High frequency of circulating {gamma}{delta} T cells with dominance of the V{delta}1 subset in a healthy population

Lars Hviid1, Bartholomew D. Akanmori2, Severine Loizon3, Jorgen A. L. Kurtzhals1,2, Christina H. Ricke1, Annick Lim4, Kwadwo A. Koram2, Francis K. Nkrumah2, Odile Mercereau-Puijalon3 and Charlotte Behr3

1 Centre for Medical Parasitology at Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet) and Institute for Medical Microbiology and Immunology, University of Copenhagen, 2000 Copenhagen, Denmark
2 Immunology and Epidemiology Units, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
3 Unité d'Immunologie Moléculaire des Parasites, CNRS URA 1960 and
4 Unité de Biologie Moleculaire du Gene, Institut Pastéur, 75724 Paris, France

Correspondence to: L. Hviid, Department of Infectious Diseases M7641, Rigshospitalet, Tagensvej 20, 2200 Copenhagen N, Denmark


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TCR {gamma}{delta}+ cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V{delta}1+ cells constitute a minority of these cells. In contrast to TCR {alpha}ß+ cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequencies of V{delta}1+ cells are found in several diseases, their function remains obscure. Here we show that the frequency of peripheral blood {gamma}{delta} T cells in healthy West Africans is about twice that of Caucasians, mainly due to a 5-fold increase in V{delta}1+ cells, which is consequently the dominant subset. No age dependency of V{delta}1 frequencies was identified and the V{delta}1+ cells in the African donors did not show preferential V{gamma} chain usage. Analysis of the CDR3 region size did not reveal any particular skewing of the V{delta}1 repertoire, although oligoclonality was more pronounced in adults compared to children. The proportions of CD8+, CD38+ and CD45RAhiCD45RO cells within the V{delta}1+ subset were higher in the African than in the European donors, without obvious differences in expression of activation markers. No significant correlations between levels of V{delta}1+ cells and environmental antigens or immunological parameters were identified. Taken together, the evidence argues against a CDR3-restricted, antigen-driven expansion of V{delta}1+ cells in the African study population. Our study shows that high frequencies of TCR {gamma}{delta}+ cells with dominance of the V{delta}1+ subset can occur at the population level in healthy people, raising questions about the physiological role of V{delta}1+ T cells in the function and regulation of the immune system.

Keywords: age, CDR3, Epstein-Barr virus, {gamma}{delta}, T cells, malaria, T cell repertoire, V{delta}1


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The vast majority of T cells present in the peripheral circulation of healthy, adult Caucasians express {alpha}ß TCR, with T cells expressing the complementary {gamma}{delta} TCR usually accounting for <5% of circulating T cells (1,2). The peripheral {gamma}{delta} T cells of healthy Caucasians are composed of two major largely non-overlapping populations. The larger of these consists of cells expressing a receptor heterodimer composed of the gene product of V{gamma}9 usually combined to the product of the V{delta}2 gene (3,4). The other, smaller, subset that encompasses essentially all remaining peripheral {gamma}{delta} T cells uses the product of the V{delta}1 gene paired to {gamma} chains other than V{gamma}9 (5). TCR {gamma}{delta}+ cells are more prevalent at other anatomical sites such as the intestinal mucosa, where V{delta}1+ cells are the predominant {gamma}{delta} T cell population (6).

Expansion of V{gamma}9+V{delta}2+ T cells is seen in many infectious diseases, whereas expansion of V{delta}1+ cells does not seem characteristic of infectious diseases in general (7,8). However, V{delta}1+ expansion has been repeatedly observed in HIV and infectious mononucleosis (9,10). In contrast, V{delta}1+ cell expansion is a feature of many auto-immune disorders (1113). The ligands recognized by V{delta}1+ cells are much less characterized than those of V{gamma}9+V{delta}2+ cells, but include the MHC class I-like molecules MICA and MICB, CD1, and antigens from Borrelia burgdorferi, Onchocerca volvulus and Epstein–Barr virus (EBV)-transformed B cells (1418).

