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International Immunology Advance Access originally published online on April 11, 2006
International Immunology 2006 18(5):637-644; doi:10.1093/intimm/dxh375
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Increased TGF-ß, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation

Qibin Leng, Zvi Bentwich1 and Gadi Borkow2

R. Ben-Ari Institute of Clinical Immunology and AIDS Center, Kaplan Medical Center, Hebrew University Hadassah Medical School, Rehovot 76100, Israel
1 Present address: Department of Virology, Center for Infectious Diseases and AIDS, Ben Gurion University, Beer Sheba 84105, Israel
2 Present address: Cupron Inc. Hameyasdim 44, Gibton 76910, Israel

Correspondence to: G. Borkow; E-mail: gadi{at}cupron.com


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In this study we investigated the mechanisms mediating T-cell hyporesponsiveness in chronically immune-activated individuals. We analyzed in healthy and persistently helminth-infected individuals the relationship between immune activation and general T-cell hyporesponsiveness, Th3/regulatory T-cell expression, transforming growth factor-ß (TGF-ß) secretion, CTL-associated antigen 4 (CTLA-4) levels, Casitas B-cell lymphoma-b (Cbl-b) (a negative regulator of T-cell activation) levels and phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinase (ERK)-1 and -2. We found a very significant increase in plasma levels of TGF-ß and intracellular pools of CTLA-4 and Cbl-b in association with immune activation, which correlates with decreased T-cell responses to anti-CD3 stimulation. We demonstrate that the impaired activity of ERK of peripheral T cells in highly immune-activated individuals is associated with increased levels of CTLA-4 and Cbl-b. Interestingly, in some, but not in all, of these immune-activated individuals, induction of Cbl-b intracellular pools occurs by TGF-ß or CTLA-4 stimulation. We suggest that the higher levels of CTLA-4 and TGF-ß, both involved in the induction of Cbl-b, point at potential mechanisms underlying general and antigen-specific immune hyporesponsiveness in chronically infected individuals.

Keywords: anergy, helminthic infections, hyporesponsiveness, signal transduction


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
It is estimated that >1 billion of the population of the world is chronically infected with the major soil-transmitted helminths (1, 2). The morbidity associated with these infections is estimated to affect 447 million people with annual mortality of 135 000 (2). The constant and lifelong infestations with such parasites result in a chronic ‘immune activation’ and unbalanced immune profile (3, 4). For example, peripheral T cells obtained from human individuals, who are chronically infected with the ‘tissue-dwelling helminths’, often fail to respond to parasite antigens in the form of proliferation or Th1-related cytokine production (5, 6), as well as to recall antigen including purified tuberculin protein derivative (PPD) (7). Recent studies support the notion that not the Th1 to Th2 shift, but Th3/regulatory T (Treg) cells, that produce the anti-inflammatory cytokines transforming growth factor-ß (TGF-ß) and IL-10 and constitutively express CTL-associated antigen 4 (CTLA-4) and CD25 (8), mediate the antigen-specific hyporesponsiveness characteristic to chronic helminthic infections (2, 9). For instance, parasite antigen-specific cellular hyporesponsiveness in patients chronically infected with filarial helminths was associated with a lack of IL-4 production and significantly lower production of IL-5 by their PBMCs compared with the same cells obtained from individuals with putative immunity. In contrast, the antigen-specific hyporesponsiveness could be reversed by the addition of anti-IL-10 and anti-TGF-ß antibodies (10–12). In addition, increased expression of TGF-ß produced by parasite antigen-specific peripheral T cells has been reported in baboons repeatedly challenged with Schistosoma mansoni as well as in Wuchereria bancrofti-infected human individuals (13–15).

