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Department of Medicine, Division of Rheumatology, Mayo Clinic and Foundation, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite extensive apoptotic death of lymphocytes after a primary response, some cells survive and differentiate into memory cells. Mechanisms underlying the survival of memory cells have been difficult to address because no convincing phenotypic differences have been described that distinguish effector and memory T cells. In addition, memory T cells appear to be able to revert to a naive phenotype (8). Members of the bcl-2 family have been implicated in the survival of activated T cells following an immune response. However, the role of these survival genes in T cell longevity is undetermined (9, 10, 11).
We have recently made the observation that most patients with rheumatoid arthritis (RA) carry, in the peripheral blood, clonally expanded CD4+ T cells that have proliferated to a large clonal size (12). In some patients, single clonotypes can represent as much as 1% of all CD4+ T lymphocytes. The expansion of such clonotypes is not a transient phenomenon. Expanded clonotypes persist over years and can be found in similar frequencies in blood samples taken as many as 5 years apart (13). The longevity of these T cell clones, as well as their clonal size, suggests that these T cells, in contrast to normal recently activated effector cells, must have developed mechanisms to prolong survival. One of these mechanisms could be chronic antigenic stimulation. Indeed, we have shown that in vivo expanded T cell clones isolated from RA patients recognize self Ags expressed on blood-derived adherent cells (14). The majority of the expanded clonotypes in the peripheral blood do not express activation markers, suggesting that continuous activation alone is not sufficient to explain the extent of the clonal expansion. Also, these T cell clones are obviously not affected by clonal exhaustion, a mechanism that counteracts clonal outgrowth in response to chronic hyperstimulation (3).
We tested the hypothesis that these T cell clones have a survival advantage independent of exogenous stimulation. These studies were facilitated by our recent observation that in vivo expanded T cell clones express a unique cell surface phenotype (14). Clonogenic CD4+ T cells lack the expression of the CD28 molecule. This subset of CD4+CD28- T cells can constitute up to one-half of the total CD4 population in some patients with RA. We compared CD4+CD28+ and CD4+CD28- T cells in short term cell lines, as well as CD4+CD28+ and CD4+CD28- T cell clones, for their ability to survive and correlated cell survival with the expression of bcl-2 gene family products. Our data show that CD4+CD28- T cells are characterized by prolonged survival following IL-2 deprivation. This prolonged survival correlated with a constitutively higher expression of bcl-2. In CD4+CD28- T cells, bcl-2 expression was relatively independent of exogenous IL-2 and was not affected by its withdrawal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Short term T cell lines were established from either PBMC or from purified CD4 T cells that contained >3% CD4+CD28- T cells. CD4 T cells were purified from PBMC by negative selection using magnetic bead separation (Miltenyi Biotec, Auburn, CA). PBMC and CD4 T cells were stimulated with immobilized anti-CD3 (OKT3; American Type Culture Collection, Manassas, VA) for 12 h and then maintained in logarithmic growth with densities between 0.5 and 2 x 106 cells/ml in RPMI 1640 (BioWhittaker, Walkersville, MD) containing 10% FBS (Summit Biotechnology, Fort Collins, CO), 2 mM L-glutamine, 50 U/ml penicillin, 5 µg/ml streptomycin (all from Life Technologies, Grand Island, NY), and 10 U/ml recombinant human IL-2 (rhIL-2) (Genzyme Diagnostics, Cambridge, MA). Medium was changed every 3 to 4 days. All assays were performed between day 14 and day 17 after the initiation of the culture.
CD4+CD28- T cell clones were established from PBMC of patients with RA, as described previously (14). Briefly, peripheral blood CD4+ T cells were screened for clonally expanded cells by TCR BV-BJ-specific PCR and were subsequently size fractionated and sequenced. CD4+ T cells expressing the appropriate BV element were activated with immobilized anti-CD3 and cloned by limiting dilution in the presence of irradiated (10,000 rad) EBV-transformed B lymphoblastoid cell lines and 20 U/ml rhIL-2. T cell clones expressing the in vivo expanded TCR sequence were identified and were maintained by restimulation in the presence of 20 U/ml rhIL-2. Control CD4+CD28+ T cell clones were isolated and maintained under the same tissue culture conditions.
