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IMMUNOBIOLOGY
From the Department of Immunology and the
Department of Pathophysiology, Medical University of Gdansk;
and the Voivodial Outpatient Geriatric Clinic of Gdansk,
Poland.
Aging is associated with modifications of T-cell phenotype and
function, leading to impaired activation in response to both new and
recall antigens. It is not known if T-cell activation results in
elimination of a number of the CD4 molecules from the cell surface, as
is the case with CD3/T-cell receptor complexes, or how aging
influences the process. The T cells of young and elderly donors with
reduced expression of CD4 were examined to see whether these cells
exhibit other phenotypic features suggesting their active
state. It was found that T lymphocytes expressing CD4 can be
divided into 2 semidiscrete subpopulations: the major (CD4+) population, in which the level of expression of CD4
is constant and high, and a minor population (CD4lo), in
which the expression of CD4 can be up to an order of magnitude lower
than on the CD4+ cells. The proportion of CD4lo
cells is age dependent and highly variable in the apparently healthy
human population, with the expression of CD4 ranging from around 10%
of all peripheral blood lymphocytes in the young to more than 30% in
the elderly. Lowered expression of CD4 is correlated with a reduced
expression of CD3, as well as with a decreased amount of CD28 and
CD95Fas. Activation of CD4lo cells is suggested by their
expression of CD25 and increased amounts of HLA-DR. Phenotypic
characteristics of the CD4lo T-cell subpopulation suggest
that it might be formed by (perhaps chronically) activated, temporarily
apoptosis-resistant cells, possibly accumulating in the elderly.
(Blood. 2001;98:1100-1107) The CD4 antigen, a major coreceptor necessary for
initiation of the T-lymphocyte activation sequence, is found on a major subpopulation of helper T cells. Cross-linking of the CD4 by the HLA
class II molecule leads to activation of associated p56lck
(lck) tyrosine kinase. Lck-dependent phosphorylation of the CD3 CD4 expression is usually believed to be constant on the surface of
human peripheral blood T lymphocytes; about 90 000 to 100 000
molecules are estimated to be expressed on a single T cell.7-9 However, it is known that many surface receptors
undergo internalization upon binding by appropriate ligands and that
the receptor-ligand complexes are eventually dissociated and/or
digested by cellular proteolytic mechanisms. Also, some observations
speak of the diminution, or even abolition, of the expression of CD4 on
the surface of human peripheral blood T cells stimulated with the
phorbol ester phorbolmyristate acetate, owing to
down-regulation of both transcription and translation.10
Until recently, neither the exact number of ligated receptors on the
surface of an activated T cell nor their fate was a subject of closer
study, mostly owing to lack of appropriate methodology. An appropriate
method, involving quantitative flow-cytometric determination of the
number of receptors on resting and stimulated T cells, has recently
been described and has allowed for thorough studies of
activation-related changes of a number of CD3-TCR
complexes.11-13 It was shown that T-cell stimulation leads
inadvertently to the reduction (detectable by flow cytometry) of a
number of the CD3-TCR molecules, predominantly owing to their
internalization.11 Depending on the presence or absence of
costimulatory signals, the number of CD3-TCR complexes required for
activation was established to be at very specific thresholds:
about 1500 and 8000 per cell, respectively.12
It is thus conceivable that other T-cell surface receptors involved in
activation follow the same fate. This should particularly be
the case for the T-lymphocyte molecules that, along with the CD3-TCR
complex, directly participate in the earliest stages of antigenic
stimulation, such as the CD4 and CD8 antigens. It was recently shown
that contact between murine CD8+ T cells and
antigen-presenting cells (APCs) loaded with a peptide recognized by the
CD3-TCR complex leads to some major histocompatibility (MHC) class I
molecules' being picked up from the APCs and ultimately internalized
by the lymphocytes concomitantly with the internalization of the
CD3-TCR complex itself.14 The authors of that study did not address the question of the fate of the CD8 molecule in the process; however, knowing the role of CD8 in class I recognition and
binding, one can speculate that CD8 could also be incorporated in the internalized complex. If this is a general phenomenon, a
subpopulation of activated CD4+ cells, in which some of the
CD4 is "spent" or exhausted simultaneously with the CD3-TCR, should
be detectable even in normal people exposed to environmental antigens
at the time of study. So far, unlike what occurs in the CD3-TCR
complex, possible changes of the actual level of CD4 expression on
single T cells have not been a subject of much study. Even the most
recent papers, some of which appeared after publication of the original
observations of modulation of the CD3-TCR complex expression, indicate
that the coefficient of variation (CV) for CD4 fluorescence
intensity was very low.7,8 Therefore, the authors of
these papers do not consider the possibility of modulation of the
expression of CD4 on the T-cell surface.