Moreover, our previous studies on the helminth-infected Ethiopian immigrants to Israel (ETH) suggest that chronic immune activation caused by chronic helminthic infection results in general hyporesponsiveness and anergy (7, 16). In these studies, the examined individuals were stratified into three categories of ‘activation’, according to the expression of HLA-DR (a marker of cell activation), within the CD3+ cell population and the expression of CD28 (a co-stimulatory receptor that dramatically enhances T cell signaling) within the CD8+ cell population. Individuals who had very high and very low expression levels of HLA-DR and CD28, respectively, were considered as highly immune-activated individuals. We found that general immune hyporesponsiveness in highly immune-activated individuals was manifested by (i) defective or lacking of early transmembrane signaling (phosphorylation and/or dephosphorylation of tyrosine kinases), suggesting malfunction of both kinases and phosphatases; (ii) lack of degradation of phosphorylated IkB{alpha}, indicating lower levels or malfunction of a cellular protease; (iii) lack or attenuated phosphorylation of downstream kinases, such as extracellular signal-regulated kinase (ERK)-1 and -2, and p38 kinase; (iv) increased expression of CTLA-4, a down-regulatory protein; (v) decreased ß-chemokine secretion of CD8+ cells following stimulation and (vi) reduced capacity to proliferate following recall antigen stimulation. These attenuated responses were not a reflection of slower kinetics, but represented significantly diminished responses. Hyporesponsiveness did not occur in only one signal transduction pathway, but in all those that we examined, indicating that this phenomenon is a more general cell deficiency. In addition, it has been shown that helminthic infections diminish the immune responses post-vaccination with polio (17, 18), rotavirus (19), diphtheria toxoid (20), tetanus toxoid (21), oral cholera (22) and tuberculosis (23) vaccines, both in humans and in mice (24). Such hyporesponsiveness and anergy could be tied to the effects of Treg/suppressor cells present in these situations. It is now >6 years that the attention of the immunological community has been increasingly focused on the role, characterization and function of Treg cells. Studies in both humans and animals demonstrated that such cells are involved in the control of the immune response to infections, neoplasia and organ and bone marrow transplantation (25–30). By now, it has become clear that Treg cells belong to a population of CD4 T cells that co-express CD25 (the IL-2 receptor alpha-chain), constitutively express CTLA-4 and often secrete IL-10, TGF-ß, IFN{alpha} and IL-5.

This study has therefore examined the relationship between general T-cell hyporesponsiveness, Th3/Treg-cell expression, TGF-ß secretion and signal transduction, with immune activation, in ETH and healthy Israeli (IS) individuals. The finding that general immune hyporesponsiveness is strongly associated with increased TGF-ß secretion, higher levels of the negative intracellular adaptor protein Casitas B-cell lymphoma-b (Cbl-b) and attenuated signal transduction, all correlated with chronic immune activation, has prompted this report.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell proliferation and culture
PBMCs were isolated from heparinized blood by standard cell centrifugation over Histopague (Sigma, Rehovot, Israel), washed and re-suspended in RPMI1640 complete medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, penicillin, streptomycin and nystatin (Biological Industries Co., Beit-Haemek, Israel). T-cell proliferation to anti-CD3 stimulation was determined by culturing PBMCs (105 cells per 200 µl) in 96-well plates (Corning, NY, USA) at 37°C under 5% CO2 for 3 days in the presence of 0.5 µg ml–1 anti-CD3 mAbs (R&D Systems, Minneapolis, MN, USA). At the last day of culture, the cells were pulsed with 0.5 µCi [3H]thymidine (Amersham Pharmacia Biotech, Buckinghamshire, UK) and 16 h later the cells were harvested on Whatman 934-AH glass microfiber filters (Whatman, Kent, UK) and the radioactivity was measured by a Packard Tri-Carb 300 ß-counter. Cells cultured in the presence of medium only served as control. All experiments were done in triplicates and the results are expressed as stimulation index (SI, mean counts per minute obtained with anti-CD3 stimulation divided by the mean counts per minute obtained for the control).