Flow cytometry
Surface staining of T cell clones and short term cell lines was performed using FITC-conjugated anti-CD4 and phycoerythrin (PE)-conjugated anti-CD28 mAbs (Becton Dickinson, San Jose, CA). Briefly, 2 x 105 to 1 x 106 T cells were incubated with mAbs or control Ig (Simultest; Becton Dickinson) for 25 min at 4°C, washed once with PBS, and fixed with 1% paraformaldehyde in PBS for 60 min at 4°C. For intracellular staining, cells were surface stained using PerCP-conjugated anti-CD4 (Becton Dickinson) and PE-conjugated anti-CD28, permeabilized with 0.05% Tween 20 (Sigma Chemicals, St. Louis, MO) in PBS for 15 min at 37°C, washed once with PBS, and then resuspended in PBS with FITC-conjugated anti-bcl-2 mAb (Dako, Carpinteria, CA) for 25 min at 4°C (15). The cells were again washed with PBS and resuspended in 1% paraformaldehyde in PBS. Flow cytometry was performed on a FACScan and data were analyzed using PC-LYSYS software (Becton Dickinson).
Apoptosis assays
T cell lines and T cell clones were cultured in the absence or presence of rhIL-2 for the indicated times. Cells were then stained with PerCP-conjugated anti-CD4 and PE-conjugated anti-CD28 mAbs, and FITC-conjugated annexin V (Clontech, Palo Alto, CA). Annexin V binds to phosphatidylserine, which is redistributed in the membranes of cells undergoing apoptosis (16). Alternatively, cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD28, fixed in paraformaldehyde, and then permeabilized with 0.05% Tween 20 and stained with 25 µg/ml 7-amino actinomycin D (7-AAD) (Calbiochem, San Diego, CA) for 30 min at room temperature. The fraction of subdiploid cells was determined by flow cytometry. To analyze AICD, T cell lines were cultured in microplates precoated with 50 µg/ml anti-CD3 for 24 h and then analyzed by three-color flow cytometry. Alternatively, AICD was induced with the anti-CD95 Ab, CH11 (Coulter/Immunotech, Westbrook, ME).
Immunoblotting
Cell lysates were prepared from 2 x 106 T cell clones in a hypotonic buffer containing 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 100 µM Na3VO4, 200 µM PMSF, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100, pH 7.4. Lysates were centrifuged at 14,000 x g for 10 min at 4°C, and protein concentrations were determined by the Bradford technique (Bio-Rad, Richmond, CA). Detergent-solubilized protein (10 µg/lane) was separated on 12% SDS-polyacrylamide gels using a MiniGel System (Bio-Rad) and then transferred to Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 4% BSA in Tris-buffered saline, the membranes were incubated with mouse anti-bcl-2 mAb (Dako) and subsequently with polyclonal rabbit anti-mouse IgG antiserum (Pierce Chemicals, Rockford, IL). The membranes were washed with 0.2% Tween 20 in Tris-buffered saline, and immunoreactive protein complexes were detected with a horseradish peroxidase-linked protein A (Amersham, Arlington Heights, IL). In some experiments, blots were reprobed with polyclonal rabbit anti-bcl-x antiserum (kindly provided by C. Thompson, Chicago, IL) and polyclonal rabbit anti-bax Ab (P-19, Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed using ECL chemiluminescence reagents (Amersham) and exposed to radiographic film (BIOMAX-MS; Kodak, New Haven, CT). The films were developed using an X-OMAT processor and scanned by optical imaging using the AMBIS 4000 system (Scanalytics, Billerica, MA). Equal protein loading was ascertained by amido-black staining and subsequent densitometry scanning.
Statistical analysis
CD28+ and CD28- T helper cells were compared for the percentage of apoptotic cells and for bcl-2 expression by Student’s t test, if appropriate, or by Wilcoxon signed rank test using SigmaStat software (Jandel, San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously described the population of
CD4+CD28- T cells as containing
oligoclonal specificities that have expanded to a large clonal size in
vivo (14). To analyze whether the ability for clonal outgrowth is
correlated with resistance to apoptosis-inducing mechanisms, short term
CD4+ T cell lines were established from PBMC that contained
a minimum of 3% CD4+CD28- T cells as
determined by flow cytometry. All lines were cultured with 10 U/ml
rhIL-2 every 3 to 4 days. The number of apoptotic cells within the
different T cell subsets was determined during logarithmic growth by
three-color flow cytometry with anti-CD4 and anti-CD28 mAbs and
7-AAD. Results from the analysis of 16 T cell lines are shown in Figure 1
. In the T cell lines studied, between
3.2 and 70.7% of all of the CD4+ T cells were deficient
for the CD28 molecule. At the time of testing, 13.1 ± 5.7% of
the CD4+CD28+, but only 2.6 ± 2.5% of
the CD4+CD28- cells, were found to be
subdiploid by 7-AAD staining (p < 0.0001).