Aging is accompanied by progressive changes in the function of the
immune system, ultimately leading to the decreased response to both
primary and recall antigens. At least some gerontologists believe that
the CD4+ T cell, acting as a central coordinator of the
immune response, is among the most affected elements of the aging
immune system.15,16 Not much is known about the fate of
the functionally important receptors, such as CD3 and CD4, in T cells
in the elderly. However, at least in the case of the CD28 molecule, its
elimination from senescent CD4+ cells is quite well
documented.17-19 On the other hand, there are
suggestions of a chronic activation of at least a subpopulation of
CD4+ cells in the elderly.18-20
In this work, we tried to answer the question of whether such a
modulation of the CD4 expression can take place under physiological conditions (eg, when the T cells of a healthy individual are responding to environmental antigens), by looking for a subpopulation of peripheral blood T lymphocytes showing lowered expression of this antigen.
Subjects
Cell preparation and antibody staining
To make sure that the apparent variation in CD4 expression was not due to a specific antibody, we compared different clones of anti-CD4 antibodies supplied by different manufacturers and conjugated to either fluorescein isothicyanate (FITC), phycoerythrin, (PE) or Cychrome (Pharmingen, San Diego, CA). The antibodies were not repurified prior to use. The following anti-CD4 monoclonal antibodies were used in the study: (1) PE-anti-CD4 (Leu-3A) (Becton Dickinson) clone SK3, immunoglobulin (Ig)-G1; (2) FITC-anti-CD4 (Dako, Glostrup, Denmark) clone MT310, IgG1; (3) PE-anti-CD4 (Sigma-Aldrich Chemie) clone Q4120, IgG1; (4) PE-anti-CD4 (Pharmingen) clone RPA-T4, IgG1; and (5) Cychrome-anti-CD4 (Pharmingen) clone RPA-T4, IgG1. If not mentioned otherwise, the results presented in the paper were obtained with the use of the PE-conjugated Leu-3A antibody. The proportion of CD4lo cells in an individual's T lymphocytes did not depend significantly on the type and manufacturer of anti-CD4 antibody conjugate used, although the Leu-3A antibody usually gave slightly lower results than the other products. Specifically, the mean percentages of CD4lo lymphocytes in the CD4+ population were 23.0% ± 5.4% (n = 13), 18.8% ± 13.5% (n = 31), and 25.7% ± 10.5% (n = 11) for FITC-anti-CD4, PE-anti-CD4 (Becton Dickinson), and Cychrome-anti-CD4, respectively. Other antibodies used in this project were the following: (1) FITC-anti-CD3 (Dako); (2) FITC-anti-CD14 (Pharmingen); (3) FITC-anti-CD28 (Pharmingen); (4) PE-anti-CD95 (Pharmingen); (5) PE-anti-CD152 (Pharmingen); (6) PE-anti-CD45RA (Dako); (7) PE-anti-HLA-DR (Becton Dickinson); (8) PE-anti-CD25 (Pharmingen); (9) Cychrome-anti-CD16 (Pharmingen). Except for CD14 and CD28, cell samples were always stained with anti-CD4 and anti-CD3 plus an additional third-color antibody, in order to clearly distinguish the CD3+CD4+ T lymphocytes and characterize the CD4lo cells within the T-cell pool only. The usual concentration of each antibody was 0.5 µg/105 cells. To examine the concentration dependency of the observed effect, PE-anti-CD4 antibody concentrations of 1.0 and 2.0 µg/105 cells were used in 3 experiments each involving young, middle-aged, and elderly donors, along with the usual concentration. Changes of the antibody concentration from 0.5 to 2 µg/105 cells did not result in significant changes of the CD4lo percentage (not shown). Irrelevant antibodies of the same class, conjugated to the corresponding fluorochrome and manufactured by the same company as the relevant antibodies, were used as isotype controls in each experiment. Flow cytometry Cytometric analysis of all samples was performed by means of an Epics XL (Coulter, Hialeah, FL) at the Department of Histology and Immunology, Medical University of Gdansk, or a FACSCalibur (Becton Dickinson, Sunnyvale, CA) at the Hematology Clinic, Medical University of Gdansk, or a FACSort (Becton Dickinson, Sunnyvale, CA) at the Voivodial Hospital for Infectious Diseases in Gdansk. To