TGF-ß expression
TGF-ß1 plasma levels were measured by ELISA basically as described by Doetze et al. (12) as follows: Immuno-Plate F96 Maxisorp (Nunc, Wiesbaden, Germany) plates were coated with 50 µl of 0.1 M NaHCO2/Na2CO3 buffer (pH 9.6) containing 1 µg ml–1 of goat anti-human TGF-ß1 mAb (R&D Systems, clone 9016.2). After overnight incubation at 4°C, the plates were blocked for 1 h with 200 µl of 1% BSA. The plates were then washed five times with PBS/0.05% Tween and incubated overnight at 4°C with 100 µl of ‘activated’ plasma samples. Activation of the plasma samples was achieved by incubating 100 µl plasma for 10 min with 100 µl of 1 N HCl and then for 10 min with 100 µl of 1.2 N NaOH/0.5 M HEPES. The plates were then washed five times with PBS/Tween/0.1% BSA. A PBS/Tween/0.1% BSA solution containing biotinylated polyclonal chicken anti-TGF-ß antibodies (R&D Systems, catalog no. BAF240) at a concentration of 0.1 µg ml–1 was then added. After 1 h, the plates were washed five times with PBS/Tween/0.1% BSA and incubated for 1 h with streptavidin–peroxidase complex (1:10 000; Boehringer Ingelheim GmbH, Germany). The wells were then washed five times with PBS/Tween/0.1% BSA and incubated for ~30 min with 100 µl per well of tetramethylbenzidine (Roth, Karlsruhe, Germany; dissolved 6 mg ml–1 in dimethyl sulfoxide) as substrate. The reactions were then stopped by adding 25 µl per well of 4 N H2SO4 and the absorbency at 450 nm of each well was determined by a spectrophotometer. As standard we used rTGF-ß1 (catalog no. 240-B-002, R&D Systems).

Cell stimulation and western blotting
For activation of ERK, PBMCs (2 x 106 cells) in 1 ml of RPMI medium were stimulated with 1 µM Ca-ionophore A23187 [GenBank] (Sigma) and 10 ng ml–1 phorbol 12-myristate 13-acetate (PMA) for various time intervals. In order to determine the relationship between TGF-ß levels and intracellular adaptor protein Cbl expression, PBMCs, re-suspended in serum-free Biotarget-1 medium (Biological Industries Co.) supplemented with 2 mM L-glutamine, penicillin, streptomycin and nystatin, were cultured overnight at 37°C under 5% CO2 with various concentrations of recombinant human TGF-ß1. To study the regulation of Cbl expression by co-stimulation via CTLA-4, PBMCs re-suspended in Biotarget-1 medium were cultured in 24-well plates immobilized with or without 0.5 µg ml–1 anti-CD3, 5 µg ml–1 anti-CD28 and/or anti-CTLA-4 antibodies for 72 h at 37°C under 5% CO2. Following culture, the above stimulated cells were washed with cold PBS, and re-suspended in 50 µl of cold 10 mM HEPES buffer (pH 7.6) containing 10 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.1 mM EGTA, 2 mM Na3VO4, 2 mM phenylmethylsulfonylfluoride, 5 µg ml–1 leupeptin, 0.3 U ml–1 aprotinin, 5 µg ml–1 pepstatin and 20 mM ß-glycerophosphate. After 15 min of incubation on ice, NP-40 was added to the cells to a final concentration of 1.0% and the cells were incubated on ice for an additional 5 min. The mixture was then centrifuged at 21 000 x g at 4°C and the supernatant was kept at –20°C until tested. The lysates were subjected to western blotting with antibodies to Cbl-b, c-Cbl (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated p42/44 ERK (1:20 000, a generous gift from Roni Seger, Weizmann Institute, Israel), Bcl-2 (1:10 000) and CTLA-4 (1:200; PharMingen, San Diego, CA, USA).