This observation suggested that CD4+CD28- T
cells have a lower rate of apoptosis in culture in the presence of
exogenous IL-2. Differences in the apoptotic rate could not be
attributed to tissue culture conditions, since
CD4+CD28+ and
CD4+CD28- T cells were maintained under
identical conditions. The percentage of
CD4+CD28- T cells in these lines remained
stable, indicating that the CD4 T cell subsets did not grossly differ
in their growth rate. However, the possibility could not be excluded
that the higher frequency of CD28+ apoptotic cells
reflected a different growth behavior and not a difference in the
expression of survival genes.
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. Three-color flow
cytometry with anti-CD4 and anti-CD28 mAbs and annexin V was
used to determine the fraction of apoptotic cells. Annexin V binds to
phosphatidylserine, which is redistributed to the outer cell membrane
during the early stages of apoptosis. The fraction of apoptotic cells
in the CD4+CD28+ subset increased from
21.0 ± 5.9% to 39.7 ± 22.5% after 3 days of culture in
the absence of exogenous IL-2 (p = 0.03), while
no significant increase in the percentage of apoptotic cells was found
among CD4+CD28- T cells.
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. The percentage of apoptotic
CD28+ clones increased from a mean of 10.2 ± 4.9% on
day 0 to 38.4 ± 8.3% 3 days after IL-2 deprivation, while there
was only a small increase in the fraction of apoptotic cells in the
CD28- T cell clones (11.0 ± 3.9% and 16.6 ±
8.4% on days 0 and 3, respectively). This difference in IL-2
withdrawal-induced apoptosis between CD4+CD28+
and CD4+CD28- T cell clones was significant
(p < 0.001).
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, demonstrated that
CD4+CD28- T cells were less susceptible to
undergoing AICD. Both cell types had a similar cell surface expression
of CD95 (data not shown). The difference in AICD is unlikely to be
related to a difference in FasL production, because both cell types
were maintained in coculture at the time of the assay. In support of
this interpretation, similar results were seen with an anti-CD95
mAb, CH11 (data not shown).
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Expression of the bcl-2 family proteins and CD4+CD28- T cells
The bcl-2 and the bcl-xLgene products have been identified as molecules that can enhance
the intrinsic ability of lymphocytes to survive. In contrast,
bcl-xS and bax have been identified
as death genes and are known to accelerate apoptosis. Alterations in
the balance of survival vs death gene expression could provide an
explanation for the enhanced survival of
CD4+CD28- T cells. In particular, dysregulated
expression of bcl-2 or bcl-xL could explain the increased
tolerance of CD4+CD28- T cells toward growth
factor deprivation. To explore this possibility, the expression of
bcl-2 was compared in CD4+CD28+ and
CD4+CD28- T cells. Intracellular bcl-2 protein
was measured in short term T cell lines composed of CD28-
and CD28+ populations. The lines were maintained in medium
containing 10 U/ml rhIL-2. A representative experiment from a series of
seven T cell lines is shown in Figure 4.
T cells were stained with anti-CD4, anti-CD28, and
anti-bcl-2. Bcl-2 expression was higher in the
CD4+CD28- T cell subset. Compared with the
CD28+ T cells from the same T cell lines, the mean
fluorescence intensity of the bcl-2 staining in the CD28-
T cell lines was higher by an average of 20%
(p = 0.03).
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, the anti-bcl-2 m2Ab identified a
band at 26 kDa that was present in CD4+CD28-
and CD4+CD28+ T cell clones. However, the level
of expression of bcl-2 protein in the two subsets of clones was
different. Concentrations of bcl-2 protein were consistently higher in
the CD28-deficient T cell clones.
