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Increased frequency of v 17-positive t cells in patients with rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM Volume 37
Number 10, October 1994, pp 1431-1440
0 1994, American College of Rheumatology
143 1
INCREASED FREQUENCY OF Vp17-POSITIVE T CELLS IN
PATIENTS WITH RHEUMATOID ARTHRITIS
GARY ZAGON, JOSEPH R. TUMANG, YIXIN LI, STEVEN M. FRIEDMAN, and MARY K. CROW
Objective. To identify the T lymphocytes that
mediate disease in rheumatoid arthritis (RA).
Methods. A panel of monoclonal antibodies reactive with T cell receptor (TCR) Vp gene products was
used to analyze the RA T cell repertoire.
Results. Of 5 TCR Vp gene products studied, only
Vpl7-positive T cells were increased in peripheral blood
and synovial fluid (SF) from RA patients, compared
with controls (P < 0.01 and P = 0.0006, respectively).
Thirty-one percent of the 49 RA SF samples and none of
the 19 non-RA SF samples contained >lo% Vf317positive T cells. Activated (Tac-positive) T cells were
enriched among Vpl7-positive synovial T cells.
Conclusion. The selective increase of Vf317positive T cells suggests a role for those T cells in the
pathogenesis of RA.
While many cell types, most notably macrophages, synoviocytes, and polymorphonuclear leukocytes, participate in the complex inflammatory response which effects joint destruction in rheumatoid
arthritis (RA) (l), several lines of evidence suggest a
central role for T lymphocytes in the initiation and
perpetuation of this process (2-1 1). The importance of
T cells in the pathogenesis of RA is strongly suggested
by the rich infiltration of activated T cells at the
primary site of disease activity, the synovial tissue
Supported by NIH research grants AR-42557, AI-32634,
and AI-28367 and by a Fellowship Award to Dr. Zagon from The
Arthritis Foundation, New York Chapter.
Gary Zagon, MD: The Hospital for Special Surgery/Comell
University Medical College, New York, New York; Joseph R.
Tumang, BS: The Hospital for Special Surgery; Yixin Li, PhD: The
Hospital for Special Surgery; Steven M. Friedman, MD: The
Hospital for Special Surgery/Cornell University Medical College;
Mary K. Crow, MD: The Hospital for Special Surgery/Comell
University Medical College.
Address reprint requests to Mary K. Crow, MD, The
Hospital for Special Surgery, 535 East 70th Street, New York, NY
10021.
Submitted for publication October 14, 1993; accepted in
revised form May 18, 1994.
(ST) (3). Genetic studies have clearly linked RA disease susceptibility to a defined amino acid sequence in
the third hypervariable region of the DRP chain of the
class I1 major histocompatibility complex (MHC) molecule ( 4 3 . It is likely that this disease susceptibility
epitope influences T cell responses by one of several
possible mechanisms: through the selection of the T
cell repertoire in the thymus, by its capacity to selectively present antigenic peptides to T cells, or by the
epitope itself acting as an antigenic peptide, presented
to autoreactive T cells in the peptide-binding cleft of
MHC molecules.
The character of the autoantibodies observed in
patients with RA also suggests a role for T cells:
anti-Ig antibodies (rheumatoid factors [RF]) within the
rheumatoid joint are often of the IgG or IgA isotype,
and sequence analysis of RF has revealed evidence for
somatic mutation, an event that usually requires T cell
help (6). In several animal models of chronic arthritis,
antigen-specific T cells are capable of transferring
disease to naive recipients (7,8). In these models, in
vivo treatment with antibody reactive with T cell
receptor (TCR) a/P,expressed on 95% of all T cells,
can prevent arthritis if given prior to the administration of arthritogenic antigen and, in some models, can
abrogate existing disease if given during the chronic
phase (9). Finally, in vivo administration of monoclonal antibody (MAb) reactive with the CDCpositive T
helper cell subset ameliorates arthritis, both in murine
models of autoimmune disease and in patients with RA
(10,ll).
In order to identify the pathogenic T cells in RA
and to characterize the relevant antigens that maintain
their chronic activation, we have utilized a panel of
murine MAb reactive with the products of particular
TCR VP gene families. Our results demonstrate a
selective increase in the percentage of T cells bearing
the VP17 TCR gene product in peripheral blood (PB),
synovial fluid (SF), and ST from patients with RA.