Lymphocyte phenotype analysis
One-, two- or three-color immunophenotyping of whole blood or PBMCs was performed by FACS (FACScan®, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) by using one, two or three of the following mAbs conjugated with either FITC, PE or peridin chlorophyll protein (PerCP): CD3, CD4, CD8, HLA-DR, CD25 (Dako, Glostrup, Denmark) and CD28 (Becton Dickinson). Intracellular labeling of CTLA-4 was carried out basically as described previously (31). Briefly, after staining whole blood (100 µl) with mAb against surface markers, the RBCs were lysed with lysis buffer (Becton Dickinson). The remaining cells were washed with PBS containing 0.01% azide and incubated with 1% PFA for 25 min at room temperature. The cells were then washed with staining buffer (PBS containing 0.1% saponin and 2.5% FCS) and re-suspended in staining buffer containing anti-CTLA-4–PE-labeled mAb (1:10 final dilution). After 45 min of incubation at room temperature, the cells were washed with staining buffer and re-suspended in PBS containing 0.01% azide. Finally, the cells were analyzed by FACS. An example of surface and intracellular staining of PBMC is shown in Fig. 1. Cells incubated with FITC-, PE- or PerCP-conjugated mouse IgG1/IgG2a (Dako) served as isotype controls. A minimum of 10 000 cells per sample were analyzed.


Figure 1
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  		Fig. 1. Example of FACS analysis of CD4, HLA-DR and CTLA-4 expression in lymphocytes. Whole blood was labeled with anti-CD4–PerCP- and anti-HLA-DR–FITC-conjugated mAb. After the RBCs were removed, the remaining cells were incubated with 1% PFA for 25 min. The cells were then washed and re-suspended in PBS containing 0.1% saponin, 2.5% FCS and anti-CTLA-4–PE-labeled mAb (1:10 final dilution). Following 45 min of incubation at room temperature, the cells were washed, re-suspended in PBS containing 0.01% azide and finally analyzed by FACS. (a) The cells were assessed by their forward- and side-scatter parameters. The cells gated in R1 (mostly lymphocytes) were further analyzed for (b) CD4 and HLA-DR expression, (c) CTLA-4 and HLA-DR expression of the CD4+ cells gated in (b) (the upper and lower right quadrates) and (d) CD4 and CTLA-4 expression of the R1-gated cells. Among the CD4+ cells, 7.38% were CTLA-4+ cells (the upper right quadrate in panel d). The percentage of cells per each quadrate is shown.

 
Statistical analysis
Student's t-tests, Mann–Whitney rank sum test and Pearson product moment correlation analysis were performed by using SigmaStat software (SPSS®, Chicago, IL, USA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Immune profile of the studied subjects
Blood samples were obtained from 42 IS and 69 ETH HIV-1 seronegative adult individuals, following their informed consent. The ETH individuals were recruited within 1 year of their immigration to Israel from Ethiopia. The male to female ratio of the studied individuals was ~1 and they were 18–65 years of age. While none of the IS had helminthic parasites, ~75% of the ETH had helminths at the time of examination (Table 1), regardless of their sex or age. The most prevalent parasites were: Necator americanus (hookworm), Ascaris lumbricoides, S. mansoni and Trichuris trichiura. Based on the immune profile of the IS control individuals, we divided the examined ETH into three categories of activation. This was done according to the proportion of HLA-DR+ cells within the CD3+ cell population and the proportion of CD28+ cells within the CD8+ cell population, similarly to what we have done previously (7). Non-activated (NA) individuals were defined as those having ‘normal’ percentage of HLA-DR+/CD3+ and CD28+/CD8+ cells (similarly to the IS group, i.e. <7% HLA-DR+/CD3+ cells and >40% CD28+/CD8+). Highly activated (HA) individuals were defined as those having >7% HLA-DR+/CD3+ cells and <40% CD28+/CD8+, while partially activated (PA) individuals were defined as those having either HLA-DR+/CD3+ or CD28+/CD8+ subsets above or below the normal range, accordingly. All IS were NA, while among the ETH, 18, 37 and 14 were NA, PA and HA, respectively. The immune profile of the four studied groups is summarized in Table 1. No correlation between the type of parasites and immune activation was observed.