check for the possibility of instrument bias, data from most of the samples were acquired on at least 2 instruments. Wherever the CD4lo population was seen, it could be discerned by analysis on any of the 3 instruments, and the differences in its proportion in the total CD4 population were negligible. Data from 20 000 mononuclear cells were acquired from each sample and stored as list modes. Off-line analysis was done by means of dedicated software (WinMDI versions 2.7 through 2.9, Joseph Trotter, Scripps, CA) and consisted of estimation of the percentages of distinct subpopulations and of the mean fluorescence intensities (MFIs). The CD4lo subpopulation usually formed a "shoulder" attached to the main CD4+ population and not a separate peak. Thus, a set of rules was adopted in order to consistently define the limits of this cell group: First, a lower limit of the total CD3+CD4+ population was set by cutting off the CD4 cells as those with the fluorescence not
different from that of appropriate isotype control (steps 1 and 2 in
Figure 1). Then, the modal value for the
entire population of lymphocytes bearing CD4 antigen was read (step 3),
and the position of marker cutting off 0.2% of cells with the highest
CD4 fluorescence was found (step 4). This marker position was then
mirrored on the other side of the modal value and served as a boundary
between the CD4+ and CD4lo cells (steps 5 and
6). We subtracted 0.2% from the proportion of CD4lo cells
in the CD4+ population to account for the cells belonging
to the lower end of the CD4+ group. In one
example, the proportion of CD4lo cells in the
total CD4+ population is 7.8% (Figure 1).
Quantification of the numbers of CD4 molecules Quantification of the numbers of CD4 molecules on the surface of cells belonging to different populations of CD4-bearing T lymphocytes was performed by means of the Cellquest Quantitative Analysis data acquisition module (Becton Dickinson, San Jose, CA) with the acquisition of signal from Quantibrite calibration beads prior to the acquisition of data from the cells. The anti-CD4 antibody used for this purpose was always PE conjugated (Leu-3A), and the cells were simultaneously stained with FITC-anti-CD3. Data analysis was performed by means of the QuantiCALC program (Verity Software, Topsham, ME), which transforms the values of fluorescence recorded from individual cells into absolute numbers of detected molecules, using a calibration curve generated by simultaneous analysis of the beads.Cell culture To investigate possible modifications of the level of CD4 expression on T cells owing to in vitro stimulation, peripheral blood lymphocytes of 4 healthy individuals were stimulated with plastic-immobilized anti-CD3 (OKT-3) (Ortho Biotech, Raritan, NJ) in 24-well plastic culture plates at 10 ng/well. Immediately before use, the antibody-containing wells were washed with 3 changes of complete culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics [penicillin/streptomycin mix, Sigma Chemical, St Louis, MO]). Cells were suspended in the same medium at 106/mL, and 1 mL cell suspension was placed in each well. Plates were then incubated for 48 hours in a mixture of 5% CO2 and 95% air at 37°C, collected, and stained with anti-CD4 and anti-CD3 antibodies. In a separate experiment, cells were cultured for 8 days before collecting, antibody staining, and analysis; in this experiment, cells were also stained with the anti-CD28 antibody.Statistics The results are shown as representative 2-dimensional flow cytometry histograms (dot plots and density plots) or as averages in bar graph form. Statistical information includes arithmetic means, SDs, SEMs, and 95% confidence intervals (CIs), as indicated in Table 1 and in the figure legends. The statistical significance of the differences was determined by means of paired or unpaired Student t test as necessary, after confirmation of the normality of data distribution. Correlation between the proportion of CD4lo cells in the total CD4+ lymphocyte population and the age of individuals was done by means of Pearson R coefficient.
