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Expansion of unusual CD4+ T cells in severe rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 6, June 1997, pp 1106-1114
0 1997, American College of Rheumatology
1106
EXPANSION OF UNUSUAL CD4+ T CELLS IN
SEVERE RHEUMATOID ARTHRITIS
PETER B. MARTENS, JORG J. GORONZY, DANIEL SCHAID, and CORNELIA M. WEYAND
Objective. The repertoire of T cells in patients
with rheumatoid arthritis (RA) is characterized by
clonal expansion of selected CD4+ T cells, which are
autoreactive and lack the expression of the functionally
important CD28 molecule. The goal of this study was to
determine the contribution of these unusual lymphocytes to the disease process.
Methods. RA patients (n = 108) and normal
controls (n = 53) were examined for the expression of
CD4+CD28- T cells by 2-color fluorescence-activated
cell sorter analysis. Clinical data were ascertained by
retrospective chart review.
Results. The frequencies of CD4+CD28- T cells
displayed a bimodal distribution, defining carriers and
noncarriers in normal subjects and RA patients. In
longitudinal studies, the noncarrier and carrier phenotypes were stable over time. Carriers of CD4+CD28- T
cells accumulated in the RA population (64% versus
45%; P = 0.02). The expansion of CD4+CD28- T cells
correlated with extraarticular involvement, but not with
disease duration, antirheumatic treatment, or severity
of joint destruction. The patient subsets with nodular
disease (P = 0.02) and rheumatoid organ disease (P=
0.04) had the highest proportion of CD4+CD28- T cell
carriers. The size of the CD4+CD28- compartment
correlated with extraarticular progression of RA (P =
0.001 in nodular RA, P = 0.003 in rheumatoid organ
disease).
Conclusion. The bimodality of distribution of
CD4+CD28- T cell frequencies is compatible with
Supported in part by grants from the NIH (R01 AR-41974
and R01 AR-42527), a Clinical Science Grant (NAF #14) and a
Biomedical Science Grant (NAF #16) from the National Arthritis
Foundation, and a grant from the Mayo Foundation.
Peter B. Martens, MD, Jorg J. Goronzy, MD, PhD, Daniel
Schaid, PhD, Cornelia M. Weyand, MD, PhD: Mayo Clinic and
Foundation, Rochester, Minnesota.
Address reprint requests to Cornelia M. Weyand, MD, PhD,
401 Guggenheim Building, Mayo Clinic, Rochester, MN 55905.
Submitted for publication September 3, 1996; accepted in
revised form January 7, 1997.
genetic control of the generation of these
cells. In RA patients, CD4+CD28- T cells
epiphenomenon of the disease process, but
patients to developing inflammatory lesions
ticular tissues.
unusual T
are not an
predispose
in extraar-
The chronic tissue-destructive process in patients
with rheumatoid arthritis (RA) has been attributed to an
ongoing immune response in which T lymphocytes play
a critical role (1). The concept that RA is a T cell-driven
disease has been supported by several lines of evidence.
T cells are the dominant cell population in the synovial
infiltrate (2). RA is genetically associated with HLA
class I1 molecules, which function by presenting antigenic peptides for recognition by the T cell receptor
(TCR) (3,4). Finally, therapeutic T cell depletion induces, at least transiently, an amelioration of disease
activity in RA patients (5,6). Most efforts directed
toward identifying disease-relevant T cells in RA have
focused on antigen-specific T cells, recognizing a putative arthritogenic antigen in the synovial tissue (7-10).
These studies have yielded conflicting results and have
not majorly advanced the understanding of the pathophysiologic role of T cells in RA (11).
