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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Hum Immunol. 2010 Jun 30;71(10):968–975. doi: 10.1016/j.humimm.2010.06.014

Impact of age, gender and race on circulating gammadelta T cells

Cristiana Cairo 2,1, Cheryl L Armstrong 2,1, Jean Saville Cummings 2,3, Carl O Deetz 2,4, Ming Tan 5, Changwan Lu 5, Charles E Davis 2, C David Pauza 2,6
PMCID: PMC2941533  NIHMSID: NIHMS218183  PMID: 20600446

Abstract

A major subset of human peripheral blood γδ T cells expresses the Vγ2Vδ2 T cell receptor (TCR) and responds to malignant or infectious diseases. We noted significant differences in the numbers of Vγ2Vδ2 T cells in blood samples from healthy Caucasian or African American (AA) donors. On average, CA donors had 3.71 ± 4.37% Vδ2 cells (as a percentage of total lymphocytes) compared to 1.18 ± 2.14% Vδ2 cells for AA donors (p < 0.0001). Age and race had the greatest impact on Vδ2 cell levels; the effect of age was similar for both racial groups. The Vδ2+ cell population was dominated, for both donor groups, by cells expressing the Vγ2-Jγ1.2 Vδ2 T cell receptor, an apparent result of strong positive selection and there was substantial overlap in the public Vγ2 clonotypes from both racial groups. Mechanisms for selection and amplification of Vδ2 cells are nearly identical for both groups, despite the significant difference in baseline levels. These data show that appropriate controls, matched for age and race, may be required for clinical studies of Vγ2Vδ2 T cells in infectious disease or cancer and raise important questions about the mechanisms regulating the levels of circulating Vδ2 cells.

INTRODUCTION

Within the U.S. population, circulating lymphocyte levels [1] and the proportions of CD4+ or CD8+ T cells [2] are nearly identical among Caucasian (CA) and African American (AA) individuals. Racial groups vary in their levels of circulating neutrophils, but these differences are modest reviewed in [1, 3]. On the other hand, differing baseline neutrophil counts may impact dosing decisions for breast cancer chemotherapy, especially among AA women [4]. It is important to understand race-dependent variation in leukocyte populations, and to determine whether matched control groups are required for clinical studies. Here, we investigate the effect of age, gender and race on the circulating levels of Vγ2Vδ2 T cells.

The T cell subset expressing the Vγ2Vδ2 T cell receptor (also designated Vδ2 cells) is distinct from the more familiar CD4+ or CD8+ T cells that express an αβ T cell receptor. Humans have three Vδ segments that generate the adult repertoire. Vδ1 chains pair with most of the Vγ chains and dominate the intraepithelial subset of mucosal γδ T cells where the TCR recognizes stress molecules on epithelial cells [5, 6]. Vδ3 cells are less well studied, are found in blood at low levels and may be associated with protective responses against herpesviruses [7]. The Vδ2 chain is mainly paired with Vγ2 in blood lymphocytes from adults, comprising 60–90% of circulating γδ T cells [8–10]. The majority of Vγ2 chains contain the Jγ1.2 segment [11, 12]; this rearrangement encodes recognition of phosphoantigens [13] that are intermediates in mammalian or prokaryotic synthesis of isoprenoids [14–16]; phosphoantigen recognition is MHC and CD1-independent [17]. It has been proposed that the chronic presence of phosphoantigens drives extrathymic selection and amplification of a Vγ2-Jγ1.2 Vδ2 subset [18] and it was reasonable to assume that these same mechanisms controlled the normal blood levels of Vδ2 cells.

Without MHC restriction, the phosphoantigen-reactive Vγ2Vδ2 repertoire is similar among most individuals. There is a strong bias toward the Vγ2-Jγ1.2 rearrangement and a high frequency of public Vγ2 chains. It is even possible to find public Vγ2 chain nucleotypes (identical CDR3 region nucleotide sequences) [19]. Thus, positive selection by phosphoantigen and the absence of MHC restriction creates a Vγ2 chain repertoire that is dominated by public clonotypes. These findings also support the view that selection and amplification by ubiquitous phosphoantigens control repertoire composition and Vγ2Vδ2 T cell levels in healthy adults. There are several examples that demonstrate the relationship between phosphoantigen levels and Vδ2 cells. Exposure to extraordinarily high levels of phosphoantigens, that occurs in Plasmodium [20], Brucella [21], Franciscella [22, 23] or Mycobacterium [24–26] infections, increase Vγ2Vδ2 T cells to more than 30% of total lymphocytes. In non-human primates, synthetic phosphoantigens elevated blood Vγ2Vδ2 cell counts [27] and exposure to mycobacterium BCG increased cell levels and altered the Vγ2 chain repertoire [28]. However, Vδ2 counts generally return to basal levels after the exposure to phosphoantigens.