ZAGON ET AL
1432
Table 1. Summary of T cell phenotypes of the rheumatoid arthritis (RA) patients and control subjects*
T cell antigen (monoclonal antibody)
Source
Peripheral blood
Normal subjects
SLE patients
Non-RA arthritis
patients
RA patients
Synovial fluid
Non-RA arthritis
patients
RA patients
Synovial tissue
RA patients
vps
CD4
(OKT4)
CD8
(OKT8)
IL-2R p55
(Anti-Tac)
Vp5.215.3
(C37)
Vfi.7a
(OT 145)
71.0 2
8.9 (14)
54.5 f
11.8 (13)
54.9 -t
14.7 (7)
70.1 -t
19.3 (27)
30.4 f
11.3 (14)
42.1 f
14.1 (13)
41.4 f
16.8 (6)
37.0 2
18.7 (27)
4.5 f
2.9 (14)
6.8 t
8.0 (13)
12.6 2
19.2 (7)
8.9 f
7.7 (27)
2.7 f
1.7 (15)
2.9 f
1.4 (11)
3.5 2
1.8 (7)
3.4 2
1.8 (22)
3.6 f
2.1 (21)
3.2 f
1.6 (25)
3.1 f
1.6 (7)
3.2 2
2.5 (28)
3.8 f
2.1 (6)
3.7 f
1.2 (12)
10.7 f
9.3 (4)
5.1 f
3.3 (18)
2.0 f
0.9 (20)
2.3 f
1.7 (21)
2.3 f
0.6 (7)
2.4 f
1.9 (29)
4.7 f
1.3 (21)
4.9 f
1.2 (25)
4.3 2
1.6 (7)
6.5 f
2.7 (28)t
62.4 -t
18.6 (15)
53.9 2
16.8 (46)
31.3 f
14.1 (15)
48.7 f
13.0 (45)
9.5 f
4.4 (15)
12.3 f
6.6 (45)
3.1 2
1.9 (17)
3.8 f
2.1 (45)
3.6 f
2.1 (17)
3.7 f
2.4 (48)
4.3 f
4.4 (12)
3.9 f
2.2 (32)
3.9 f
4.2 (15)
3.4 f
3.8 (47)
5.3 f
2.0 (19)
8.5 f
4.1 (49)$
63.2 -+
15.2 (19)
41.6 f
23.1 (19)
9.7 '.
7.0 (20)
2.6 ?
2.2 (17)
3.8 2
2.7 (21)
4.1 f
2.5 (15)
3.3 f
4.5 (19)
6.7 f
2.2 (21)8
( 16G8)
vp12
(S511)
Vp17
(C1)
* Isolated T cells were analyzed by indirect immunofluorescence on a cytofluorograph. Values are the mean f SD percentage of CD3-positive
cells expressing each T cell surface antigen; values in parentheses are the number of individuals analyzed. IL-2R = interleukin-2 receptor; SLE
= systemic lupus erythematosus.
t P < 0.01 versus peripheral blood of normal subjects, SLE patients, and non-RA arthritis patients (t-test).
$ P = 0.0006 versus synovial fluid of non-RA arthritis patients (Mann-Whitney U test).
0 P = 0.001 versus peripheral blood of normal subjects (t-test).
PATIENTS AND METHODS
Subjects. Normal control subjects consisted of 21
healthy volunteers (femalemale ratio 2.4, mean age 43.3).
Disease controls included patients with systemic lupus erythematosus (SLE) (n = 25; femalemale ratio 10.0, mean age
40) or with n o n - U inflammatory arthritis (n = 19), including
osteoarthritis, gout, Reiter's syndrome, and monarticular
arthritis (femalemale ratio 2.0, mean age 59.5). RA patients
(n = 54; femalemale ratio 2.7, mean age 49.3), as well as
disease controls, were recruited from the outpatient and
inpatient rheumatic disease and orthopedic surgery services
at The Hospital for Special Surgery. RA was defined according to the American College of Rheumatology (formerly, the
American Rheumatism Association) criteria (12), and all
patients were seropositive. RA patients were not selected
with respect to medical therapy (which included aspirin,
nonsteroidal antiinflammatory drugs, corticosteroids, methotrexate, gold, hydroxychloroquine, and sulfasalazine).
RA serum samples and HLA typing. Serum samples
from RA patients were assayed by latex fixation for RF
activity, in the clinical laboratories of The Hospital for
Special Surgery. Mononuclear cells (MNC) from the peripheral blood of 4 patients with marked expansions of Vp17positive T cells were characterized for HLA-DR haplotype
by Dr. Peter Gregersen (North Shore University Hospital,
Manhasset, NY), using standard serologic reagents.