More recent data have introduced a new aspect
of pathologic T cell function in RA patients. Molecular
analysis of the repertoire of circulating CD4+ T cells
has led to the observation that RA patients carry CD4+
T cell populations that have undergone in vivo clonal
expansion. Accumulating evidence shows that these
expanded clonotypes are detected very early in the
disease and are not a consequence of synovial inflammation. Phenotypic examination of expanded clonotypes
has demonstrated that the clonally proliferating CD4 +
lymphocytes express a unique spectrum of cell surface
markers (12). Specifically, they lack expression of the
CD28 molecule, a very unusual feature for a mature
CD4+ T cell. T cell function has been intimately linked
to the CD28 molecule. Receptor-ligand interaction involving CD28 facilitates T cell costimulation, a necessary
CD4+ T CELL EXPANSIONS IN SEVERE RA
component of T cell activation (13). Thus, clonally
expanded T cells in RA patients are characterized not
only by abnormal growth behavior but also by unusual
functional properties. T h e presence of large numbers of
these unusual T cells in RA patients is likely to influence
immune responsiveness and alter mechanisms of inflammation which depend on T cell regulation.
T h e finding that clonogenic T cell populations
have a distinct phenotype opened the possibility of
utilizing CD28 phenotyping as a surrogate marker to
screen large cohorts for the presence of these unusual
cells. T h e current study was designed to explore whether
CD4+CD28- T cells are a requirement for developing
RA or whether they can b e associated with certain
disease manifestations of RA. A recent study in RA
multiplex families has described oligoclonal T cell populations in unaffected siblings of RA patients, raising
the interesting possibility that CD4 clonality is an intrinsic defect in RA families, possibly attributable to
genetic factors (14). It can therefore be expected that
t h e phenomenon of oligoclonal proliferation of
CD4+CD28- T cells is present in t h e general population but accumulates in RA families. Combined with
other genetic risk determinants, t h e presence of clonotypic T cell populations could increase the risk of
developing RA.
Here we have compared t h e frequencies of
CD4+CD28- T cells in RA patients and normal subjects and have found that a marked expansion of the
CD28- compartment is characteristic of RA patients
who present with a defined disease pattern. Increased
frequencies of these oligoclonal CD4+ T cells is a
feature of patients who develop extraarticular manifestations. These data suggest that CD4t-CD28- T cells
with abnormal growth behavior have a tendency to
induce the most severe form of rheumatoid disease,
rheumatoid lesions in major organ systems, and may be
useful as a predictive marker for identifying this category of RA patients before the onset of rheumatoid
organ disease.
PATIENTS AND METHODS
Patients and controls. One hundred eight patients
diagnosed as having RA based on the 1987 criteria of the
American College of Rheumatology (formerly, the American
Rheumatism Association) (15) and 53 controls were entered
into the study. Patients were obtained from the outpatient and
inpatient populations of Mayo Medical Center. Controls were
either healthy subjects (n = 34) or ambulatory outpatients
without inflammatory rheumatic disease (n = 19) and were the
same age range as the RA patients. Ambulatory outpatients
1107
had the following main diagnoses: osteoarthritis (n = 8);
tendinitis, bursitis, or back pain (n = 5); fibromyalgia (n = 2);
coronary artery disease (n = 1); recurrent genital herpes
simplex infection (n = 1); migraine headaches (n = 1); and
colon polyps (n = 1).All patients and controls were Caucasian.
Flow cytometry. Peripheral blood mononuclear cells
(PBMC) were obtained by Ficoll gradient centrifugation.
PBMC (300,000-500,000) were stained with fluorescein isothiocyanate (F1TC)-conjugated anti-CD4 and phycoerythrin
(PE)-conjugated anti-CD28 monoclonal antibodies (Becton
Dickinson, San Jose, CA). FITC-conjugated IgGl and PEconjugated IgG2a (Simultest; Becton Dickinson); and FITCconjugated anti-CD4 and PE-conjugated anti-CD3 (Becton
Dickinson) were used as negative and positive controls, respectively. Flow cytometry was performed on a FACS-Vantage;
results were analyzed with PC-lysis software (Becton Dickinson). The fraction of cells within the CD4+ population that
was CD28- was calculated by gating on the CD4+CD28+ and
CD4+CD28- populations.
Clinical evaluation. Histories of 108 patients were
reviewed, with attention to the disease, pattern of joint involvement, presence of nodules and other extraarticular features
such as vasculitis or organ involvement, and laboratory features such as Westergren erythrocyte sedimentation rate
(ESR) and rheumatoid factor (RF).