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shows a comparison of six
CD4+CD28+ and four
CD4+CD28- T cell clones. All T cell clones
were unstimulated and cultured in 1 U/ml rhIL-2. Cell lysates were
adjusted for protein content, and the blots were sequentially developed
with anti-bax, anti-bcl-x, and anti-bcl-2 reagents. Neither
the CD28+ nor the CD28- T cell clones
expressed significant amounts of bax (data not shown). The
anti-bcl-x Ab bound to a protein of about 32 kDa, consistent with
bcl-xL. No bcl-xS was detected. Results are
summarized in Table II and are given as arbitrary density units (ADU)
corrected for total protein staining. Bcl-xL expression
varied widely from 8 to 82 ADU. T cell clones with low
bcl-xL expression were found among CD28- as
well as among CD28+ T cell clones. In contrast, four of the
five T cell clones with high expression of bcl-2 protein lacked
CD28.
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CD4+CD28- T cells are characterized
by prolonged survival following IL-2 withdrawal. To analyze the
relationship between bcl-2 expression and stimulation with IL-2,
CD4+CD28+ and
CD4+CD28- T cell clones were cultured for 2
days with varying concentrations of IL-2, and the expression of bcl-2
protein was determined by immunoblotting. Results of two experiments
are shown in Figure 6. Exogenous IL-2
induced the expression of bcl-2 in the CD28+ T cell clones.
In contrast, bcl-2 expression in CD28- T cell clones was
only minimally affected, if at all, by changes in the IL-2
concentrations. At low concentrations of exogenous IL-2, bcl-2
expression was lower in the CD4+CD28+ T cell
clones. However, with increasing amounts of IL-2, bcl-2 protein
expression was enhanced to levels constitutively found in the
CD28-deficient clonotypes.
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A demonstrates a
representative experiment. As in Figure 6
, the addition of 10 U/ml
rhIL-2 on day 0 enhanced bcl-2 expression in the
CD28+CD4+ T cell clones to a much greater
extent than in the CD28- T cell clones. Culture in the
absence of IL-2 was associated with a progressive decline in bcl-2
expression of the CD28+ T cells. This decline preceded the
appearance of increased numbers of apoptotic cells. On days 0 and 2,
24.5 and 18.7%, respectively, of the CD4+CD28+
T cell clone JF1-48 were apoptotic as determined by 7-AAD staining for
subdiploid cells. This number increased to 35.7% on day 3. In
contrast, bcl-2 expression was maintained at a high level in the
CD4+CD28- T cell clone H1-67 despite IL-2
depletion. In parallel, the fraction of apoptotic cells remained
constant at 17.0, 12.3, and 16.2% over the 3-day period.
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, however, indicated some variability in the sensitivity of
CD4+CD28- T cell clones to IL-2 withdrawal.
For instance, the clones H1-10 and KD1.1 appeared to be more sensitive
to apoptosis compared with the average CD28- clone.
Western blotting analysis showed that upon IL-2 withdrawal, the levels
of bcl-2 expression in both clones were, in fact, significantly lower
than in the apoptosis-resistant clones K9 and KP5 (Fig. 7B). However, the levels of bcl-2 in both of these
CD28- clones were still higher than in the average
CD4+CD28+ T cell clone, T3-3, and were
equivalent to the clone PP5, which had the highest bcl-2 expression of
all of the CD28+ clones (Fig. 5). These data suggest that
bcl-2 expression is an important, but not the only, factor explaining
the longevity of CD4+CD28- T cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4+CD28- T cells are an unusual subset of T
lymphocytes. The majority of healthy individuals has <1% circulating
CD4+CD28- T cells (17). Given the important
costimulatory functions of the CD28 molecule, the constitutive
expression of CD28 on CD4 T cells is not unexpected (18). Triggering of
the CD28 pathway is required to complete the activation of a T cell
stimulated through the Ag receptor, and
CD4+CD28- cells should therefore not be able
to function properly. Also, CD28-mediated signaling is critical in
preventing anergy, probably through its effects on IL-2 production
(19). Finally, the activation-induced up-regulation of
bcl-xL, which is essential for T cell survival during and
shortly after activation, is dependent on the CD28 pathway (20, 21).
Given the lack of CD28 expression, CD4+CD28- T
cells should be anergic or at least have a short life span and be
highly susceptible to apoptosis. In addition, we have recently shown
that CD28-deficient CD4+ T cells cannot maintain a
sustained expression of the IL-2R 
-chain after stimulation (22).