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  		Table 1. Immune profiles of the studied groups

 
The NA-ETH individuals studied had a similar immune profile as the IS group, with the exception of higher percentages of CD4+ expressing CD25+ and CTLA-4+ (P < 0.01). In contrast, the PA-ETH and HA-ETH groups, which by definition had higher percentages of HLA-DR+CD3+ cells and lower percentages of CD28+CD8+ cells than the IS individuals, had also significantly decreased CD4 and increased CD8 levels, significantly lower CD4/CD8 ratios and higher percentages of CD25+CD4+ and CTLA-4+CD4+ cells, than the IS group (Table 1). An example of CTLA-4+ expression in CD4+ cells in a highly immune-activated individual (18% HLA-DR+CD3+ cells) is shown in Fig. 1. While the levels of CD25+CD4+ cells were not correlated with the levels of HLA-DR+CD3+ cells, there was a positive correlation between the percentages of CTLA-4+CD4+ cells and HLA-DR+CD3+ cells (r = 0.56, P < 0.001).

Proliferation of T cells after stimulation with anti-CD3 mAb
We have recently shown that PBMCs obtained from immune-activated ETH proliferated poorly to recall antigens, such as PPD (7). In order to determine if this impairment is a result of a deficiency in antigen-specific T-cell response, or whether it is a result of a general state of immunosuppression, in the present study we compared the capacity of T cells obtained from NA-, PA- and HA-ETH and IS individuals to respond to TCR ligation by stimulation with 0.5 µg ml–1 anti-CD3 mAbs. As shown in Fig. 2(a), there was a clear trend of decrease in the proliferation of PBMCs with the ‘activation status’ of the examined group. While the SIs of the IS and NA-ETH groups were not significantly different (43.9 ± 35.2 and 35.4 ± 33, respectively), those of the PA-ETH and HA-ETH were significantly lower: 23.1 ± 19.5 and 17.6 ± 14, respectively (P < 0.05). The lower proliferation following anti-CD3 stimulation of PBMCs, obtained from HA-ETH, could not be augmented, even if the concentration of the anti-CD3 mAbs was increased by 10-fold (Fig. 2b). Overall, among the ETH, the response to CD3 stimulation was negatively correlated with the percentage of HLA-DR+CD3+ cells (r = –0.36, P < 0.05), but not with the percentage of CD28+CD8+, CD25+CD4+ or CTLA-4+CD4+ cells.


Figure 2
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  		Fig. 2. Decrease proliferation to anti-CD3 stimulation in association with immune activation. (a) PBMCs were incubated for 3 days at 37°C with medium alone or with 0.5 µg ml–1 anti-CD3 antibodies. The SI for each group and the statistical differences (t-test) between the IS group and the ETH groups (P) are shown. The boxes represent the middle 50% of the data values. The horizontal line across the box marks the median value. The error bars show the 10th and 90th percentiles of the population. Individual data points falling beyond these boundaries are shown as dots. (b) PBMCs taken from a non-activated Israeli individual (closed circles), with 1%, 5.4% and 47.7% HLA-DR+CD3+, CTLA-4+CD4+ and CD28+CD8+ cell subsets, respectively, and PBMCs taken from a PA-ETH individual (open circles), with 6.2%, 15.3% and 28.5% HLA-DR+CD3+, CTLA-4+CD4+ and CD28+CD8+ cell subsets, respectively, were stimulated with various concentrations of anti-CD3 antibodies for 3 days. The data represent the mean ± SD of triplicates.