Cell preparations. PB samples were obtained by
venipuncture, and SF samples were obtained at the time of
therapeutic arthrocentesis. ST specimens were obtained
from the Department of Pathology at The Hospital for
Special Surgery, following therapeutic arthroscopic syno-
vectomy, open synovectomy, or total joint replacement. ST
was minced under sterile conditions and incubated in 20 ml
of an enzyme preparation containing RPMI 1640 (Gibco,
Grand Island, NY), 20% fetal calf serum (Whittaker Bioproducts, Walkersville , MD), 1% penicillinlstreptom ycin ,
1% glutamine (Gibco), 0.5 mg/ml collagenase, 0.15 mg/ml
DNase, and 0.1 mg/ml hyaluronidase (Sigma, St. Louis,
MO), in 95% air-5% C 0 2 for 2-4 hours at 37°C. Tissue was
then mechanically disrupted using forceps and scalpel and
pressed through a mesh sieve.
MNC were isolated from PB, SF, or ST digest on a
Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). In
some cases, T cells were selectively enriched by rosetting of
MNC with sheep red blood cells followed by incubation at
4°C for 16 hours and subsequent fractionation of rosetted
and nonrosetted cells over Ficoll-Hypaque.
Monoclonal antibodies. MAb used included OKT3
(anti-CD3, pan-T), OKT4 (anti-CD4, helperlinducer subset),
and OKT8 (anti-CD8, suppressorlcytotoxic subset) (American Type Culture Collection, Rockville, MD). Phycoerythrin-labeled antLCD4 and antLCD8 MAb were obtained
from Coulter Immunology (Hialeah, FL). MAb anti-Tac,
reactive with the p55 chain of the interleukin-2 receptor
(IL-2R), was obtained from Dr. Thomas Waldmann (NIH,
Bethesda, MD). Phycoerythrin-labeled anti-IL-2R MAb was
obtained from Becton Dickinson (Mountain View, CA).
MAb reactive with T cell surface epitopes encoded by
defined TCR pchain variable genes included MAb pV3
(reactive with TCR Vp3; T Cell Diagnostics, Cambridge,
MA), C37 (reactive with TCR Vp5.2b.3) (13), MAb OT145
(reactive with TCR Vp6.7a) (14,15), MAb 16G8 (reactive
1433
INCREASED Vp17-POSITIVE T CELLS IN RA
with TCR Vp8; T Cell Diagnostics), MAb S511 (reactive with
TCR Vp12) (16), and MAb C1 (reactive with TCR Vp17) (17).
Indirect immunofluorescence. MNC or T cells (1-2 x
lo’) were incubated with buffer alone or with a saturating
concentration of MAb at 4°C for 30 minutes. Cells were then
washed 3 times and incubated with a saturating concentration of fluorescein-labeled F(ab’), fragments of goat antimouse IgG (Tago, Burlingame, CA) at 4°C for 30 minutes.
After 3 washes in buffer, the cells were analyzed on a
cytofluorograph. In some cases, 2-color immunofluorescence analysis was performed. The above procedure was
followed by a blocking step, with cells incubated at 4°C for
30 minutes with an irrelevant murine MAb (anti-trinitrophenol). After 3 washes, the cells were incubated with a
phycoerythrin-labeled murine MAb, washed, and prepared
for analysis on a cytofluorograph. Cell fluorescence was
analyzed on an Ortho (Raritan, NJ) 11s cytofluorograph, with
gating on the small, nongranular lymphocyte population.
The percentage of cells that were fluorescent with buffer or
irrelevant control murine MAb and fluorescein-labeled goat
anti-mouse IgG alone was subtracted from the total. Cytofluorographic histograms of cells stained with anti-TCR MAb
exhibited a peak of fluorescence distinct from the negative
peak and with fluorescence intensity approximating that of
cells stained with anti-CD3 MAb.
Statistical analysis. Mean f SD values were determined for each subject group. Patient and control populations were compared and analyzed for statistically significant
differences using Student’s t-test (if the data were compatible with a normal distribution and if standard deviations
were small) or the Mann-Whitney test (for nonparametric
distributions). P values shown are unadjusted for multiple
tests. An a level of 0.01 was used to determine statistical
significance.
0
0
T
0
normal
RA
non-RA
arthritis
Vp-17
0
20
-
0
0
0
0
0
0
&
RESULTS
TCR VP repertoire in peripheral blood. MNC
were isolated from the peripheral blood of healthy
subjects and from patients with seropositive RA,
non-RA inflammatory arthritis, or SLE, and the percentage of CD3-positive cells expressing the TCR Vp
gene products was identified using our panel of antiTCR MAb and indirect immunofluorescence analysis.