Rapid down-regulation of IL-2R expression should also limit clonal
expansion of this subset after antigenic stimulation in vivo. However,
a vast majority of patients with RA and a small subset of normal
individuals have increased numbers of
CD4+CD28- T cells. In some patients, this can
be as high as 50% of the CD4 compartment (17).
Despite the impact of CD28-mediated signals on several aspects of T
cell stimulation, CD4+CD28- T cells are not
anergic in vivo and are, in fact, characterized by clonal expansion and
longevity of single T cell specificities (13, 14). The
CD4+CD28- compartment is dominated by a
limited number of T cell clones (23). These T cell clones may reach
frequencies of 0.1 to 1.0% of the total CD4 compartment, corresponding
to clonal sizes of 
109 T cells. They are long lived and
are detectable at constant frequencies over at least several years
(14). These in vivo features of CD4+CD28- T
cells suggest that these clonogenic cells must have developed
mechanisms that allow them to survive despite the absence of
CD28-mediated signaling. Dysregulation of survival proteins may protect
these cells from undergoing apoptosis and allow them to clonally expand
in vivo.
Members of the bcl-2 gene family have been shown to influence the ability of lymphocytes to undergo apoptosis (24). The bcl-2 gene was first identified at the breakpoint of the t(14:18) chromosomal translocation, which is characteristic of the majority of human follicular lymphomas (25). Transfection of bcl-2 is able to render hemopoietic cells relatively resistant to growth factor withdrawal-induced apoptosis, as shown in bcl-2 transgenic mice, in which the T cells have a prolonged life span (26, 27). Also, the rapid downsizing of activated T cells at the end of an immune response is attenuated. Additional evidence for a protective role of bcl-2 in apoptosis has come from experiments demonstrating that the life span of lymphocytes in mice with a targeted disruption of the bcl-2 gene is markedly reduced (28).
Lymphocytes express at least four bcl-2-related proteins (29). One of
these, bcl-x, exists in long (bcl-xL) and short
(bcl-xS) forms. Bcl-xL provides protection from
cell death, while bcl-xS expression is associated with
increased susceptibility to apoptosis (30). Our data would suggest that
bcl-2 and not bcl-xL is the major protein that protects
CD4+CD28- cells. The central role for bcl-2 in
regulating survival distinguishes CD4+CD28- T
cells from the subset of activated CD45RO+CD4+
T cells (31). While it has been shown that bcl-2 and
bcl-xL can substitute for each other in their
effect on prolonging T cell survival and protecting T cells from
undergoing apoptosis after IL-2 withdrawal, the two genes are
differentially regulated (9). Expression of bcl-2 is higher in resting
peripheral T cells. It can be up-regulated by the addition of exogenous
IL-2, but it is only slightly increased immediately after T cell
activation (9, 10, 32). Bcl-2 levels rapidly decrease in activated T
cells immediately after a viral infection (32). In contrast, the
expression of bcl-xL is closely associated with T cell
activation (20). Broome et al. (9) have shown that IL-2 starvation of
activated T cells results in a decrease of bcl-xL
expression preceding IL-2 withdrawal-induced apoptosis, while the
expression of bcl-2 is maintained. Mueller et al. (10) have proposed a
critical role of bcl-xL in the survival of Ag-primed T
cells in vivo. Memory T cells may remain resistant to apoptosis because
of intermittent contact with Ag, which results in up-regulation of
bcl-xL, secretion of IL-2, and finally IL-2-induced
up-regulation of bcl-2 (10, 32). In contrast to memory T cells, the
survival of CD4+CD28- T cells appears to be
relatively resistant to fluctuations in bcl-xL but is
mainly determined by the high expression of bcl-2. Notably, bcl-2
expression in CD4+CD28- T cells is sustained
even in the absence of exogenous IL-2 and IL-2R -chain expression.
Experiments shown in Figures 5 and 6 were done at time points when the
IL-2R -chain was undetectable in CD4+CD28-
T cell clones by flow cytometric analysis but was still expressed on
CD4+CD28+ T cells. These data would suggest
that CD4+CD28- T cells are best mimicked by T
cell lines with forced overexpression of bcl-2. High expression of
bcl-2 in T cells from bcl-2 transgenic animals or T cells
transfected with bcl-2 can override fluctuations in
bcl-xL expression (9).
