part by the Edmond and Marianne Blaauwfonds for Ophthalmology.

Submitted for publication January 31, 2000; revised May 30, 2000; accepted May 31, 2000.

Commercial relationships policy: N.

Corresponding author: Marc D. de Smet, Department of Ophthalmology, University of Amsterdam, Rm G2-217, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands. m.d.desmet{at}amc.uva.nl


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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