The group with rheumatoid organ involvement included patients with vasculitis (n = 6), lung (n = 2), and
peripheral nerve (n = 2) involvement. Rheumatoid vasculitis
was diagnosed by histomorphology or angiogram in all but 1
patient. This patient had nodular RA and recurrent lower
extremity skin ulcers without clinically evident atherosclerosis.
Periungual nailfold infarcts were not considered sufficient for
the diagnosis of vasculitis, and sicca syndrome was not considered sufficient for the diagnosis of organ disease.
Rheumatologic medications at the time of blood collection were also recorded, as well as the surgical history. Joint
arthroplasty or reconstruction were considered significant in
the surgical history, while synovectomy and carpal tunnel
release were not.
Statistical analysis. To address the question whether
the observed distribution of CD4+CD28- frequencies was a
mixture of 2 or 3 underlying distributions, mixture models were
fit to the data as follows. The data were transformed by natural
log of CD4+CD28- frequencies to remove skewness of the
observed frequencies, and these transformed data were fit to a
mixture of either 1, 2, or 3 normal diqtributions. The parameters of the mixture model were the means of the underlying
distributions, a common variance, and the mixing proportions
of the underlying distributions. Observations below a threshold
of 0.1% CD4+CD28- T cells were truncated, and the mixture
model accounted for truncation by fitting a cumulative normal
distribution for theqe truncated data, the normal density was fit
for nontruncated data. Parameters were estimated by maximum likelihood, and models were compared with likelihood
ratio statistics using the SAGE computer package (16).
The distribution of CD4+CD28- percentages was
compared between controls and RA subgroups, using the
nonparametric Mann-Whitney test. Since the mixture models
described above suggested an underlying bimodal distribution
of CD4+CD28- subset frequencies, a cutoff point was determined at the intersection of the bimodal distribution curves to
MARTENS ET AL
1108
Table 1. Demographics of the study population
Rheumatoid
arthritis
patients
(n = 108)
Age, mean -t SD years
Female, %
Disease duration, mean t SD years
Rheumatoid factor, %
Bony erosions, %
Nodular disease, %
Major organ involvement, %
Control
subjects
(n = 53)
56.6 2 12.8
62
11.2 ? 12.7
81
54.3 t- 16.0
66
30
9
-
66
-
-
separate the control and RA groups into 2 subgroups. This
point occurred at a CD4+CD28- subset size of 1.04% in the
RA group, and allowed division into subjects who essentially
lacked CD4+CD28- cells (51.04%; “noncarriers”) and those
who possessed CD4+CD28- cells (>1.04%; “carriers”). Using this cutoff, the probability of misassignment to the lower
subpopulation is 8.3%, and the probability of misassignment to
the upper category is 5.6%. Proportions of carriers and
noncarriers were compared in different subgroups using chisquare analysis. The Mann-Whitney test and chi-square analysis, as well as box plots and confidence intervals were calculated with the Systat software package (Systat, Evanston, IL).
RESULTS
Frequencies of unusual CD4+CD28- T cells in
normal subjects and RA patients. Fluorescenceactivated cell sorter (FACS) analysis was used to determine the frequency of the unusual CD4+CD28- T cell
population in 108 R A patients and 53 controls without
clinical evidence of a chronic inflammatory disease. The
demographics of the study populations are shown in
Table 1.
Both cohorts were not significantly different in
age and sex distribution. In a normal immune system,
CD4+ T cells lacking the CD28 molecule should be
infrequent. Accordingly, in most healthy donors,
CD4+CD28- T cells were hardly detectable. The median frequency among controls was 0.7% (Figure 1).
Expanded frequencies of CD4+CD28- cells were often
encountered among R A patients, but expansion of these
cells was a feature of only a subset of RA patients and
was not detectable in all patients. The median
CD4+CD28- frequency of 2.6% in the patient population was significantly different from that in the normal
population (P = 0.009), indicating that a substantial
portion of RA patients had increased frequencies of
these unusual lymphocytes. In some patients,
CD4+CD28- cells accounted for 40-50% of the entire
CD4 compartment.
