Download Enumeration of autoreactive helper T lymphocytes in uveitis.

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
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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).
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No. 9
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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.
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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-
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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.
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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.
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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
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