The mean percentage of T cells reactive with MAb C37
(Vp5.2/5.3), OT145 (Vp6.7a), 16G8 (VpS), or S511
(Vp12) was similar in each of the groups tested (Table
l), a result consistent with findings of previous studies
of the T cell repertoire in autoimmune disease in which
these monoclonal reagents were used (18,19). In contrast, analysis of CD3-positive PB cells reactive with
the more recently available MAb C1, specific for the
Vp17 TCR gene product, demonstrated a significant
increase in the mean percentage of Vpl7-positive cells
in RA patients when compared with the normal sub-
SLE
?9
RA
non-RA
arthritis
Vp-17
Figure 1. Increased percentage of Vpl7-positive T cells in peripheral blood lymphocytes (PBL) (upper panel) and synovial fluid
(SF) (lower panel) of patients with rheumatoid arthritis (RA).
Mononuclear cells from normal subjects, patients with seropositive
RA, patients with non-RA inflammatory arthritis, and patients with
systemic lupus erythematosus (SLE) were analyzed by indirect
immunofluorescence for expression of Vp17 T cell receptor (TCR)
gene products. Results are expressed as the percentage of cells
reactive with anti-TCR Vp monoclonal antibody (MAb) divided by
the percentage of cells reactive with anti-CD3 MAb. Horizontal bars
represent the means.
jects ( P < 0.01) or control patients (P < 0.01) (Table 1
and Figure 1). No significant increase in the percentage of Vpl7-positive cells was observed in the non-RA
ZAGON ET AL
1434
I
I’US
CD3
CDS
Tac
CD4
I
c1
I~luoresceiiccliilciisily
Figure 2. T cell phenotype of SF rnononuclear cells (MNC) from a patient with RA. MNC were isolatedfrom
SF and analyzed by indirect immunofluorescence for expression of the T cell surfaceepitopes indicated.
Fluorescence is demonstrated on the abscissa (log scale) and cell number on the ordinate (linear scale) of each
cytofluorographic histogram. PBS = phosphate buffered saline; see Figure 1 for other definitions.
arthritis or SLE patients when compared with the
normal controls. Taken together, these results demonstrate a selective, statistically significant expansion of
the Vpl7-positive T cell population in the peripheral
blood of RA patients.
TCR Vp repertoire in synovial fluid. To characterize the T cell repertoire at the site of disease in
patients with RA, S F T cells were isolated from 49
patients with seropositive RA and analyzed for TCR
Vp gene usage by indirect immunofluorescence staining (Table 1 and Figure 1). The mean SD percentage
of CD3-positive cells reactive with the Vpl7-specific
MAb C1 was significantly elevated in the RA patients
(8.5 2 4.1%) when compared with the percentage of
Vpl7-positive cells in the 19 S F specimens from patients with non-RA inflammatory arthritis (5.3% %
2.0%) (P = 0.0006). Strikingly, 31% of the RA S F
samples (15 of 49) and none of the 19 control samples
contained >lo% Vpl7-positive T cells (Figure 1). In
contrast, no significant differences were noted be-
*
tween RA and non-RA arthritis control S F in the
percentages of Vp5.2/5.3-, Vp6.7a-, VpS-, or Vp12positive T cells.
A representative study of SF T cells from an
RA patient with an elevated percentage of Vp17positive T cells is presented in Figure 2. Analysis of
the TCR repertoire in this patient showed that 17.3%
of the cells expressed the Vp17 gene product, but only
2.8%, 2.4%, and 1.9% expressed the Vp5.215.3,
V/36.7a, and Vp12 products, respectively. Thus, of the
5 TCR Vp gene families studied, only Vp17 was significantly increased in expression at the site of disease in
the RA patients. Although HLA typing data were not
available for all of the subjects, an increased percentage of Vpl7-positive T cells in RA S F did not appear to
correlate directly with expression of the DR4 RA
susceptibility allele in these patients. To date, 4 of the
15 patients with >lo% Vpl7-positive SF T cells have
been HLA typed, and their DR haplotypes are DR4,7;
DR2,3; DRwl3,-; and DR5,7.
1435
INCREASED VP17-POSITIVE T CELLS IN RA
23 1
0
0
0 .
0
0
=0
10
c
P
+
o
A
'
8
I
VB 5 2 . 5 3
Vp 6 7
V8 8
v p I2
Vp 17
TaC
Figure 3. Distribution of T cells expressing particular TCR Vp
genes between CD4 and CD8 T cell subsets. SF T cells from RA
patients were isolated and prepared for 2-color immunofluorescence
analysis. T cells were incubated with either MAb reactive with TCR
Vp5.215.3, 6.7a, 8, 12, or 17 gene products or anti-Tac MAb,
followed by fluorescein isothiocyanate-labeled goat anti-mouse IgG.
After a blocking step, the T cells were incubated with either
phycoerythrin-labeled anti-CD4 or anti-CD8 MAb. Results are expressed as the percentage of CDCpositive (open symbols) or CD8positive (closed symbols) T cells positive for the indicated TCR Vp
gene product or for Tac. See Figure I for definitions.