Expansion of the CD4+CD28- compartment, a
discrete phenomenon in a subset of individuals. The
observation that the CD4+CD28- compartment was
either extremely small or reached a substantial size
suggested that not all subjects are able to generate
CD28- cells in measurable frequencies. Emergence of
unusual CD28- lymphocytes might be regulated by a
genetic or environmental component and only be possible in selected individuals. To address the question
whether the observed distribution of CD4+CD28- frequencies was a mixture of 2 or 3 underlying distributions,
implicating the existence of a genetic or environmental
component, mixture models were fit to the data.
Figure 2 shows the fit of a mixture of 2 populations; this had a significantly better fit for both the
control (P = 0.009) and the R A patient ( P = 0.0008)
groups than a model assuming 1 population. A model of
3 populations was not statistically different from that of
2 populations (data not shown). Age was not a confounding variable in the R A population, and correction
for age did not change the mixture of 2 populations. The
analysis could not exclude the possibility that age played
a role in the cohort of normal individuals.
The bimodal distribution among R A patients
resulted from the finding that not all patients carry high
frequencies of CD4 + CD28 - cells, and both the RA and
control groups could be divided into 2 subgroups, one
with very low frequencies and another with higher
frequencies of CD4+CD28- cells. In the RA group, the
separation occurred at the CD4+CD28- value of
“I
.
.
.
40
$ 1
E-
y
.*.
e
20
1 -
.*=*,
*.-***
0
Controls
i
..*a.
**a:
:*.
Patients with rheumatoid
arthritis
Figure 1. Increased frequencies of CD4+CD28- T cells in rheumatoid arthritis (RA) patients. Peripheral blood mononuclear cells from
108 patients with R A and 53 normal controls were analyzed by 2-color
fluorescence cytometry. The patients had a significantly higher frequency of CD4+CD28- T cells ( P = 0.009).
CD4+ T CELL EXPANSIONS IN SEVERE RA
Controls
001
““9
109
Patients with Rheumatoid Arthritis
1353ioooo
C D 4 t CD28- T Cells (“A)
ooi
009
CD4+
104
,35310000
CD28- T Cells (%)
Figure 2. Bimodal distribution of CD4+CD28- T cell frequencies in
rheumatoid arthritis (RA) patients and normal controls. Mixture
models of 1, 2, or 3 normal distributions were fitted to the observed
frequencies of CD4+CD28- T cells after log transformation. Results
are shown for the model of 2 underlying distributions, which was
significantly better for the RA patients ( P = 0.0008) and controls ( P =
0.009). Individuals with low CD4+CD28- T cell frequencies were
considered noncarriers. The cutoffs between carriers and noncarriers
were 1.04% in the RA cohort and 1.09% in the normal cohort.
1.04% (Figure 2), the point at which the curve of the
lower population intersected the curve of the upper
population. Patients from the group with very low
frequencies of CD4-tCD28- cells (51.04%) essentially
lacked this subset and were considered “noncarriers” of
CD4+CD28- cells. Patients from the second group
possessed higher frequencies of these cells and were
considered “carriers.”
The parameters describing the mixture of 2 subpopulations differed significantly between RA patients
and controls ( P = 0.004). CD4+CD28- T cell carriers
were enriched among the RA cohort. Sixty-four percent
of RA patients versus 45% of controls were classified as
carriers ( P = 0.02). The mean CD4+CD28- T cell
frequencies in noncarriers were very similar (0.27% in
controls versus 0.23% in RA patients). In contrast, the
RA group not only included a higher proportion of
carriers of CD4+CD28- T lymphocytes, but the frequencies in this subset (fitted mean of 7.1%) were also
expanded compared with the controls (fitted mean of
4.1%) (P = 0.02).
Longitudinal analysis of CD4+CD28- lymphocytes in individual patients. The distinction between
patients who carry unusual lymphocytes and those who
essentially do not raises the question of whether the
presence of such lymphocytes is an inherent feature of a
subset of patients or whether unusual CD4+ cells can be
acquired over time. To explore this possibility, RA
patients with low and high numbers of CD4+CD28lymphocytes were monitored longitudinally and the size
1109
of the compartment was determined repeatedly. Again,
the cutoff of 1.04% (from Figure 2) was used.