It would be most interesting to understand the mechanism by which bcl-2
expression in CD4+CD28- T cells is induced and
maintained. Since bcl-2 is known to be regulated through the
action of the common 
-chain of the IL-2R (33), cytokines are obvious
candidates with potential roles of inducing high expression of bcl-2 in
CD4+CD28- T cells. In addition to IL-2, the
cytokines IL-4, IL-7, IL-13, and IL-15 could all be involved in
modulating bcl-2 expression. However, we do not have any evidence that
cytokines are facilitating the high and sustained expression of bcl-2
in these unusual lymphocytes. CD4+CD28- T
cells have been assigned to the Th1 category because they have the
ability to produce IL-2 and IFN- but secrete little IL-4. The
consideration that IL-2 provided in an autocrine fashion could be
regulating bcl-2 expression is not supported by our experimental data.
Bcl-2 expression and apoptotic susceptibility were tested in T cell
clones 7 days following stimulation, and thus, long after
activation-induced IL-2 production. More importantly, as shown in
Figure 2, CD4+CD28- T cells were characterized
by resistance to apoptosis as compared with CD28+ T cells,
even when both cell types were cocultured. These data are not
compatible with a cytokine-mediated mechanism regulating bcl-2
expression in CD4+CD28- T cells. Finally, we
attempted to study to what extent exogenous IL-2 could up-regulate
bcl-2 expression. Whereas the concentration of bcl-2 protein
synthesized in CD4+CD28+ T cells was directly
related to the amount of exogenous IL-2, this was not the case for
CD4+CD28- T cell clones. Rather, their
expression of the bcl-2 protein appeared to be independent of the
levels of IL-2 in the culture medium, consistent with their low
expression of the IL-2R -chain (22).
Alternatively, signaling through cell surface molecules could be involved in the induction of high and sustained expression of bcl-2. It is well established that cross-linking of CD40 on the surface of B cells by CD40 ligand (CD40L)-expressing T cells up-regulates bcl-2 and thus prevents apoptosis in Ag specific B cells (34). Spontaneously high levels of bcl-2 was a characteristic of CD4+CD28- T cell clones that were isolated from any other cells. Thus, both components of a putatively important receptor-ligand pair would have to be available on CD4+CD28- T cells. CD4+CD28- T cell clones do express low levels of CD40 as demonstrated by flow cytometry. However, CD40L mRNA cannot be detected by RT-PCR, suggesting that a CD40-CD40L interaction is not involved (data not shown).
In summary, expression of the survival protein bcl-2 is constitutively high in CD28-deficient CD4+ clonotypes. The well-established dependence of bcl-2 on the T cell growth factor, IL-2, is dissociated in these unusual lymphocytes. In that sense, CD4+CD28- T cells break the rule of cytokine-controlled T cell survival. It is likely that the dysregulation of bcl-2 expression and subsequent resistance to apoptosis in CD28-deficient T cells is related to their clonogenic potential in vivo and their longevity in patients with RA. These cells constitute a major fraction of the oligoclonal T cell populations in the synovial tissue (35). The resistance to undergoing apoptosis distinguishes them from tissue-infiltrating CD4+CD28+ T cells and from synovial fluid T cells, which have been shown to be exquisitely sensitive to Fas-mediated apoptosis (36, 37). Firestein et al. (38) have addressed the question of whether apoptosis occurs in the synovial tissue and have found a surprisingly low number of apoptotic T cells in situ. One possible explanation is that the CD4+CD28- T cell clones are an important functional subset in the synovial tissue, while the majority of synovial T cells are bystander T cells maintained through the action of cytokines such as IL-15 and IFN-ß (39, 40). In addition to their role in synovial inflammation, CD4+CD28- T cells are involved in the extra-articular spreading of RA. We have recently described the highest frequencies of CD4+CD28- T cells as found in patients with nodular RA or rheumatoid vasculitis (17). Defects in the regulation of their physiologic death should therefore have a major impact on the disease process in RA. Understanding the mechanisms up-regulating bcl-2 expression or rendering CD4+CD28- cells susceptible to apoptosis might provide novel therapeutic strategies in the management of RA.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J.J. Goronzy, 401 Guggenheim Building, Mayo Clinic, Rochester, MN 55905. E-mail address:
3 Abbreviations used in this paper: AICD, activation-induced cell death; rhIL-2, recombinant human IL-2; 7-AAD, 7-amino actinomycin D; ADU, arbitrary density units; RA, rheumatoid arthritis; PE, phycoerythrin; CD40L, CD40 ligand.