Caccamo and colleagues reported gender and age effects on blood Vδ2 levels, with women having lower levels compared to age-matched men [29]. Hviid's group reported inversion of the Vδ2:Vδ1 cell ratio in healthy volunteers in Ghana who were negative for malaria and HIV disease [30]. In a control group for our HIV studies in China, we observed Vδ2 cell levels nearly identical to Caucasian populations in the United States [31]. These studies begin to show that Vδ2 cell levels may be subject to multiple control mechanisms and there may be population differences.

Now, we have examined the levels, phenotypes and functional responses of Vδ2 cells in healthy, adult African Americans (AA) living in and around the city of Baltimore, MD, and compared them to age and gender-matched healthy Caucasian (CA) donors. We uncovered substantial differences between AA and CA donors. To test the hypothesis that Vδ2 populations are controlled by responses to phosphoantigens, we characterized the spectrum of public Vγ2 chain clonotypes in both donor pools and performed in vitro antigen stimulations. These data provide a comprehensive view of Vδ2 cell differences between AA and CA donors, and suggest that simple positive selection models will not account for the overall control of Vδ2 cells in humans.

MATERIALS AND METHODS

PBMC Isolation and Vγ2Vδ2 Stimulation

Heparinized venous blood was collected from 33 self-reported Caucasians and 32 self-reported African American HIV-negative and healthy volunteers. HIV status was self-reported and confirmatory tests were not administered. The study was approved by the Institutional Review Board at the University of Maryland, Baltimore, and written informed consent was obtained from each donor. Peripheral blood mononuclear cells were purified and cultured as described previously [32].

Stimulation index (SI) represents the proportional increase in Vδ2 cells following IPP stimulation compared to control with IL2 and no IPP. SI is a ratio of the absolute number of Vδ2 lymphocytes on day 14 of the IPP expansion to the absolute number of Vδ2 lymphocytes on day 14 of the IL2 alone expansion.

Flow cytometry

Ex vivo PBMC or expanded Vγ2Vδ2 T lymphocytes were resuspended in PBS (Gibco)-10% FBS (Gibco) and stained at 4°C with directly conjugated monoclonal antibodies for the detection of cell surface markers. Granzyme B was detected by intracellular staining using the Cytofix/Cytoperm Kit from BD Biosciences according to manufacturer's instructions.

Flow cytometry data were collected on a FACSCalibur (BD Biosciences, San Diego CA). At least 3 ×104 lymphocytes (gated on the basis of forward and side scatter profiles) were collected for each sample and results were analyzed with FlowJo software (Tree Star, San Carlos CA). The following monoclonal antibodies were used for four-color staining: anti-Vδ2 FITC, clone B6 (BD); anti-CD95 PE, clone DX2 (BD); anti-CD3 APC, clone UCHT1 (BD); anti CD45Ra PC5, clone HI100 (BD); anti-CD16 PC5, clone 3G8 (BD); anti-CD3 PerCP, clone SP34-2 (BD); anti CD56 PE and anti CD56 APC, clone N901 (Beckman Coulter); anti-Granzyme B, clone GB12 (Caltag, Burlingame CA); anti-CD27, clone O323 (eBioscience, San Diego CA); and the appropriate isotype controls (all purchased from BD Biosciences).

RNA extraction, RT- PCR, PCR

RNA extraction and cDNA synthesis were performed as described [33] using the following primers for the Vγ2 (Vγ9 according to the IMGT nomenclature) chain: oligo Vγ2 (5' ATC AAC GCT GGC AGT CC 3') and oligo Cγ-1 (5' GTT GCT CTT CTT TTC TTG CC 3').

Run-off reaction

Primer extension reactions and analysis of the length distribution of the Vγ2 chain CDR3 region were performed as described [32]. Run-off product lengths were corrected by adding the length of the known mRNA coding region outside the run-off primer-binding site. According to our convention [32] the Vγ2 chain population has a peak length at 993 nucleotides.

Repertoire analysis

We evaluated the Vγ2 chain repertoire among representative AA donors. mRNA were prepared from PBMC and individual Vγ2 chain sequences were cloned into plasmids as described [34]. DNA sequences surrounding and including the CDR3 regions were determined for 150–300 plasmid clones from each donor. Sequences were aligned, we determined how many sequences were repeated in the library from each donor, and we generated amino acid sequences for comparison. Data were expressed as the proportion of Vγ2-Jγ1.2 rearranged sequences among all Vγ2 chains, with average values calculated for 8 AA donors and 6 CA donors. We also identified public clonotypes within the AA group and deduced the frequency of appearance for individual public clonotypes in each donor. Data for CA donors were derived from our existing database of nearly 50,000 human Vγ2 chain sequences. The CA and AA donors were matched for age and gender.

Statistical analysis

The student t test was used to compare the mean difference when a Gaussian distribution was present; otherwise (e.g., when there were outliers), the Wilcoxon rank sum test was used to compare two groups. The multi-variable analysis was performed with the mixed procedure using SAS 9.2 software (SAS Institute Inc., Cary, NC). Non-normal data were normalized (e.g., log-transformed) before analysis. P < 0.05 was considered to be significant.