Results from 9 patients with frequencies of
CD4+CD28- cells 51.04% and 7 patients with higher
frequencies, assessed over an interval range of 5-57
months, are shown in Figure 3. Noncarriers were consistently negative and did not develop higher frequencies
on followup. In patients who were carriers, the numbers
of CD4+CD28- cells varied, but the assignment of a
patient to either the low or high frequency population did not change. These findings are best compatible
with the interpretation that formation of these unusual
lymphocytes is an inherent characteristic of certain
individuals. Such individuals are enriched among patients who have developed RA.
Lack of correlation between duration of RA and
presence of CD4+CD28- lymphocytes. RA patients
have an ongoing immune response in the inflammatory
lesions that could result in the emergence of unusual
lymphocyte populations. If CD4+ CD28- cells represent an epiphenomenon of chronic inflammation, the
frequencies of these cells should increase with progressing disease. To investigate the relationship between
CD4+CD28- T cells and disease chronicity, cell frequencies were correlated with disease duration.
As demonstrated in Figure 4, the size of the
CD4+CD28- compartment was not determined by the
duration of the inflammatory response. Expanded pools
Non-carriers
Carriers
Time points
A
B
A
Time points
B
Figure 3. Carrier and noncarricr status of CD4+CD28- T cells is a
stable feature. Nine patients with low CD4+CD28- T cell frequencies
(noncarriers) and 7 with frequencies higher than the cutoff defined in
Figure 2 (carriers) were reanalyzed at a second time point 5-57 months
after the initial analysis. None of the noncarriers developed higher
frequencies of CD4+CD28- T cells. Conversely, none of the patients
with increased frequencies lost the carrier status, although considerable variations in the frequencies were seen.
MARTENS ET AL
1110
50
.
.
40
h
s
v
.
30
0
CA
N
F! 20
0
10
20
30
40
50
Disease duration (years)
Figure 4. The frequencies of CD4+CD28- T cells do not correlate
with disease duration. The year in which the diagnosis of rheumatoid
arthritis was established was determined by a retrospective chart
review. The disease duration was compared with the frequencies of
CD4+CD28- T cells. No correlation was seen.
of CD4+CD28- cells to >35% were detected in patients who had had RA for only a few months. Likewise,
patients with longstanding RA did not necessarily express increased numbers of the unusual lymphocytes. In
addition, no correlation was found between markers of
disease activity and the size of the CD4+CD28- T cell
compartment; the ESR, which was obtained in 79 of the
patients at the time of the FACS study, did not correlate
with CD4+CD28- T cell frequencies (Pearson's correlation coefficient -0.10). Based on these results, it is
unlikely that these CD4+ T lymphocytes are generated
by the disease process.
Correlation of the presence of unusual
CD4+CD28- T cells with extraarticular disease. The
accumulation of individuals with expanded CD4+ CD28compartments among RA patients implicates these unusual lymphocytes as playing a direct role in the disease.
If these cells contribute to pathologic events, they might
modulate the clinical presentation. Patients were subsetted according to their disease pattern. Disease categories included patients with seronegative disease, patients
with seropositive disease but without extraarticular manifestations (limited RA), patients with subcutaneous
nodules, and patients with rheumatoid organ disease. The
proportion of patients with expanded CD4+CD28- T cell
compartments in each of these disease categories are
shown in Table 2.
In the group of controls, 45% were carriers of
CD4+CD28- cells. In patients with limited RA, the
proportion was similar (52%). The subset of seronega-
tive RA patients included a higher fraction of patients
with CD4+CD28- T cells. More than 70% of the
patients who had developed extraarticular disease manifesting as nodulosis could be categorized as carriers,
with expanded CD28- lymphocytes (P = 0.02 versus
controls). The highest rate (80%) of patients with increased frequencies of unusual lymphocytes was found
among the patient subset with rheumatoid organ disease
(P = 0.04 versus controls). This analysis demonstrated a
relationship between the expansion of the CD4+ CD28compartment and progression of RA to extraarticular
disease.