Analysis of SF V'l7-positive T cells. To determine the distribution of Vpl7-positive SF T cells
between the CDCpositive and CDS-positive T cell
subsets, SF T cells were simultaneously assessed for
Table 2.
expression of TCR Vp gene products and CD4 or CDS,
using 2-color immunofluorescence analysis (Figure 3).
A relative increase of CD8-positive T cells has been
previously noted in SF samples from patients with RA
(20,21), an observation confirmed in our SF analysis
(Table 1). The CD4:CD8 ratios of the SF samples
studied were variable, but were noted to be reversed
(<1.0) in 5 of 10 individuals. Overall, the mean percentages of CD4 versus CDS T cells expressing VP5.2/
5.3, 6.7a, 8, 12, or 17 were comparable. In 6 of the 10
SF samples tested, including 1 containing 19.4% Vp17positive T cells, those cells were equivalently distributed between the CD4-positive and CD8-positive subsets. In 2 samples, the Vpl7-positive T cells were
skewed toward the CD4 population and in 2 samples
they were skewed toward the CD8-positive subset.
Analysis of paired PB and SF samples demonstrated
that some patients with increased percentages of Vp17positive T cells in SF also had expansions of these
cells systemically, as reflected in the PB T cell repertoire (data not shown). Therefore, the distribution of
SF VpI7-positive T cells in both T cell subsets is to be
expected, since the T cell repertoire in this compartment is likely to reflect, at least to some extent, the
peripheral T cell pool.
We next investigated whether the SF Vp17positive T cells were selectively activated in vivo, by
analyzing IL-2R (Tac) expression. Although the mean
percentage of Tac-positive T cells in RA SF specimens
was only modestly higher than the percentage of those
Distribution (%) of Tac-positive cells among synovial fluid and tissue T cell subpopulations*
T cell population
Source
RA synovial fluid
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
RA synovial tissue
Patient 6
Non-RA arthritis synovial fluid
Patient 7
Patient 8
Patient 9
CD4
CDS
Vp3
Vp5.215.3
Vfi.7a
Vpl2
Vp17
79.3
80.0
81.6
3.4
8.7
6.8
4.8
6.6
6.9
4.3
1.9
10.9
13.1
3.4
4.3
3.9
6.8
8.2
48.3
21.7
10.7
18.4
100
3.4
10.0
18.4
0
90.3
11.1
0
4.3
ND
3.4
ND
78.4
21.6
ND
0
0
0
25.2
86.2
59.3
70.7
50.8
14.8
27.6
ND
ND
ND
3.1
3.7
3.4
6.9
11.1
3.4
4.6
3.7
5.2
4.6
3.7
3.4
18.0
~
* Two-color immunofluorescence analysis was performed on synovial fluid T cells from patients with
rheumatoid arthritis (RA) or non-RA arthritis and on synovial tissue T cells from a patient with RA,
using phycoerythrin-labeled anti-interleukin-2 receptor monoclonal antibody (anti-IL-2R MAb) and
either anti-CM, anti-CDS, or anti-T cell receptor (anti-TCR) Vp MAb, followed by fluorescein isothiocyanate-labeled goat anti-mouse IgG. The distribution of the total Tac-positive T cells between CD4 and
CD8 subsets, and among the 5 TCR Vp populations tested, was calculated by determining the
percentage of cells positive with both anti-IL-2R and anti-T cell MAb divided by the percentage of
IL-2R-positive cells. ND = not determined.
ZAGON ET AL
1436
IE
ze
38
48
c1-
70
ie
90
Figure 4. Two-color immunofluorescence analysis of S F T cells. S F T cells were stained with anti-Vp3 (left
panel) or anti-Vpl7 (right panel) MAb and fluorescein isothiocyanate (FITCblabeled goat anti-mouse IgG,
followed by phycoerythrin-labeled anti-interleukin-2 receptor antibody (ILZR-PE) (anti-p55-Tac), and
immunofluorescence was assessed by cytofluorography. The histograms show that of the Vp3-positive T cells
(5.8%), <lo% were also IL2R positive, while of the Vpl7-positive T cells (4.5%), -45% expressed IL2R. The
bold dashed lines indicate the distribution of the T cells that were reactive with either anti-Vp3 or anti-Vpl7
MAb, as well as with the anti-IL2R MAb. See Figure 1 for other definitions.
cells in control SF (Table I), as has been reported
previously (22,23), we wished to determine if Vp17positive SF T cells selectively expressed this activation antigen. Two-color immunofluorescence analysis
was performed on 5 RA SF samples and on 3 non-RA
inflammatory arthritis SF samples, all of which contained <lo% Vpl7-positive T cells (Table 2). Tacpositive T cells were increased among the Vp17positive T cells from the RA SF, but not from the
non-RA arthritis SF. Moreover, the Tac-positive cells
were found almost exclusively among the CD4positive T cells from the RA patients (Table 2 and
Figure 3). Cytofluorographic histograms demonstrating Tac expression on a high proportion of Vp17positive T cells, but not Vp3-positive T cells, from an
RA SF specimen are shown in Figure 4. Thus, even
RA SF that do not contain a markedly expanded
Vpl7-positive T cell population showed evidence for
preferential activation of the CDCpositive, Vp17positive T cell fraction, when compared with T cells
expressing the other Vp gene products tested.