Although {gamma}{delta} T cells have been studied for the last 10 years in different models and species, their functional role as well as the selection of their antigenic repertoire selection are not fully understood. Besides an important role in the early immune response to certain bacterial and parasitic infections as evidenced by data from studies of {gamma}{delta} T cell-deficient mice, recent data suggest a more general immuno-regulatory role of these cells (19,20). In contrast to TCR {alpha}ß+ cells, which are selected in the thymus, the {gamma}{delta} T cell repertoire is mainly generated and selected extrathymically (21,22). At birth, Caucasians have 5-fold fewer TCR {gamma}{delta}+ cells than adults and V{delta}1+ cells dominate among {gamma}{delta} T cells early in life. The increase in {gamma}{delta} T cells during the first decade of life is largely limited to the V{gamma}9+V{delta}2+ population, causing a shift towards the V{gamma}9 dominance seen in Caucasian adults (21). This expansion appears to be the result of exposure to environmental antigens recognized by V{gamma}9+V{delta}2+ cells, such as microbial phospho-antigens or alkylamines (23,24).

During studies of the T cell response to Plasmodium falciparum malaria in individuals living in coastal Ghana, we have previously observed high frequencies of {gamma}{delta} T cells both in malaria convalescents and in healthy individuals (25). In the present report we have investigated in detail the {gamma}{delta} T cell subset in healthy individuals from this area, with special emphasis on children in the age group most susceptible to malaria. While the prevalence of HIV is relatively low in Ghana (26,27) and O. volvulus is completely absent from our study area, Ghana has a high prevalence of other microorganisms known to stimulate {gamma}{delta} T cells, such as Mycobacteria, malaria parasites and EBV. Consequently, we have searched for correlations between the high numbers of TCR {gamma}{delta}+ cells observed in both the children and adults of our study population and a number of possible causative agents. To our knowledge, this is the first systematic study of its kind in a non-Caucasian population.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Donors
Healthy adult donors were recruited among laboratory staff at either Rigshospitalet or Noguchi Memorial Institute for Medical Research. The children were recruited from the village of Dodowa, situated outside Accra in an area of seasonal, hyperendemic transmission of P. falciparum malaria parasites (28). All children were clinically healthy at the time of the blood collection. Informed consent was obtained from all donors or their parents or guardians. The Ministry of Health, Ghana approved the study.

Sample processing
Venous blood samples (1–5 ml) were collected in heparinized test tubes and were processed for analysis within 3 h or cryopreserved for later analysis by PCR. Plasma was isolated following centrifugation and stored frozen at –20°C until analysis of antibody levels. Sample volume was then reconstituted by PBS (CellWash; Becton Dickinson, Mountain View, CA), followed by antibody labeling of 100 µl aliquots for flow cytometry (FCM, see below).

FCM
Two- and three-color FCM was applied, using mAb to the determinants listed below, either as unconjugated or biotinylated mAb, or as direct FITC, phycoerythrin or Cy5 conjugates: CD3 (UCHT1; Dako, Glostrup, Denmark), CD4 (MT310; DAKO), CD8 (DK25; Dako), CD38 (HIT2; PharMingen, San Diego, CA), CD45RA (HI100; PharMingen), CD45RO (UCHL1; Dako), CD69 (L78; Becton Dickinson), HLA-DR (L243; Becton Dickinson), V{gamma}I (aka V{gamma}2,3,4, 23D12; ImmunoTech, Marseilles, France), V{gamma}4 (4A11; BIOAdvance, Emerainville, France), V{gamma}5 [56.3 (29); a kind gift from Dr Dieter Kabelitz], V{gamma}8 (R4.5; ImmunoTech), V{gamma}9 (BIOAdvance), V{delta}1 (TS-1; BIOAdvance) and TCR {gamma}{delta} (11F2; Becton Dickinson).

Unconjugated mAb were detected using rabbit anti-mouse F(ab)2 fragments conjugated to either FITC or Cy5 (Dako) as second-step reagent. Biotinylated mAb were detected using Cy5-conjugated streptavidin (Dako) as second-step reagent. Relevant isotype controls were always included.