The finding that RA patients are often carriers of
CD4+CD28- T lymphocytes does not necessarily imply
that the frequency of these cells is markedly expanded.
To examine whether the presence of high frequencies of
unusual lymphocytes in some RA patients was predictive
of disease manifestations, the size of the CD4+CD28subset in patient subgroups (defined by their clinical
presentation) was compared. Median frequencies in
patient groups stratified for disease pattern are shown in
Figure 5. The median size of the CD4+CD28- subset
was 1.1% in patients with seropositive disease limited to
joint manifestations, but increased to 7.0% in patients
with nodulosis (P = 0.001) and to 16.5% in those with
major organ lesions (P = 0.002) (Figure 5). These data
suggest that the number of CD4+CD28- lymphocytes
circulating in the blood is directly correlated with the
development and the severity of extraarticular RA.
Besides a possible contribution of these unusual
lymphocytes to extraarticular manifestations, they might
play a role in the synovial inflammation. The degree of
joint disease in RA is difficult to assess. The proportion
of patients requiring joint arthroplasty was taken as an
indirect measure for the extent of disease-related destruction. Among patients with negative or very low
Table 2. Correlation of CD4+CD28disease patterns
T cell carrier status with
Carriers of
CD4+CD28T cells (%)*
Control subjects
RA patients
Seropositive RAT
Seronegative RA
Nodular RA
Rheumatoid organ disease
P
(versus controls)
45
52
70
72
80
0.90
0.06
0.02
0.04
* Carrier status was defined as expressing >1.04% CD4+CD28- T
cells, as shown in Figure 2. Values are percentages.
t Seropositive rheumatoid arthritis (RA) without extraarticular manifestations.
CD4+ T CELL EXPANSIONS IN SEVERE RA
Rheumatoid organ diseas
1111
Table 4. Proportions of carriers and noncarriers of CD4+CD28- T
cells taking antirheumatic medications
Nonsteroidal antiinflammatory
drugs, %
Hydroxychloroquine, %
Methotrexate, %
Prednisone, %
Nodular RA
CD4+CD28noncarriers
CD4+CD28carriers
P
79
76
0.71
21
50
39
24
40
55
0.74
0.34
0.12
Seronegative RA
Limited RA
A I,I
Controls
Figure 5. Correlation between extraarticular disease and frequencies
of CD4+CD28- T cells. Rheumatoid arthritis (RA) patients were
grouped into the following subsets by retrospective chart review:
patients with seropositive disease limited to joint manifestations
(limited RA), patients with seronegative RA, patients with nodular
disease, and patients with major organ involvement. Sicca syndrome
alone was not sufficient to group patients into the subset with
rheumatoid organ disease. Results are shown for the different subsets
as box plots of CD4+CD28- T cell frequencies, displaying medians,
25th and 75th percentiles as boxes, and 10th and 90th percentiles as
whiskers. Frequencies in patients with nodular disease and with major
organ disease were significantly different from those in patients with
seropositive disease limited to joint manifestations (P= 0.001 and P =
0.002, respectively).
concentrations of unusual lymphocytes, 38% had undergone joint surgery. This rate did not differ significantly
from the 26% of patients with a history of joint surgery
among the patients with expanded numbers of unusual
CD4+CD28- lymphocytes (Table 3). Similarly, the
mean R F concentrations were almost identical in both
of the patient subsets defined by the absence or presence
of unusual lymphocytes. Taken together, no association
was detected between the presence of clonally expanded
and phenotypically abnormal T cells and the joint manTable 3. Lack of correlation between carrier status of CD4+CD28T cells and rheumatoid factor or need for joint surgery
Joint surgery, %
(95% CI)
Rheumatoid factor,
mean 2 SD IU
Noncarriers of
CD4+ CD28T cells
Carriers of
CD4+ CD28T cells
P
38 (21-57)
26 (15-39)
0.18
271 2 436
292 t 400
0.70
ifestations of RA, whereas extraarticular disease was
tightly linked with the presence and the frequencies of
such T cells.