Received for publication September 15, 1997. Accepted for publication March 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. S. Longhi, M. J. Hussain, R. R. Mitry, S. K. Arora, G. Mieli-Vergani, D. Vergani, and Y. Ma Functional Study of CD4+CD25+ Regulatory T Cells in Health and Autoimmune Hepatitis J. Immunol., April 1, 2006; 176(7): 4484 - 4491. [Abstract] [Full Text] [PDF]
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 X. Zhang, A. Niessner, T. Nakajima, W. Ma-Krupa, S. L. Kopecky, R. L. Frye, J. J. Goronzy, and C. M. Weyand Interleukin 12 Induces T-Cell Recruitment Into the Atherosclerotic Plaque Circ. Res., March 3, 2006; 98(4): 524 - 531. [Abstract] [Full Text] [PDF]
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 D. Baeten, S. Louis, C. Braud, C. Braudeau, C. Ballet, F. Moizant, A. Pallier, M. Giral, S. Brouard, and J.-P. Soulillou Phenotypically and Functionally Distinct CD8+ Lymphocyte Populations in Long-Term Drug-Free Tolerance and Chronic Rejection in Human Kidney Graft Recipients J. Am. Soc. Nephrol., January 1, 2006; 17(1): 294 - 304. [Abstract] [Full Text] [PDF]
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 C. Duftner, R. Seiler, P. Klein-Weigel, H. Gobel, C. Goldberger, C. Ihling, G. Fraedrich, and M. Schirmer High Prevalence of Circulating CD4+CD28- T-Cells in Patients With Small Abdominal Aortic Aneurysms Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1347 - 1352. [Abstract] [Full Text] [PDF]
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V. Taneja, N. Taneja, M. Behrens, M. M. Griffiths, H. S. Luthra, and C. S. David Requirement for CD28 May Not Be Absolute for Collagen-Induced Arthritis: Study with HLA-DQ8 Transgenic Mice J. Immunol., January 15, 2005; 174(2): 1118 - 1125. [Abstract] [Full Text] [PDF]
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M. R. Snyder, T. Nakajima, P. J. Leibson, C. M. Weyand, and J. J. Goronzy Stimulatory Killer Ig-Like Receptors Modulate T Cell Activation through DAP12-Dependent and DAP12-Independent Mechanisms J. Immunol., September 15, 2004; 173(6): 3725 - 3731. [Abstract] [Full Text] [PDF]
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 A. Canton, E. M. Martinez-Caceres, C. Hernandez, C. Espejo, J. Garcia-Arumi, and R. Simo CD4-CD8 and CD28 Expression in T Cells Infiltrating the Vitreous Fluid in Patients With Proliferative Diabetic Retinopathy: A Flow Cytometric Analysis Arch Ophthalmol, May 1, 2004; 122(5): 743 - 749. [Abstract] [Full Text] [PDF]
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Y.-G. Hur, Y. Yun, and J. Won Rosmarinic Acid Induces p56lck-Dependent Apoptosis in Jurkat and Peripheral T Cells via Mitochondrial Pathway Independent from Fas/Fas Ligand Interaction J. Immunol., January 1, 2004; 172(1): 79 - 87. [Abstract] [Full Text] [PDF]
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A. N. Vallejo, H. Yang, P. A. Klimiuk, C. M. Weyand, and J. J. Goronzy Synoviocyte-Mediated Expansion of Inflammatory T Cells in Rheumatoid Synovitis Is Dependent on CD47-Thrombospondin 1 Interaction J. Immunol., August 15, 2003; 171(4): 1732 - 1740. [Abstract] [Full Text] [PDF]
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G. Strauss, I. Knape, I. Melzner, and K.-M. Debatin Constitutive Caspase Activation and Impaired Death-Inducing Signaling Complex Formation in CD95-Resistant, Long-Term Activated, Antigen-Specific T Cells J. Immunol., August 1, 2003; 171(3): 1172 - 1182. [Abstract] [Full Text] [PDF]
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 G. Rajagopalan, M. K. Smart, E. V. Marietta, and C. S. David Staphylococcal enterotoxin B-induced activation and concomitant resistance to cell death in CD28-deficient HLA-DQ8 transgenic mice Int. Immunol., July 1, 2002; 14(7): 801 - 812. [Abstract] [Full Text] [PDF]