All antibody labeling of cells was done in full blood, followed by lysis of red blood cells (FACS Lysing Solution; Becton Dickinson). After each labeling step (20 min, room temperature) and after red blood cell lysis (10 min, room temperature), the cells were washed twice in PBS. All samples were analyzed on a FACScan flow cytometer (Becton Dickinson). Analysis of FCM data was done using WinList software (Verity, Topsham, ME).

PCR analysis of CDR3 size distribution of V{delta}1–C{delta} rearrangements
Peripheral blood mononuclear cells (PBMC) were isolated by density-gradient centrifugation (Lymphoprep; Nyegaard, Oslo, Norway) and stored in liquid N2 in RNAzol (Bioprobe Systems, Montreuil, France) until use. Total RNA was prepared from 1–5x106 RNAzol-preserved PBMC following the instructions of the manufacturer. Single-strand cDNA was subsequently synthesized from the RNA following the procedure described by the manufacturer (Gibco/BRL, Gaithersburg, MD). The analysis of CDR3 size distribution of V{delta}1–C{delta} rearrangements was done following the Immunoscope technique (30,31). In brief, cDNA was amplified by PCR using a V{delta}1 primer and a C{delta} antisense primer. Amplification products were subsequently subjected to a run-off elongation cycle using an internal C{delta} primer with a fluorescent dye bound to the amino-2 group of the primer. Each labeled run-off product was loaded onto a 6% acrylamide sequencing gel for size and fluorescent intensity determination using an Applied Biosystems 373A DNA sequencer and analyzed with Immunoscope software (C. Pannetier, Paris, France).

Plasma antibody levels
Plasma levels of total IgG were measured by radial immunodiffusion assays (Serotec, Oxford, UK). Plasma levels of anti-EBV nuclear antigen (EBNA) IgG and EBV early antigen (EA)-specific IgG and IgM were measured by ELISA at the Department of Clinical Microbiology, Rigshospitalet, Copenhagen.

Statistical analyses
Pair-wise comparison between groups and subsets were done by Student's t-test (t), unless the normal distribution and equal variance assumptions were violated, in which case the Mann-Whitney rank-sum test (T) was used. Confidence intervals for median differences were calculated as described by Campbell and Gardner (32). Parameter association was analyzed by Pearson product-moment correlation (r). Simultaneous comparison of more than two donor groups was done by the Kruskal–Wallis test (KW), followed by Dunn's test as appropriate. Values of P < 0.05 were considered significant. SigmaStat software (Jandel Scientific, San Rafael, CA) was used for all statistical calculations.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The frequency of {gamma}{delta} T cells is higher in Africans than in Caucasians, mainly due to an increased frequency of V{delta}1+ cells
To confirm our previous finding of high frequencies of peripheral {gamma}{delta} T cells in healthy Africans (25), we first analyzed the prevalence of this cell population in two groups of healthy adults. One group consisted of 10 individuals of Caucasian descent with permanent residence in Denmark and the other group consisted of eight individuals of African descent with permanent residence in Ghana. As shown in Fig. 1Go, the frequency of CD3+ cells expressing TCR {gamma}{delta} was approximately twice as high in Ghanaian compared to Danish adults [P(T) = 0.05, median difference: 4.8, 95% CI: –0.3–9.3]. This difference was largely due to an ~5-fold higher frequency of V{delta}1+ cells in the Ghanaian compared to the Danish donors [P(T) = 0.001, median difference: 2.1, 95% CI: 0.7–8.8], whereas the frequency of V{delta}1 {gamma}{delta} T cells was similar in both groups of adults [P(T) = 0.4, median difference: 1.4, 95% CI: –2.2–3.8]. The absolute number of CD3+ cells was similar in the two groups of adults (data not shown). Thus, the increased frequencies of {gamma}{delta} T cells and V{delta}1+ cells in the African donors were not simply due to decreased numbers of V{gamma}9+V{delta}2+ cells in the African compared to the Caucasian donors.