Antirheumatic therapy and unusual CD4+CD28T lymphocytes. The possibility exists that CD4+CD28T lymphocytes develop as a consequence of antirheumatic drug therapy. Patients with extraarticular progression have more aggressive disease, and thus could have
received more intense treatment. To address whether
the emergence of T cells with abnormal phenotype and
growth behavior could result from the use of corticosteroids or other medications, the antirheumatic drug
therapy was compared between noncarrier and carrier
patients (Table 4). The cutoff of 1.04% CD4+CD28- T
cells was again used to divide patients into carrier and
noncarrier groups.
The majority of patients in both groups, 76% and
79% of the carriers and noncarriers, respectively, took
nonsteroidal antiinflammatory medications. Similarly,
no difference was found in the proportions of patients
taking hydroxychloroquine or methotrexate. A slightly
higher proportion of CD4+CD28- carriers took prednisone compared with noncarriers (55% versus 39%),
but this did not reach statistical significance.
DISCUSSION
T lymphocytes have been postulated to be disease
mediating in RA (1). Prevailing theories have proposed
that persistent synovial inflammation results from failure
of the immune system to eliminate a foreign antigen or
from breakdown of self-tolerance initiated by an immune response directed against an arthritogenic antigen
(17). T lymphocytes specific for the arthritogenic antigen
would thus represent disease-causative cells. A different
view of T cell function in RA patients emerged when
TCR repertoire studies revealed that RA patients harbor T cell populations that have undergone considerable
clonal growth (14). The finding that these clonogenic T
cells were present in the blood as well as the synovia
1112
argued against the interpretation that such T cell populations resulted from an antigen-specific response in the
synovia (18). Rather, the size of these clonogenic T cell
populations was consistent with the concept that the
primary defect related to a disturbance in clonal downsizing (11).
Mechanisms underlying clonal expansion of
CD4+ T cells in RA are not understood. Possibilities
include lymphoproliferation as well as lymphoaccumulation. In the first scenario, monoclonal populations
would emerge as the result of overshooting proliferation, possibly initiated or maintained by antigenic stimulation. Lymphoaccumulation could be caused by a
defective regulation of cell death. Recognition of specific antigen by the TCR initiates a cascade of events
which includes entry into the cell cycle and proliferation
of the clone (19). At the same time, antigen contact also
induces programmed cell death, activation-induced apoptosis (20). Thus, the growth of antigen-specific T cells
is coupled to pathways of clonal downsizing, ultimately
preventing the development of clonal lymphocyte
subsets.
Both aspects of T cell function, antigen-induced
activation as well as apoptosis, are dependent on the
CD28 molecule (13,21). If antigen presentation occurs
without costimulation, T cells become anergic and do
not proliferate or undergo apoptosis (22). Signaling
through the CD28 molecule is therefore necessary to
allow the continuous survival and function of T cells.
The finding that clonogenic CD4+ T cell populations
isolated from RA patients lacked surface expression of
CD28 classified these cells as unusual not only in their
growth pattern, but also in their phenotype and functional capacity.
The identification of a unique phenotype made it
possible to rapidly detect these unusual cells in vivo and
to screen a large patient cohort. Determining the frequency of CD4+CD28- T cells in peripheral blood
demonstrated that the distribution of CD4+CD28frequencies followed a bimodal pattern in RA patients
as well as in normal controls. The bimodal distribution is
best compatible with a mixture of 2 subpopulations, and
therefore supports the model that the CD4+CD28- T
cell subset is under regulatory control of genetic or
environmental factors that are either absent or present.
The phenotype defined as carrier of CD4+CD28- T
cells could be assigned to most but not all RA patients.
Thus, these unusual lymphocytes are not an absolute
requirement for the disease, but are associated with
the disease. In this regard, the property of having
CD4+CD28- T cells resembles other well-established
MARTENS ET AL
risk factors or disease phenotypes such as diseaseassociated HLA-DRBl polymorphisms or the production of RF.