The proportion of human T lymphocytes with lower-than-average expression of the CD4 increases in the elderly In addition to the typical, major CD4+ population with small CV, found within the lymphocyte window on the basis of low forward scatter (FS) and side scatter (SS) parameters (Figure 2A, inset R1), a smaller population, which we designated CD4lo, could be detected among the T lymphocytes of every person examined. This population presented a CD4-staining intensity intermediate between the isotype control or CD4 cells and the normal CD4+ cells. The CV
of CD4 fluorescence in this population was higher than in the
CD4+ cells. CD4lo lymphocytes could be
distinguished from the monocytes by their lower FS and SS (Figure 2A)
and their positive CD3 (Figure 2B-C) and negative CD14 (Figure 2D)
expression. The monocytes present in the mixture consistently showed a
CD3CD14+ phenotype in addition to high SS and
low CD4 expression detectable on their surfaces (Figure 2). For the
subjects as a group, the mean percentage of CD4lo cells was
18.0% ± 3.38% (mean ± 95% CI, n = 55) of the total CD4+ population, with a range of 1.8% to 46.1%. The
corresponding proportion of the CD4lo lymphocytes among the
total peripheral blood lymphocytes (PBLs) was 9.2% ± 1.95% (range,
1.4% to 27.9%). Thus, both in total PBLs and in the CD4+
population, the proportion of CD4lo cells greatly varied
among individuals. However, the proportion was quite stable if one
individual was tested more than once. Thus, cells were obtained from 4 different donors aged 34 to 65 years at least twice at monthly
intervals; for one of these donors, the analysis was also performed
after another month. The results from each individual varied about 6%
to 15% (1.9% vs 2.2% vs 2.0%; 3.1% vs 3.3%; 4.5% vs 4.2%; and
16.3% vs 18.9%). We did not observe significant correlation between
the number or proportion of cells with the CD4lo phenotype
and those with the CD4+ phenotype (r2 = 0.16,
P > .1). Considering possible causes of this variability, we analyzed the proportion of CD4lo lymphocytes in relation
to the sex and age of donors. We found no difference in the percentages
of the CD4lo cells in women (26.5% ± 5.9%, n = 34)
and men (20.84 ± 4.62%, n = 21, P = .15) in the
group under study.
When the absolute numbers of CD4 receptors were compared on
CD4lo and CD4+ T cells by means of Quantibrite
beads, the numbers on the major CD4+ populations were about
95 000 CD4 molecules per lymphocyte, whereas CD4lo cells
expressed on average fewer than 50 000 CD4 molecules each, and this
difference was highly statistically significant (Figure 3).
Compared with the young, the elderly group significantly showed an
almost 2-fold higher proportion of CD4lo T cells.
Interestingly, the middle-aged group did not show such a tendency
(Table 1). Next, we performed a
regression analysis on the whole set of data, including proportions of
CD4lo T cells among CD4 lymphocytes and among PBLs on one
side and the age of each donor on the other. We found a significant
positive correlation between age and the proportion of
CD4lo cells among CD4+ cells (r = 0.42,
P < .005) and among PBLs (r = 0.49,
P < .05). We then tested for a possible relation between
an increased proportion of CD4lo lymphocytes and a
decreased proportion of the CD4+ T cells in the elderly. In
the group under study, no such correlation could be demonstrated
between the age of donor and the proportion of the CD4+
cells in the PBL (r = In principle, anti-CD4 antibody molecules could, like any other immunoglobulin, be picked up and bound nonspecifically by the non-CD4 cells having enough Fc receptors on their surface, especially some of the CD16+ natural killer (NK) cells. To exclude the possibility that CD4lo cells belong to FcR+ cells (say, of NK or CD8+ phenotype), we stained the cells with anti-CD4, anti-CD3, and either anti-CD8 or anti-CD16. We found that the CD4loCD3+ cells bore neither CD8 nor CD16 on their surface (not shown). Also, CD4lo lymphocytes did not bind more isotype control antibodies than the rest of the CD4 population. We concluded from these experiments that the binding of anti-CD4 to the CD4lo cells is specific and does not depend on the binding of antibody via its Fc portion. Modulation of expression of activation-related antigens indicates the activated status of CD4lo T cells Our hypothesis of an activation-related association between a decrease of CD3 and CD4 expression is based on the data relating T-cell activation to a loss of some surface CD3.3,11-13 To this end, we compared the levels of CD3 expression on T cells showing different levels of expression or a complete lack of CD4, as well as on non-T cells from the PBMC pool. A representative flow cytometry data plot and a graph of MFIs of FITC-anti-CD3 on the CD3+CD4 (presumably CD8+
lymphocytes), CD3+CD4lo, and
CD3+CD4+ cells as well as the MFI of
CD3 PBLs (presumably a mixture of B and NK cells) are
shown in Figure 4A. Although the level of
CD3 expression on CD4 (CD8+) lymphocytes and
CD4+ T cells did not differ significantly, the MFI of
FITC-anti-CD3 bound to CD4lo cells was, on average, 1.2 to
2.1 times lower than that seen on the CD4+ lymphocytes, yet
very significantly higher than that of
CD3CD4 cells (Figure 4A).