TCR Vp repertoire in synovial tissue. Although
access to synovial membrane specimens was more
limited, we were able to study ST T cells from 21
patients with seropositive RA (Table 1). The mean t
SD Vp17 expression among the ST T cells (6.7 t 2.2%)
was in the same range as that observed in RA PB T
cells (6.5 t 2.7%) and significantly higher than that in
normal PB T cells (P= 0.001). There were insufficient
T lymphocytes from most control (osteoarthritis) ST
Table 3. Sequential study of the phenotype of peripheral blood, synovial fluid, and synovial tissue T cells from a patient with rheumatoid
arthritis
Synovial fluid T cells
Time 1
Time 2
Time 3
Time I t
Time 2t
Time 3
Synovial tissue
T cells,
time 1
30.4
32.6 (9.9)
0.5 (40.0)
13.5 (19.8)
58.8
ND
2.5 (48.8)
10.7 (20.3)
30.6
37.0 (4.5)
0
11.0 (25.6)
37.3
3.7 (17.6)
3.5 (27.8)
9.9 (40.4)
77.0
3.0 (71.0)
0.5 (52.0)
1 .O (67.0)
53.4
6.6 (48.5)
3.9 (40.5)
5.2 (48.1)
71.0
5.0 (30.0)
2.6 (31.2)
7.6 (89.0)
Peripheral blood T cells
% CD4+
% Vp3+ (% CD4+)
% Vp.5.2+ (% CD4+)
% Vp17+ (% CD4+)
* Peripheral blood, synovial fluid, and synovial tissue T cells were isolated from a patient with rheumatoid arthritis on multiple occasions over
1Vz years. The percentage of T cells expressing the indicated T cell receptor Vp gene product was determined by indirect immunofluorescence
analysis. Two-color immunofluorescence was performed using phycoerythrin-labeled anti-CD4 monoclonal antibody, and the percentage of T
cells that expressed the Vp gene product and were also CD4-positive is shown in parentheses. ND = not done.
t Methylprednisolone (40 mg) was injected into the patient's knee joint at the time S F sample 1 was obtained. SF sample 2 was obtained from
the same joint 3 weeks later.
1437
INCREASED VP17-POSITIVE T CELLS IN RA
specimens for immunofluorescence analysis or statistical comparison with the RA ST T cells. Two-color
immunofluorescence analysis of T cells from 1 RA ST
sample was performed (Table 2). As was observed in
the 2-color analysis of SF T cells, the Tac-positive cell
population was enriched among the Vpl7-positive RA
ST T cells.
Detailed analysis of the T cell repertoire phenotype in an RA patient. To explore the pathogenic
significance of the expanded Vpl7-positive T cells in
RA, we analyzed these cells in detail in a representative patient with longstanding active RA (Table 3).
This patient was RF positive at a high titer, HLADR4,7 positive, and, most striking, demonstrated a
persistent (>1.5 years) alteration in the peripheral T
cell repertoire, characterized by elevation of both Vf33and Vpl7-positive T cells. The large V,3 population,
comprising one-third of the PB T cell repertoire, was
almost exclusively CD8-positive, while the SF and ST
Vp3 populations, representing 3.0-6.6% of the local T
cell repertoire, were distributed between both CD4
and CD8 subsets.
Markedly expanded oligoclonal CD8-positive T
cell populations have been described in the PB of some
RA patients (24) and, more recently, normal individuals. Molecular analysis of this patient’s PB Vp3 TCR
suggested the presence of a monoclonal expansion (Li
Y, Sun G-R, Tumang JR, Crow MK, Friedman SM:
unpublished observations). The dramatic and persistent expansion of Vp3-positive, CD8-positive T cells in
the blood, but not the SF or ST, suggests that these
cells are unlikely to represent an important pathogenic
T cell population at the site of joint inflammation. They
may have an immunoregulatory or cytotoxic functional role, or may be a manifestation of a benign
lymphoproliferative disorder.