The bimodality of the frequencies of unusual
CD4 cells in normal subjects and RA patients can
be explained assuming that the carrier status for
CD4+CD28- T cells represents an inherited phenotype. Support for that notion comes from studies in RA
families. We have shown that an expansion of the
CD4-t CD28- compartment is generally associated with
oligoclonality and that unaffected siblings of RA patients have a higher rate of CD4+ clonality than control
donors (14). Provided that the ability to generate clonal
CD4 populations is determined by a genetic mechanism,
the underlying gene would not be infrequent in the
general population. The proposed “clonality gene”
would cluster in RA cohorts and could thus add to the
threshold liability assumed for the disease.
From a clinical point of view, the most interesting question is if and how these unusual cells contribute
to disease. RA is not a single entity, but includes a
wide spectrum of clinical phenotypes, from mild synovitis to systemic, life-threatening disease. We have previously proposed that extraarticular RA is not simply a
more aggressive form of RA, but represents a new
dimension of the disease requiring a distinct set of
disease risk factors and pathomechanisms (23). Data
presented here strongly support the concept that
CD4+CD28- T cells have a role in the extraarticular
manifestations of the disease. In patients presenting with
seropositive RA limited to the joint, the carrier phenotype was not enriched. Interestingly, the subset of seronegative RA patients included a higher percentage of
individuals with these unusual lymphocytes. In these
patients, other risk factors might be less frequent and
CD4+ CD28- lymphocytes could compensate to reach
the threshold of disease susceptibility. In the absence of
these additional risk factors, the patients apparently did
not progress to extraarticular disease.
The highest proportion of CD4+CD28- T cell
carriers and the highest frequencies of unusual lymphocytes were found in patients with subcutaneous nodule
formation and, in particular, in patients with rheumatoid
organ disease. The organ involvement can possibly be
explained by the distribution of the antigen recognized.
Evidence supporting that notion comes from the demonstration that isolated clonotypes proliferate in response to autologous monocytes (12). These unusual
cells are self-reactive and have escaped self-tolerance
mechanisms. The stimulating self-antigen does not appear to be restricted to the synovial inflammation. While
CD4+ T CELL EXPANSIONS IN SEVERE RA
these T cell clones can be detected consistently in the
inflamed synovial membrane, they do not enrich in the
joint but are expressed at about similar frequencies in
the peripheral blood and in the joint. In this model, the
presence of CD4+CD28- cells may not be specific for
RA but may also be found in other diseases with features
of systemic autoimmunity. Alternatively, we have recently demonstrated that CD4+CD28- T cells utilize
an alternate costimulatory pathway (24). The cell surface molecules involved in costimulation have not yet
been identified. The tissue distribution of the ligands of
the costimulatory molecules used by these cells could
also determine their tissue tropism and thus provide a
molecular basis for the patterns of disease seen in RA
patients.
We and others have previously reported that
HLA genes, in particular the combination of 2 RA
associated HLA-DR alleles, is highly associated with
severe disease (25-28). Detailed correlations of disease
phenotype and genotype have revealed a risk hierarchy
determined by HLA gene products. Genotyping of
rheumatoid vasculitis patients documented that these
patients were frequently homozygous for HLADRB1*0401,the most powerful HLA variant associated
with RA (29). The inheritance of HLA-DRB1*0401
does not render an individual susceptible for CD4
clonality. Rather, we have shown that the carrier status
for CD4+CD28- T cells is independent from the
HLA-DR polymorphisms of the donor (12). In that
sense, RA resembles other common genetic diseases in
which multiple genetic elements are suspected to act in
concert to confer disease risk. More detailed studies
could explore whether HLA-DR genes and CD4 clonality are additive or synergistic in modulating the phenotype of RA toward extraarticular manifestations. Using
both parameters should provide a very powerful tool for
predicting the risk of an individual patient to develop
major organ complications of RA. The ability to predict
the disease pattern could facilitate the development and
the application of treatment strategies which might be
able to prevent rheumatoid organ disease that typically
manifests later in the disease process.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the help of Drs.
Jonathan M. Evans, Robert M. Valente, and Eric L. Matteson
for recruiting patients for this study and Toni L. Higgins for
secretarial support.
1113
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