The majority (on average, more than 52.6% ± 8.9%) of the CD4lo cells display high levels of expression of CD25 antigen (Figure 4B). Contrarily, its expression can be seen only on a small proportion (around 18.4% ± 3.4%) of other CD4+ cells and is significantly lower (average MFI, 29.0 vs 495.2 for CD4lo). The expression of HLA-DR, believed to be present only on the activated and not on the resting CD4+ T cells, could be seen both in the CD4+ and CD4lo populations. However, similarly to the pattern of CD25 expression, all of the CD4lo lymphocytes expressed HLA-DR and the level of expression of this antigen was higher in this population than in the CD4+ population (Figure 4C). Monocytes (known to express high amounts of HLA-DR) were eliminated from the analysis by simultaneous staining with the anti-CD3 antibody and analyzing only the CD3+ population in addition to the usual gating based on forward and side scatter. It is believed that the expression of CD45RO delineates T cells that
are activated. The proportion of naive
(CD45RO We then compared the expression of 3 other surface antigens known
either to directly participate in antigenic stimulation of T cells
(CD28, CD152) or to be involved in termination of this response (CD95).
We found that unlike the CD4+ population, in which on
average 83.6% cells expressed CD28, only 59.5% ± 4.9% of
CD4lo cells were CD28+ (Figure
5A), and the difference in the proportion
of CD4+CD28+ and
CD4loCD28+ cells was strongly significant. The
amount of CD28 expressed on single CD4lo T cells was
minimally lower than that seen on CD4+ lymphocytes (Figure
5A). Reciprocally, the percentage of CD4lo lymphocytes
bearing CD152 was higher than that observed in the population showing
high expression of CD4 (Figure 5B). The actual difference, although
statistically significant, was not big. There was no difference between
CD4+ and CD4lo cells in the levels of
expression of CD152. Finally, the proportion of CD4lo cells
expressing the CD95 receptor was significantly higher than was observed
for the CD4+ population. However, the level of expression
of CD95 on CD4lo cells was lower than on CD4+
lymphocytes, with the MFI values at 43.7 ± 4.2 compared with 76.1 ± 8.4 (Figure 5C).
Stimulation in vitro leads to increased proportion of CD4lo T cells When human PBLs were stimulated in vitro with an optimal concentration of immobilized anti-CD3 antibody, the proportion of CD4lo cells among CD4+ T lymphocytes increased after only 48 hours of stimulation in 3 of 4 individuals tested. Interestingly, this increase was proportional to the age of the donor, with the highest percentage of CD4lo cells exhibited by the oldest person (60 years old) (Figure 6A). In a separate experiment, when stimulation with immobilized anti-CD3 was prolonged to 8 days, the proportion of CD4lo cells increased from 1.2% in unstimulated control to 38.9% in the anti-CD3-treated sample (Figure 6C-D). We then asked if stimulation led to any change in the number of CD4 molecules on the surface of predefined CD4lo and CD4+ populations with the use of quantitative calibration with Quantibrite beads. No clear tendency could be seen in either population. In fact, in 2 young donors (donors a and b), there was an increase in the number of CD4 molecules on stimulated CD4+ cells with a simultaneous (but proportionally smaller) decrease in the CD4lo population. On the other hand, the number of CD4 molecules on the surface of both CD4lo and CD4+ cells of a middle-aged (donor c) and an elderly individual (donor d) did not change upon stimulation (Figure 6B).