 
Plasma TGF-ß expression
As shown in Fig. 3(a), the plasma levels of TGF-ß of the IS and NA-ETH groups were not statistically different, while those found in the plasma of the PA-ETH and HA-ETH were significantly higher than those of the IS group and NA-ETH group. Of note, the plasma TGF-ß expression of 41% of IS individuals was under the detectable level of the assay, in contrast to only one (an NA-ETH) out of 69 ETH (1.45%) examined. The expression of plasma TGF-ß (in those individuals with detectable plasma TGF-ß levels) was positively correlated with the percentage of HLA-DR+CD3+ cells (Fig. 3b).


Figure 3
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  		Fig. 3. Increase levels of TGF-ß in association with immune activation. (a) The TGF-ß1 plasma levels of each group and the statistical differences between the IS or NA-ETH groups and the PA- and HA-ETH groups are shown. The Mann–Whitney test was used to compare between the IS and the other groups, as the TGF-ß levels in the IS group were not normally distributed. The Student's t-test was used to compare between NA-ETH and PA- and HA-ETH groups. (b) The correlation (r) between HLA-DR expression on CD3+ cells and the plasma levels of TGF-ß obtained from all ETH and IS studied who had detectable plasma TGF-ß levels, determined by using SigmaPlot software (SPSS®), and their statistical significance (P) obtained by using the Pearson product moment test, are shown.

 
Activation of ERK is correlated to CTLA-4 and Cbl expression
CTLA-4 and the intracellular adaptor protein Cbl-b play pivotal roles in down-regulating signaling events during T-cell activation (32, 33). It was shown that CTLA-4 engagement selectively shuts off activation of downstream TCR/CD28 signaling events, such as phosphorylation of ERK-1/2 (33, 34), and phosphorylation of Cbl-b was associated with impaired activation of ERK-1/2 signaling (35, 36), together leading to decreased IL-2 production and maintenance of T-cell anergy.

We have found increased expression of CTLA-4 (7) as well as signal induction impairments, including phosphorylation of ERK-1/2 (24), in immune-activated ETH individuals. In the present study, we examined whether the impaired ERK activation in chronically immune-activated individuals is related to increased Cbl and CTLA-4 levels. As shown in some representative examples in Fig. 4, which are consistent with our previous findings (7), phosphorylation of ERK-1 or ERK-2 of PBMCs, following PMA and Ca++-ionophore stimulation, was significantly lower in cells obtained from HA-ETH individuals than PA-ETH and NA-ETH. The decreased phosphorylation of ERK-1 or ERK-2 upon stimulation was clearly associated with increased expression of Cbl-b and CTLA-4 expression in seven out of 10 cell samples examined.


Figure 4
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  		Fig. 4. Decreased phosphorylation of ERK-1 and ERK-2, increased Cbl-b and CTLA-4 expression, associated with immune activation. PBMC extracts obtained from two HA-, one PA- and two NA-ETH individuals were not stimulated (–) or stimulated for 6 min (+) with PMA and Ca++-ionophore. Lysates of these cells were resolved on SDS-PAGE and immunoblotted with anti-phosphorylated p42/44 ERK, anti-Cbl-b, anti-CTLA-4 and anti-Bcl-2.

 
Cbl-b expression regulated by TGF-ß and CTLA-4
TGF-ß is a potent inhibitor of T-cell-mediated immune responses, both in vitro and in vivo (37). Since we found increased levels of TGF-ß and Cbl-b in PA-ETH and HA-ETH (Figs 3a and 4 and data not shown), we investigated the relationship between these two immunosuppressor factors. We cultured PBMCs, obtained from healthy IS individuals, in serum-free medium containing various concentrations of recombinant human TGF-ß. After overnight incubation at 37°C, cell lysates were prepared and subjected to western blotting with antibodies against Cbl proteins. We found that addition of TGF-ß significantly increased Cbl-b expression, without affecting c-Cbl expression, in three out of four experiments (Fig. 5a). In one of these experiments, the levels of c-Cbl were significantly up-regulated, while those of Cbl-b were only slightly increased.