In contrast to the distribution of the Vp3positive T cells, the compartmentalization of the Vp17positive T cell fraction in this patient (patient 2 in
Table 2) is consistent with the notion that those cells
play a pathogenic role at the primary site of disease,
the synovial membrane. While the expanded Vp17positive PB and SF T cells were distributed between
the CDCpositive and CDB-positive T cell subsets,
among the ST T cells, 89% of the Vp17 population was
distributed to the CD4-positive subset. Moreover,
among the SF T cells, the Tac-positive cells were
selectively represented among the Vpl7-positive cells.
This RA patient, then, demonstrated the persistent
high-level expression of PB Vpl7-positive T cells in
both the CD4 and CD8 subsets, while the Vp17 T cells
at the primary site of disease, the synovial membrane,
were almost exclusively found among the CD4positive, helperhducer subset. Of interest, 3 weeks
after the therapeutic administration of intraarticular
steroids effected clinical improvement in knee synovitis, the expanded population of Vpl7-positive SF T
cells (9.9%) was reduced to 1.0% (Table 3).
DISCUSSION
Investigators at a number of laboratories, using
a variety of molecular techniques, have analyzed the T
cell repertoire in RA with the goal of identifying the
pathogenic T cells. Initial studies used Southern blot
analysis of DNA isolated from RA SF or ST T cells or
T cell clones to detect evidence of oligoclonal T cell
populations (25-33), and several investigators reported
dominant TCR rearrangements (25,26,30,33). However, rarely were identical rearrangement patterns
observed, and the pathogenic significance of T cell
clones that may have been “selected” for survival in
vitro remains unclear.
Polymerase chain reaction (PCR) analysis of
TCR Vp messenger RNA (mRNA), using nucleotide
primers specific for each of the known Vp gene families, has been used to identify, and in some studies to
quantitate, the T cells expressing each of the TCR Vp
gene families (3447). Sottini et a1 amplified complementary DNA (cDNA) synthesized from SF T cell
RNA samples of 3 patients with RA (43). In 2 of the 3
patients, Vp7 was heavily expressed. Paliard et a1 used
quantitative PCR to document relative depletion of
Vpl4-specific RNA in PB, compared with SF, from 9
patients with RA (44). The VplCpositive T cell lines
derived from SF tended to be dominated by several
clones and showed evidence for restricted Jp gene
usage. Howell et al reported oligoclonal Vp3, 14, and
17 mRNA transcripts among IL-2R-positive, presumably in vivo-activated ST T cells (45). A recent study
also showed oligoclonally expanded populations of T
cells expressing Vp14 TCR in SF and ST samples from
patients with juvenile rheumatoid arthritis (42).
Because the assessment of TCR V gene usage
by molecular techniques is, at best, semiquantitative
(481, we have taken a more direct approach to T cell
repertoire analysis by enumerating the percentage of
freshly isolated T cells that are reactive with the
available panel of murine MAb specific for particular
human TCR Vp gene products. In this report, we
present the first documentation of a selective increase
in the percentage of Vpl7-positive T cells in RA.
1438
Vpl7-positive T cells were increased in the PB of RA
patients, while the proportions of T cells expressing
products of the other TCR Vp gene families analyzed
were similar to those in control subjects. Moreover, a
highly significant increase in the percentage of Vp17positive T cells was observed in SF specimens from
RA patients when compared with joint effusions from
patients with gout, osteoarthritis, Reiter’s syndrome,
and monarticular arthritis. While approximately onethird of the RA SF studied contained >lo% Vp17positive T cells, it is of interest that even in RA SF
with <lo% Vpl7-positive T cells, these cells were
enriched among the IL-2R-positive fraction. These
findings suggest the selective in vivo activation of
Vpl7-positive, CDCpositive T cells, and, by extension, a potential pathogenic role for these cells.
A recent analysis of the T cell repertoire in RA
PB and SF using anti-TCR MAb confirms the normal
expression of Vp5, 6.7a, 8, and 12 gene products (19).
Those investigators did not study Vp17 expression.
Documentation of the reactivity of RA synovial cells
with an MAb reactive with Vp14 gene products, as yet
unavailable to us or others, will be of considerable
importance.
Our analysis of paired RA PB and SF samples
showed a moderately or significantly increased percentage of Vpl7-positive T cells in PB of patients with
increased Vpl7-positive SF T cell expression. These
data are analogous to reports of expanded populations
of T cells expressing particular V gene products at the
site of disease, and a moderate increase of these cells
in the peripheral blood, in Crohn’s disease and sarcoidosis (49,50). The results contrast with those of
Paliard et al, who noted a reduction in Vpl4-specific
RNA in the PB of RA patients, with a concomitant
relative increase among joint fluid T cells (44). The
reasons for this discrepancy are not clear, but may
relate to differences in the techniques employed (PCR
versus monoclonal antibody staining) or differences in
the response of Vpl7-positive versus Vpl4-positive T
cells to the pathogenic antigen or superantigen (see
below).