We have found a population of human peripheral blood lymphocytes
bearing significantly lower amounts of CD4 than the major CD4+ population. Usually, these CD4lo cells
seen in the cytometric histogram form a "shoulder" or a "tail"
between the CD4+ and the CD4 To make sure we were not chasing after an artifact, we applied extensive variations to the methodology, including the use of 4 different clones of anti-CD4 antibody from different manufacturers, different conjugates of these clones, changes in the antibody concentration, and the use of 3 different methods of sample preparation and staining. We did not detect significant differences in the proportion of CD4lo T cells with any of these variations in methodology. We did not attempt to specifically block the Fc receptors on the analyzed cells. However, CD4lo T cells did not differ from the rest of the CD4+ population (or from other lymphocytes) in the level of nonspecific staining with the PE-conjugated isotype control containing irrelevant antibodies (not shown). Thus, we can assume that the level of FcR expression on CD4lo cells does not differ from that on other CD4+ cells and has no influence on their lower staining with anti-CD4. To understand the genesis of CD4lo cells, we hypothesized that CD4 could either become down-regulated on these cells owing to activation or be in a different stage (perhaps the terminal stage) of differentiation. The proportion of CD4lo cells in the peripheral blood varied greatly among apparently healthy individuals, from typically only a few percentage points to (in rare cases) more than 40% of all T cells expressing CD4. Why, then, have CD4lo cells not been described before? First, except for the works of Lanzavecchia's group,12,13 the possibility of even partial modulation of the level of expression of CD4 on T cells is not considered, even in the most recent papers on the subject.7,8,22 One of these recent papers actually assumes absolute constancy of the numbers of CD4 molecules on T cells and uses this constant as a calibration gauge for estimation of the numbers of CD38 molecules.8 Second, in healthy young subjects (being studied for the normal values of the CD4 expression), the percentages of CD4lo cells are usually low (not exceeding 10% to 15% of all CD4+ cells) and are seen in cytometric histograms mostly in the form of an inconspicuous shoulder at the main CD4+ population. They stand out more clearly in the analysis of material from elderly people, who are usually excluded from non-aging-oriented studies. The papers cited above do not characterize their subjects other than as "a small sample of healthy donors"7 or as "healthy HIV-1 uninfected men"8 and do not give the number of individuals studied or their ages. Neither of the just-mentioned papers contains plots of actual flow-cytometric data; thus, it is hard to predict if the CD4lo cells could be seen in their specimens. In our study, the percentage of CD4lo lymphocytes was positively correlated with the donor's age. This suggests that the physiological level of CD4 expression on the T lymphocytes may be dependent on age and possibly the functional status of the cell. There are currently only a very few papers that approach the problem of age-dependent changes of CD4 receptor density, and all of them look at the changes in the total pool of CD4-bearing T cells.23-25 Also, different authors report either an increase,23 a decrease,24 or no change25 in the CD4 density. In one instance, a decrease in average expression of CD4 is described in late-passage (the in vitro approximation of aging) human T-cell clones.26 Few lines of reasoning indicate that activation plays a role in the genesis of the CD4lo T cells. The first one comes from analogy to the recently shown activation-related decrease of the CD3-TCR complex on the T-cell surface.3,11-13 In our study, the expression of CD3 on the CD4lo lymphocytes was significantly lower than on the other CD4+ T cells. This observation may indicate that the lower level of expression of CD4 is characteristic for activated T cells, which express lower amounts of CD3. The hypothesis of activated status of the CD4lo cells is also supported by our observation of higher frequency of activation-related antigens CD25 and HLA-DR on their surface. To further validate our hypothesis, we examined the CD4 expression on T cells stimulated with immobilized anti-CD3. We were able to show an increased proportion of CD4lo T cells after just 48 hours; this tendency was strengthened after 8 days of culture, when the proportion reached almost 40%. Interestingly, the numbers of CD4 molecules did not seem to change significantly within any of the 2 CD4-bearing populations upon stimulation. This may indicate that the process of activation-dependent down-regulation of the CD4 molecule is rapid and involves a quasi-standard number of molecules that have to be eliminated from the surface during effective stimulation, similar to what happens to the CD3-TCR complex.3,11-13 The frequency of CD4lo cells is similar in the memory-activated CD45RO+ and naive CD45RA+ population; in fact, they tend to express more CD45RA than the rest of the CD4+ lymphocytes. This may reflect the reversion of the CD45RO+ phenotype to the CD45RA+ after the acute phase of stimulation is over27 and suggests that the CD4lo population in fact contains T cells at later stages of activation. In the CD4lo population, the