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Fig. 1. Levels and subset composition of circulating {gamma}{delta} T cells. (A) Frequencies of circulating TCR {gamma}{delta}+ and V{delta}1+ cells in 10 healthy European (EA) and eight African (AA) adults, and in 77 healthy African children (AC). (B and C) Flow cytometry profiles of a European (B) and an African (C) healthy adult.

 
Subsequent analysis of a group of 77 healthy Ghanaian children showed that the frequency of TCR {gamma}{delta}+ cells was similar in African adults and children (Fig. 1Go), whereas the proportion of V{delta}1+ cells in the children (mean: 64.5% of TCR {gamma}{delta}+ cells) was significantly higher [mean difference: 19.2, P(t) < 0.001, 95% CI: 8.0–30.4] than in the Ghanaian adults (mean fraction of TCR {gamma}{delta}+ cells expressing V{delta}1: 45.2%). The frequencies of TCR {gamma}{delta}+ cells or V{delta}1+ cells in the Ghanaian children were not significantly correlated to the age of the donors [P(r) = 0.27 and P(r) = 0.07 respectively (Fig. 2Go)]. In conclusion, the high proportion of TCR {gamma}{delta}+ cells among peripheral CD3+ cells is mainly due to expansion of the V{delta}1+ cell population, which is the dominant {gamma}{delta} T cell subset in both children and adults from our African study population.



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Fig. 2. Relationship between donor age and frequency of TCR {gamma}{delta}+ cells (A) and V{delta}1+ cells (B) in a cohort of 77 healthy Ghanaian children. Individual data points (B: two data points missing), first-order regression lines and 95% CI for the regression lines are shown.

 
V{delta}1+ T cells from healthy African individuals use all expressed V{gamma} gene products
We next analyzed the expression of V{gamma} gene products by V{delta}1+ cells to determine if the expansion of this subset in the African donors involved all V{delta}1+ cells or just a subpopulation (29). Six of the children and five of the Ghanaian adults were analyzed this way in addition to four Caucasian control donors (Fig. 3Go). There was considerable variation in V{gamma} usage by V{delta}1+ cells between donors (Fig. 3AGo), but no significant difference was detectable between the three donor groups [P(KW) > 0.07 in all cases]. V{gamma}4 was the most commonly used V{gamma} chain in both groups (median: 41% of V{delta}1+ cells), followed by V{gamma}2 (24%), V{gamma}9 (11%) and V{gamma}8 (10%). V{delta}1+ cells rarely expressed V{gamma}3,5 (2%). When a cocktail of all V{gamma} antibodies was used, >95% of the V{delta}1+ cells were labeled, demonstrating that the entire expressed V{gamma} repertoire was included in the analysis (Fig. 3BGo).




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Fig. 3. Expression of V{gamma} gene products by V{delta}1+ T cells from healthy donors. Individual data points and median values of five Ghanaian adults ({circ}; V{gamma}2,3,4 and V{gamma}4: four adults), six Ghanaian children (• V{gamma}8: five children) and four Caucasian adults ({triangledown}) (A), and flow cytometry profile of a representative donor (B, child 69) are shown.

 
V{delta}1+ T cells in children are less oligoclonal than in adults
To investigate whether the expansion of V{delta}1+ cells was driven by antigen recognized by V{delta}1, we assessed the V{delta}1 repertoire diversity by Immunoscope size spectratyping of the V{delta}1–C{delta} CDR3 region (33). As shown in Fig. 4Go, the children generally showed many CDR3 size classes, often with a size class distribution approximating the normal distribution. In contrast, essentially all the adults examined showed a markedly restricted number of CDR3 size classes in the V{delta}1–C{delta} region, indicating a higher degree of oligoclonality among these cells in the adults compared with the children. The oligoclonality appears significantly lower in the Ghanaian adults compared to the children, although higher than that generally seen in European adults (data not shown). Our analysis did not show any marked skewing of the V{delta}1 CDR3 size distribution in the children and it thus unlikely that the expansion of the V{delta}1 population is the result of selection in early childhood by a specific antigen.