Figure 5
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  		Fig. 5. Induction of Cbl-b by TGF-ß. PBMCs taken from an Israeli individual were (a) cultured overnight at 37°C with various concentrations of TGF-ß1 and (b) stimulated with immobilized antibodies against CD3 and CTLA-4 for 72 h at 37°C. Lysates of these cells were resolved on SDS-PAGE and immunoblotted with anti-Cbl-b, anti-c-Cbl and anti-Bcl-2.

 
To examine the role of CTLA-4 in regulating Cbl expression, PBMCs obtained from healthy IS individuals were stimulated with immobilized antibodies against CD3 and CTLA-4 for 72 h at 37°C and then cell extracts were prepared and subjected to immunoblotting with anti-Cbl-b antibodies. As shown in Fig. 5(b), in some cases (three out of five), CTLA-4 engagement up-regulated Cbl-b expression.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
It has been suggested that chronic immune activation results in general hyporesponsiveness and anergy of the host lymphocytes, and thereby contributes to the incapacity of these individuals to cope with infections (4, 7, 10, 12, 24, 38–41) and to generate cellular protective immunity after vaccination (23, 42). In order to further elucidate how overall immunosuppression occurs in association with immune activation, in the present study, we analyzed the relationship between immune activation and general T-cell hyporesponsiveness, Th3/Treg-cell expression, TGF-ß secretion and signal transduction in ETH infested with helminthic parasites (43–45; Table 1) within 1 year of their immigration. We divided the ETH individuals into different immune activation categories, not only according to the expression of HLA-DR, a universal indicator of T-cell activation, but as we have done previously (7), also according to the levels of expression of CD28, a co-stimulatory receptor that dramatically enhances T cell signaling. Although this self-made definition of immune activation may by itself influence the outcome of the results, this definition is originated on the clear inverse correlation that we found between HLA-DR expression and CD28 expression (7, 46). Based on IS healthy individuals who served as the control reference population, we thus classified the ETH into (i) NA-ETH, which did not differ statistically from the IS group in the percentages of HLA-DR+CD3+, CD28+CD8+, CD4+ and CD8+ sub-populations of cells; (ii) HA-ETH, which had a very disturbed and immune-activated profile, in comparison with the IS and NA-ETH groups and (iii) PA-ETH, which had an immune activation profile that could be classified as significantly different from the IS and NA-ETH groups, but not as extremely altered as the HA-ETH group.

Interestingly, all ETH individuals had, irrespectively to their immune activation status, similar higher percentages (~10%) of CD25+CD4+ cells, i.e. Th3/Treg cells (8), than the IS control individuals. Two reasons may account for this: (i) there are genetic constitutive differences between Israeli and Ethiopian populations and (ii) persistent, sometimes lifelong, exposure to helminthic parasites, results in increased populations of Th3/Treg cells. We have already found elevated expression of CCR5 and CXCR4 on CD4+ cells of ETH, whether immune activated or not, than in an IS group (47), supporting the first possibility. There are, however, reports showing increased expression of CD25+CD4+ cells in association with helminthic infections (48–50), supporting the second possibility. In any case, the higher levels of the Th3/Treg cells by themselves cannot account for the decreased proliferation seen in cells obtained from PA-ETH and HA-ETH.

Similar to CD25+CD4+ cells, all ETH groups had higher CTLA-4+CD4+ cells than the IS control group, which may be explained partially by the increase of CD25+CD4+, as Th3/Treg cells are the only lymphocyte sub-populations in humans that express CTLA-4 constitutively (8). However, as opposed to CD25+CD4+ cells, the levels of CTLA-4+CD4+ cells were correlated with the levels of activated cells, and the HA-ETH group had significantly higher CTLA-4+CD4+ cells than the other two ETH groups, indicating that immune activation contributes significantly to the increase of this immunosuppressor factor. We have already found increased CTLA-4 expression in association with immune activation in helminthic infections (7, 24), HIV-1 infection (31) and aging (51). However, again, the increased levels of CTLA-4+CD4+ cells by themselves could not account alone for the decreased proliferation to anti-CD3 stimulation associated with immune activation, since the levels of these cells were not significantly higher in the PA-ETH than in the NA-ETH.