percentage of CD28+ cells was significantly lower than in the main CD4+ pool, and the level of expression of the CD28 molecule was marginally lower than on the CD4+ lymphocytes. This in turn may suggest that the process of activation leading to a lowered expression of CD4 (and CD3) on CD4lo cells eliminates (or, at least, reduces) the CD28 antigen. In fact, significant down-regulation of both CD28 messenger RNA and surface expression in activated T cells upon ligation by the B7 molecule has been shown to last up to 48 hours following stimulation.28 As we show here, in anti-CD3-stimulated PBMCs, a population of CD3+CD4lo cells with visibly lower (albeit not null) expression of CD28 could be clearly discerned (Figure 6D). This observation seems to be in line with the earlier reports. Alternatively, CD4lo, CD3lo, and
CD28lo cells could form a stable subpopulation of T cells
originally endowed with this phenotype and having as-yet-unknown
functional properties. Whatever its genesis, this population Thus, CD4lo phenotype can be due either to down-regulation at the transcription or translation level, to reduction from the resting state by activation-related shedding and/or internalization, or to intrinsically lower expression of the molecule on a separate population of T cells. The last explanation seems the least possible though, in light of our observations showing stimulation-dependent increase in the proportion of CD4lo cells in vitro. The discovery of membrane "rafts" as specific sites in the T-cell membranes important for initiation of signal transduction may offer some clues to understanding the apparent concerted down-regulation of surface molecules. It was recently suggested that T-cell activation is accompanied by dimerization (or even oligomerization) of some (MHC-II-ligated) CD4 molecules.31 This observation may have something in common with the existence of rafts containing CD3-TCR complex, CD4, CD28, and LAT.32 It is conceivable that the ultimate fate of all transmembrane molecules of such a raft after ligation of at least some of them and propagation of the activating signal into the cytoplasm is common and leads to their simultaneous disappearance from the cell surface. In any case, CD4lo cells would form an activated
subpopulation of the CD4 T cells that were not (or not yet) able to
rebuild their CD4 surface complement. It is a current paradigm that
activation promptly leads to an increased expression of CD95 on the
T-cell surface. Our finding of a uniformly intermediate level of CD95 on the CD4lo lymphocytes may thus be attributed to their
prolonged (rather than recent) activated state. Decreased CD95 may
confer some level of protection from the too-early onset of apoptosis,
before the activated lymphocyte completes its task, or it may be an
indication of decreased ability of the immune system to eliminate
"used" T cells at the end of an immune response, especially in the
elderly. The proportion of CD4lo cells found in any single
individual would then tell about the donor's prolonged contact with
T-cell-activating antigens, even if the person did not manifest any
pathological symptoms at the time of investigation. If our theory is
correct, it should be expected that situations leading to chronic
activation and eventually functional exhaustion of the CD4 cells should
lead to the accumulation of CD4lo lymphocytes. Our recent
observations suggest that this may be the case for rheumatoid
arthritis. T cells of rheumatoid arthritis patients are believed to
undergo chronic activation and precocious proliferative
senescence.33 Chronic activation involves multiple rounds
of cell division, leading, inter alia, to telomere
shortening. This was recently exemplified in the paper showing that
clonally expanded CD8+CD28
Submitted August 17, 2000; accepted April 13, 2001.
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Reprints: Jacek M. Witkowski, Department of Pathophysiology, Medical University of Gdansk Debinki 7 80-211 Gdansk, Poland; e-mail: jawit{at}amedec.amg.gda.pl.
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  B. H. Lemster, J. J. Michel, D. T. Montag, J. J. Paat, S. A. Studenski, A. B. Newman, and A. N. Vallejo Induction of CD56 and TCR-Independent Activation of T Cells with Aging J. Immunol., February 1, 2008; 180(3): 1979 - 1990. [Abstract] [Full Text] [PDF]
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 K. J. Claycombe, D. Wu, M. Nikolova-Karakashian, H. Palmer, A. Beharka, K. E. Paulson, and S. N. Meydani Ceramide Mediates Age-associated Increase in Macrophage Cyclooxygenase-2 Expression J. Biol. Chem., August 16, 2002; 277(34): 30784 - 30791. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
chain of the CD3/T-cell receptor (TCR) complex
(simultaneously triggered by the antigenic epitope presented by the
class II molecule) is thus at the beginning of the cascade of
events leading to lymphocyte activation.
0.16, P > .2), indicating no
decrease in the proportion of CD4+ cells in our elderly group.
) and CD4+
lymphocytes (
; *P = .022) and between CD4lo
and CD3
; ***P = .00004). (B-C)
Lowered expression of CD4 is associated with high expression of
activation antigens CD25 and HLA-DR (B and C insets show representative
density plots). Bar graph in panel B shows statistically significant
difference between the expression of CD4 on cells from the R2 region of
the inset expressing CD25 (
) and the CD25
in our
opinion