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Fig. 4. V{delta}1 CDR3 characterization of T cells ex vivo from healthy donors from Ghana. Individual profiles from five adults and five children are shown.

 
V{delta}1+ cells in healthy Africans have a phenotypically immature phenotype
To examine whether the high frequency of V{delta}1+ cells in the African donors was associated with a particular phenotype of these cells, we analyzed the phenotype of the V{delta}1 subset in six European and 10 Ghanaian adults, and in 10 Ghanaian children (Fig. 5Go). Only few V{delta}1+ cells expressed CD4. A higher proportion of V{delta}1+ cells expressed CD8, albeit most at lower levels than TCR {alpha}ß+ cells, and the proportion of CD8+ V{delta}1+ cells was significantly higher in both Ghanaian adults and children compared to European adults. A very large proportion (~80%) of the V{delta}1+ cells from Ghanaian donors were phenotypically immature, i.e. CD45RAhiCD45RO. In contrast, only ~30% of V{delta}1+ cells in the European donors had this phenotype, as the majority of V{delta}1+ cells from these donors had the intermediate phenotype CD45RAloCD45ROlo (data not shown). Only a few V{delta}1+ cells had the mature CD45RACD45ROhi phenotype in either donor group (Fig. 5Go). In agreement with the high proportion of phenotypically immature V{delta}1+ cells in the Ghanaian donors, significantly more V{delta}1+ cells expressed the early differentiation marker CD38 in the African compared to the European donors. Expression of early (CD69) and late (HLA-DR) activation markers did not differ significantly between children and adults or between the African and the European donors (Fig. 5Go).



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Fig. 5. Phenotypic analysis of V{delta}1+ T cells from Danish adults (open bars), and Ghanaian adults (narrow-hatched bars) and children (wide-hatched bars). Medians and interquartile range are shown. Figures above error bars indicate the number of individuals tested. In addition, the overall level of significance of one-way ANOVA are shown [P(KW)] as well as pair-wise differences significant at the 0.05 level (light bars) and 0.01 level (heavy bars) by Dunn's post-hoc test (horizontal bars).

 
Levels of TCR {gamma}{delta}+ and V{delta}1+ cells are not related to malaria parasitemia or plasma antibody levels
Finally, the high prevalence in our study area of infectious diseases known to induce {gamma}{delta} T cell expansion prompted us to search for correlations between the frequency of TCR {gamma}{delta}+ and V{delta}1+ cells and evidence of exposure to such microorganisms.

Several studies have reported increased frequencies of TCR {gamma}{delta}+ T cells following malaria (34,35) and these cells have been shown to proliferate in response to malaria antigens in vitro (36,37). Although all individuals examined in the present study were healthy, three-quarters of the children had patent, asymptomatic P. falciparum parasitemia (geometrical mean 244 parasites/µl; range 30–120,000 parasites/µl) at the time of blood sampling. However, we did not detect any significant association between parasitemia and the frequency of either TCR {gamma}{delta}+ or V{delta}1+ cells [N = 77, r = –0.1 and –0.01, P(r) = 0.5 and 0.9 respectively].

Increased proportions of V{delta}1+ {gamma}{delta} T cells have been reported in a number of diseases characterized by polyclonal B cell activation, e.g. HIV infection and infectious mononucleosis (9,10). Furthermore, inhabitants of areas of intense malaria transmission have markedly increased Ig synthesis (38). We thus investigated the relationship between increased levels of V{delta}1+ T cells and levels of plasma total IgG or EBV-specific antibodies in the Ghanaian children. Indeed, the mean plasma level of IgG (19 g/l) was about twice that reported in healthy, European children, and some of the children had very high plasma levels of IgG (up to 64 g/l) (data not shown). However, there was no significant correlation between plasma total IgG and frequencies of TCR {gamma}{delta}+ or V{delta}1+ cells [N = 72, r –0.2–0 and P(r) > 0.15 in both cases]. All but eight of 72 plasma samples examined (89%) had high titers of anti-EBNA IgG, indicating past exposure to EBV, 14 (19%) had positive plasma titers to EA and 30 (42%) had positive anti-EA IgM plasma titers. There was no statistically significant relationship between the responder status to either EBNA, EA IgG or EA IgM and frequencies of either TCR {gamma}{delta}+ or V{delta}1+ cells [N = 72, P(T) > 0.1].