The potential role of joint-localized Vp17positive T cells in RA was suggested by the analysis of
a patient who demonstrated persistent expansion of
that T cell fraction. Comparison of the T cell repertoires in PB and joint specimens showed essentially
equal representation of Vpl7-positive T cells in the
CD4-positive and CD8-positive subsets within PB and
SF, but selective distribution of the Vpl7-positive T
cells to the CD4 subset in the ST. In the SF, activated
ZAGON ET AL
(Tac-positive) T cells were enriched among the Vp17positive cells. Molecular analysis of mRNA from this
patient’s freshly explanted SF and ST specimens has
demonstrated several oligoclonal Vpl7-positive T cell
populations with highly homologous antigen-binding
complementarity-determining region-3 (CDR3) sequences, while the TCR CDR3 sequences from PB
Vpl7-positive cDNA were heterogeneous. Taken together, these results suggest that antigens localized in
the joint clonally expand some of the Vpl7-positive T
cells (51). Additional studies show that Vpl7-positive
ST T cell lines from this DR4,DR7-positive RA patient
selectively proliferate in response to DRCpositive
Epstein-Barr virus-transformed B cell line cells, demonstrating that Vpl7-positive T cells expanded in the
synovial membrane may be specific for or restricted by
the RA-associated class I1 MHC antigen.
Selective expansion of T cells bearing products
of particular Vp gene families is consistent with either
a superantigen-driven or conventional antigen-driven
process. In this regard, superantigens are, by definition, Vp-selective T cell mitogens, while soluble antigens derived from human pathogens can also generate
TCR VFrestricted T cell proliferative responses
(52,53). The data presented here, which document a
significant increase in the percentage of Vpl7-positive
T cells in PB and joint samples from patients with RA,
together with the PCR data from Paliard et a1 (44),
Howell et a1 (49, and Grom et a1 (42) implicating Vp14
and V,3, are consistent with a potential role for a
superantigen-like molecule preferentially reactive with
TCR encoded by subgroup IV of TCR Vp gene families. Vp3, 14, and 17 are members of subgroup IV,
based on a high level of sequence homology (54). An
analysis of sequence-relatedness in a 15-amino acid
stretch of the third hypervariable region, proposed to
contribute to superantigen binding, revealed that Vp3,
12.1, and 14 are most homologous to Vp17 (45).
Additional data implicating subgroup IV TCR gene
products in RA are provided by our analysis of SF
samples from both knees of an RA patient which
contained a modest increase in Vpl7-positive (7.6%
and 7.4%), but a marked elevation in Vpl2-positive
(18.3% and 18.9%), T cells (data not shown).
Of the described microbial superantigens, the
Mycoplasma arthritidis-derived superantigen, MAM,
has specificity for TCR subgroup IV Vp determinants
(17,55). In addition, MAM has the special capacity to
preferentially induce T cell-dependent B cell differentiation, while stimulating weak T cell proliferative
responses, by human peripheral blood lymphocytes
INCREASED VP17-POSITIVE T CELLS IN RA
(56). An MAM-like superantigen might account for the
initial expansion of Vpl7-positive T cells documented
here, as well as the polyclonal B cell activation that is
characteristic of RA (57).
While the selective, but polyclonal, expansion
of Vpl7-positive T cells in the PB of RA patients is
consistent with a superantigen trigger, the presence of
oligoclonal populations of Vpl7-positive synovial
membrane T cells supports a concomitant role for
specific antigenic peptides in the persistent localization, activation, and expansion of those cells. Cloned
populations of CDCpositive, Vpl7-positive synovial T
cells and panels of potentially relevant superantigens
and classic antigens are being used to characterize the
specificity of pathogenic T cells in RA.
ACKNOWLEDGMENTS
The authors are indebted to many physicians, both
rheumatologists and orthopedic surgeons, at The Hospital
for Special Surgery for their interest, cooperation, and
generosity during the course of this study. In particular, we
would like to thank Drs. Charles Christian, Theodore Fields,
Mark Figgie, Matthew Kraay, Timothy Lovell, Stephen
Paget, Marcos Rivelis, and Sergio Schwartzman. We appreciate the expertise and cooperation of Dr. Peter Gregersen,
who assisted with HLA typing, Dr. David Posnett, for
helpful comments and review of the manuscript, Mr. Carl
Triscari, who provided assistance with cytofluorographic
analysis, Mr. Zhongqiang Chu, who provided technical
assistance, Dr. Margaret Peterson, who assisted with statistical analysis of data, and Ms Venus Te Eng Fo, who helped
in the preparation of the manuscript.
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