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 J. B. Smith and M. K. Haynes Rheumatoid Arthritis--A Molecular Understanding Ann Intern Med, June 18, 2002; 136(12): 908 - 922. [Abstract] [Full Text] [PDF]
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M. Saurwein-Teissl, T. L. Lung, F. Marx, C. Gschosser, E. Asch, I. Blasko, W. Parson, G. Bock, D. Schonitzer, E. Trannoy, et al. Lack of Antibody Production Following Immunization in Old Age: Association with CD8+CD28- T Cell Clonal Expansions and an Imbalance in the Production of Th1 and Th2 Cytokines J. Immunol., June 1, 2002; 168(11): 5893 - 5899. [Abstract] [Full Text] [PDF]
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M. R. Snyder, L.-O. Muegge, C. Offord, W. M. O'Fallon, Z. Bajzer, C. M. Weyand, and J. J. Goronzy Formation of the Killer Ig-Like Receptor Repertoire on CD4+CD28null T Cells J. Immunol., April 15, 2002; 168(8): 3839 - 3846. [Abstract] [Full Text] [PDF]
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J.-H. Lai, L.-J. Ho, K.-C. Lu, D.-M. Chang, M.-F. Shaio, and S.-H. Han Western and Chinese Antirheumatic Drug-Induced T Cell Apoptotic DNA Damage Uses Different Caspase Cascades and Is Independent of Fas/Fas Ligand Interaction J. Immunol., June 1, 2001; 166(11): 6914 - 6924. [Abstract] [Full Text] [PDF]
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J. Zhang, T. Bardos, K. Mikecz, A. Finnegan, and T. T. Glant Impaired Fas Signaling Pathway Is Involved in Defective T Cell Apoptosis in Autoimmune Murine Arthritis J. Immunol., April 15, 2001; 166(8): 4981 - 4986. [Abstract] [Full Text] [PDF]
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A. N. Vallejo, M. Schirmer, C. M. Weyand, and J. J. Goronzy Clonality and Longevity of CD4+CD28null T Cells Are Associated with Defects in Apoptotic Pathways J. Immunol., December 1, 2000; 165(11): 6301 - 6307. [Abstract] [Full Text] [PDF]
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 G. Liuzzo, J. J. Goronzy, H. Yang, S. L. Kopecky, D. R. Holmes, R. L. Frye, and C. M. Weyand Monoclonal T-Cell Proliferation and Plaque Instability in Acute Coronary Syndromes Circulation, June 27, 2000; 101(25): 2883 - 2888. [Abstract] [Full Text] [PDF]
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A. N. Vallejo, L. O. Mugge, P. A. Klimiuk, C. M. Weyand, and J. J. Goronzy Central Role of Thrombospondin-1 in the Activation and Clonal Expansion of Inflammatory T Cells J. Immunol., March 15, 2000; 164(6): 2947 - 2954. [Abstract] [Full Text] [PDF]
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K. Ina, J. Itoh, K. Fukushima, K. Kusugami, T. Yamaguchi, K. Kyokane, A. Imada, D. G. Binion, A. Musso, G. A. West, et al. Resistance of Crohn's Disease T Cells to Multiple Apoptotic Signals Is Associated with a Bcl-2/Bax Mucosal Imbalance J. Immunol., July 15, 1999; 163(2): 1081 - 1090. [Abstract] [Full Text] [PDF]
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A. N. Vallejo, J. C. Brandes, C. M. Weyand, and J. J. Goronzy Modulation of CD28 Expression: Distinct Regulatory Pathways During Activation and Replicative Senescence J. Immunol., June 1, 1999; 162(11): 6572 - 6579. [Abstract] [Full Text] [PDF]
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 A. N. Vallejo, C. M. Weyand, and J. J. Goronzy Functional Disruption of the CD28 Gene Transcriptional Initiator in Senescent T Cells J. Biol. Chem., January 19, 2001; 276(4): 2565 - 2570. [Abstract] [Full Text] [PDF]
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