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Most (>75%) peripheral {gamma}{delta} T cells from healthy Caucasians express a disulfide-linked TCR composed of V{gamma}9 and V{delta}2, while most of the remaining {gamma}{delta} T cells (<1% of CD3+ cells and ~10% of TCR {gamma}{delta}+ cells) express a receptor encoded by V{delta}1 non-disulfide-linked to V{gamma} chains other than V{gamma}9 (5,39). In marked contrast to this, as many as half or more of the {gamma}{delta} T cells obtained from healthy Ghanaians in general, and from children in particular, expressed V{delta}1. Thus, the difference in TCR {gamma}{delta}+ frequencies between Europeans and Africans was largely due to the much higher proportion of V{delta}1+ cells in the latter group. Although high proportions of V{delta}1+ cells have previously been noticed in some healthy African individuals (40), the present study is the first to provide systematic population level evidence of high levels of circulating TCR {gamma}{delta}+ and V{delta}1+ cells in the absence of disease. This finding is in line with previous studies showing differences in other T cell phenotypes between Caucasian and African populations (41,42).

In Caucasians, the size of the circulating pool of {gamma}{delta} T cells is small at birth, but expands during the first decade of life from <2 to 5–10% (21). The increase is limited to V{gamma}9+V{delta}2+ cells and presumably occurs in response to environmental stimuli such as microbial phospho-antigens or alkyl-amines derived from edible plants (23,24,43). This causes the V{delta}1+ cells, which are the dominant {gamma}{delta} T cell phenotype at birth, to gradually become a minor subset, while the initially small V{gamma}9+V{delta}2+ subset dominates by 10 years of age (21). It is striking that we found no evidence of a similar age-dependent change in our study population, despite the fact that it is continuously exposed to agents such as P. falciparum, one of the infectious microorganisms known to contain antigens specific for V{gamma}9+V{delta}2+ cells (43).

Elevated frequencies of circulating {gamma}{delta} T cells have been reported in a number of infectious diseases (9,10,35). Although all the Ghanaian individuals examined here were healthy, we considered the possibility that the high levels of TCR {gamma}{delta}+ and V{delta}1+ cells could be related to infection by malaria parasites, EBV or HIV. In vivo expansion of both V{gamma}9+ and V{delta}1+ T cells following malaria has been reported (40,44), but in vitro activation of {gamma}{delta} T cells by malaria parasite antigens is limited to V{gamma}9+V{delta}2+ T cells (36,37). In the present study we did not observe any relationship between the presence of malaria parasites and frequencies of either TCR {gamma}{delta}+ or V{delta}1+ T cells. This suggests that if the frequency of TCR {gamma}{delta}+ and V{delta}1+ cells is reflecting exposure to malaria parasites it requires clinical disease rather than parasitemia per se. A detailed study of the {gamma}{delta} T cell response to P. falciparum malaria is in progress.

The proportion of V{delta}1+ cells within the TCR {gamma}{delta}+ subset is markedly increased in HIV+ individuals with or without general {gamma}{delta} T cell expansion (9,45,46). HIV tests were not done in the present study, and we can thus not formally exclude a relationship between the observed high frequencies of TCR {gamma}{delta}+ and V{delta}1+ T cells and HIV infection. However, all the donors included in the study were clinically healthy at the time of blood collection and the prevalence of HIV is only ~1–4% in adults from the same area (27), and presumably even lower in children. While antigens from O. volvulus can stimulate V{delta}1+ cells in vitro, this infection is absent from our study area (47).

Analysis of the V{gamma} usage did not reveal preferential expansion of a particular subset of V{delta}1+ cells. In particular, we did not observe preferential use of the V{gamma}8 chain, a chain mainly expressed by V{delta}1+ cells from the gut (48,49). It is thus unlikely that the high frequency of peripheral V{delta}1 cells in our donor population is the consequence of migration of intestinal V{delta}1 T cells to the peripheral blood.