In contrast to CD25 and CTLA-4 expression, both PA-ETH and HA-ETH groups had significantly higher levels of TGF-ß than both the NA-ETH and IS groups, and the increase of TGF-ß was correlated to the percentage of HLA-DR+CD3+ cells. Thus, the general cellular hyporesponsiveness, i.e. lower proliferation to CD3 stimulation, could be associated with the increased levels of the inhibitory cytokine TGF-ß. This is in agreement with reports involving TGF-ß in antigen-specific cellular hyporesponsiveness in chronic human or primate helminthic infections (12, 13, 52–54). Th3/Treg cells, as well as other T-cell populations, and even non-lymphoid cells, such as epithelium in the process of healing, have been shown to secrete TGF-ß (8, 37). Thus, it is clear that the higher levels of TGF-ß, seen in the PA-ETH and HA-ETH individuals, could not result solely from higher levels of CD25+CD4+ cells. Another possible source of TGF-ß may be macrophages, since it has been shown that apoptotic cells trigger TGF-ß production by macrophages (55), and we have previously found increased lymphocyte apoptosis in heavily helminth-infected ETH (46).

TGF-ß plays an essential role in T-cell regulation, including anti-proliferative effects on T cells, acquisition of effector functions by naive T cells, inhibition of Th differentiation and inhibition of pro-inflammatory cytokine production by macrophages, B cells and T cells, both in vitro and in vivo [reviewed in (37)]. One possible way, through which TGF-ß down-regulates T-cell responses, is via up-regulation of the intracellular adaptor protein Cbl-b. Cbl-b plays pivotal roles in down-regulating signaling events during T-cell activation (32, 33), and as we have shown here, stimulation of PBMCs with TGF-ß increases in most cases the intracellular pools of Cbl-b. This, together with the increased levels of CTLA-4, that raise the threshold for effective T-cell activation (56), may explain the reduced proliferation, following anti-CD3 stimulation, and reduced phosphorylation of ERK-1/2, following PMA and Ca++-ionophore stimulation, of PBMCs obtained from PA-ETH and HA-ETH. In addition, we have also found that in several cases stimulation of PBMCs with immobilized anti-CTLA-4 antibodies enhances Cbl-b expression. Enhancement of Cbl-b expression may result indirectly from the effect of TGF-ß, since CTLA-4 engagement leads to TGF-ß expression in T cells (57). Thus, the higher levels of CTLA-4, involved in the induction of TGF-ß, together with the higher levels of TGF-ß, involved in the induction of Cbl-b, all associated with immune activation, may significantly contribute to the general and antigen-specific hyporesponsiveness, characteristic to chronic helminthic infections, also observed in this study and in our previous studies of cellular immune responses in the ETH population (7, 24).


   Acknowledgements
 
The work was supported by the Horowitz Foundation, granted to the Kaplan AIDS Center.


   Abbreviations
 
Cbl-b, Casitas B-cell lymphoma-b
CTLA-4, CTL-associated antigen 4
ERK, extracellular signal-regulated kinase
ETH, Ethiopian immigrants to Israel
HA, highly activated
IS, Israeli
NA, non-activated
PA, partially activated
PMA, phorbol 12-myristate 13-acetate
PPD, purified tuberculin protein derivative
SI, stimulation index
TGF-ß, transforming growth factor-ß
Treg, regulatory T

   Notes
 
Transmitting editor: S. Romagnani

Received 7 September 2003, accepted 25 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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