To test the possibility that the expansion was driven by antigens recognized by the V{delta}1 chain, we analyzed the CDR3 region size distribution. This region corresponds to the V–D–J junctional region in Ig, thus presumably reflecting the antigenic specificity of the encoded V{delta}1 chains. However, this analysis did not show any obvious dominance of particular CDR3 sizes. Rather, our data showed a pattern similar to that previously described in Caucasians, with age-dependent progression from polyclonality towards oligoclonality (50). Taken together, these data do not suggest a conventional antigen-driven process in explaining the high V{delta}1 frequency.

No evidence of activation of peripheral V{delta}1+ cells was found, as expression of CD69 and HLA-DR was similar to that observed in healthy Caucasians. However, the proportions of CD8+ and CD38+ cells within the V{delta}1+ subset were increased as has previously been seen in studies of HIV-infected individuals (46). The proportions of V{delta}1+ cells in general and V{delta}1+ cells expressing CD8 in particular are higher in the spleen than in the peripheral blood (51,52). CD38 can be involved in T cell activation and proliferation, but CD38 expression is mainly a feature of immature cells and thymocytes (53,54). We did not find evidence of V{delta}1+ T cell activation in our population, and it thus appears that the high levels of circulating V{delta}1+ cells reflect altered recirculation between the spleen and the peripheral blood and/or increased turnover of this cell subset. Our observation of an increased proportion of V{delta}1+ cells having the naive/unprimed phenotype CD45RAhiCD45RO supports this idea.

The conditions where increased frequencies of V{delta}1+ cells have been reported are often characterized by polyclonal B cell activation (HIV infection, onchocerciasis as well as numerous auto-immune disorders). Studies of the impact of endemic malaria transmission on the immune system show much higher rates of Ig synthesis in Africans than in Europeans, suggesting persistent B cell activation in malarious areas (38,55). Indeed, plasma samples from some of the children in our study showed very high IgG levels. However, we did not detect significant relationships between levels of total IgG or of EBV-specific IgG or IgM and the frequencies of either TCR {gamma}{delta}+ or V{delta}1+ cells. Whatever is causing the high levels of V{delta}1+ T cells in our Ghanaian study population, it is not due to expansion of V{delta}1+ cells expressing particular V{gamma} chains and the CDR3 region of V{delta}1+ cells from these individuals does not appear very restricted. Taken together, the increased frequency of V{delta}1+ in these people does not seem to be the consequence of peripheral CDR3-dependent antigenic selection and, in this respect, our data conform to previous studies of the V{delta}1-specific expansion seen in HIV+ individuals (46,56).

In conclusion, our study demonstrates that marked expansion of V{delta}1+ T cells can occur at the population level and in the absence of clinical disease. The study thus raises profound questions regarding the physiological role of this {gamma}{delta} T cell subset in host defence and regulation of the immune system, and points to a need for a more detailed knowledge about the ligands recognized by V{delta}1+ T cells.


    Acknowledgments
 
Enid Owusu, Abdelrahman Hammond, Gitte Pedersen and Ernestina Dwamena are thanked for excellent technical assistance. Dieter Kabelitz (Paul-Erlich Institute, Langen, Germany) is thanked for his kind gift of the 56.3 mAb. Philippe Kourilsky (Institut Pastéur, Paris, France) is thanked for access to the Immunoscope facility. The continued support of the inhabitants of Dodowa is gratefully acknowledged. This study received financial support from the ENRECA program of the Danish International Development Assistance (Danida) and the INCO-DC program of the European Union. L. H. is supported by the Danish Medical Research Council (SSVF) and the Danish Research Council for Development Research (RUF).


    Abbreviations
 
EA EBV early antigen
EBNA EBV nuclear antigen
EBV Epstein–Barr virus
FCM flow cytometry
PBMC peripheral blood mononuclear cell

    Notes
 
Transmitting editor: S. Kaufmann

Received 8 November 1999, accepted 10 February 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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