RESULTS

Characteristics of the donor population

We obtained blood specimens from 32 African American (AA) donors and 33 Caucasian (CA) donors. The mean age was 43.6 years (19 – 63 years) and 41.8 years (26 – 64 years) for AA and CA donors, respectively. The AA group included 19 females and 13 males; the CA group included 19 females and 14 males. Data for all donors are presented in Tables 1 and 2.

Table 1.

Caucasian Donors

Sample ID Sex Age
ND001 M 54
ND003 M 27
ND004 M 57
ND005 M 37
ND006 F 32
ND007 F 28
ND008 M 30
ND010 F 32
ND014 M 34
ND015 F 26
ND016 F 57
ND017 M 32
ND018 F 27
ND019 F 29
ND020 F 29
ND038 F 64
ND039 M 44
ND040 M 43
ND041 M 40
ND042 M 41
ND043 M 47
NB23 F 43
RF32 F 43
AT49 F 57
AD58 F 48
LH61 M 52
CB62 F 54
MH66 F 41
KH68 F 39
ME71 F 50
SE72 M 53
DW74 F 50
MV50 F 41
Mean 41.88
St.Dev 11.28
Median 41.00
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Table 2.

African-American Donors

Sample ID Sex Age
ND023 F 24
ND024 F 45
ND025 F 26
ND026 F 58
ND027 F 41
ND028 M 50
ND029 M 57
ND030 M 62
ND031 M 39
NVS101 M 55
NVS102 F 44
NVS104 M 59
NVS105 F 49
NVS107 F 47
NVS109 F 48
NVS112 M 43
MP063 F 63
MP068 F 51
MP089 M 53
CH46 F 35
EG41 F 52
KP42 F 59
HR45 M 49
LR33 F 31
CF37 M 47
SF48 F 19
YL43 F 47
ND036 M 35
ND037 M 25
ND044 F 29
ND045 M 30
ND046 F 24
Mean 43.63
St.Dev 12.49
Median 47.00
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Baseline values for Vγ2Vδ2

Baseline values were measured in PBMC specimens. We evaluated by flow cytometry, the percentage of Vδ2 T cells among total lymphocytes. Total lymphocyte counts are very similar among AA and CA donors in the U.S. [1, 35, 36]. Thus, percentage should be valid for our comparisons. The range for all donors was 0.03% to 21.3% Vδ2 cells in total lymphocytes; with ranges of 0.16–21.3% for CA donors and 0.03–11.4% for AA donors (Fig. 1). Mean and median, for the percentage of Vδ2 cells in total lymphocytes were 3.71% and 2.25% for CA donors, with a standard deviation of 4.37% (Fig.1); and 1.18% and 0.43% for AA donors, with a standard deviation of 2.14% (Fig.1). The mean and median were 3.1 fold and 5.6 fold higher, respectively in CA compared to AA. Three outliers were identified in the boxplot analysis. The Wilcoxon test revealed a significant difference in the distribution of Vδ2 numbers between CA and AA (P < 0.0001). If outliers were excluded, the difference was still significant (P < 0.0001).

Figure 1.

Figure 1

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Vδ2 cell levels are lower in AA donors than in CA donors. Vδ2 frequencies were compared for 33 Caucasian (CA) and 32 African American (AA) donors. The scatter-plots show individual values as well as mean ± standard deviation for each group of donors. The Vδ2 frequency was 3.71% ± 4.37% (median 2.25%) for CA donors and 1.18% ± 2.14% (median 0.43%) for AA donors. P value is the result of Wilcoxon test.

Multivariate analysis by mixed effect models

We next sought to determine whether the racial difference in Vδ2 cell frequency is significant after adjusting for age and gender. Race (P < 0.0001) was still a significant predictor of log(Vδ2) values after adjusting for age and gender. Age itself was also associated with log(Vδ2+) (P = 0.022), whereas gender was not (P = 0.34). There were no interactions between race and age (P = 0.28). The percentage of Vδ2+ cells declined as age increased, and rates of decline were the same for CA and AA donors.

We extended the multivariate analysis to examine whether phenotype markers or the Vγ2 chain repertoire depended on race, age and gender. CD56 is a marker identifying the sub-population of Vγ2Vδ2 T cells that is most potent in cytotoxicity assays [19, 37]. Neither gender (P = 0.41) nor age (P = 0.24) affected the proportion of CD56+ Vδ2 cells. However, race was a significant predictor of CD56 cell levels (P = 0.0031). There were no significant interactions between race and gender (P = 0.64). CD56 was expressed on 45.3% ± 16.1 of Vδ2 cells from CA donors (Fig. 2) compared to 29.8% ± 18.3 from AA donors (P = 0.0013).

Figure 2.

Figure 2

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The fraction of CD56+ Vδ2 cells is lower for AA donors than for CA donors. The frequencies of CD56+ Vδ2 cells were compared between Caucasian and African American donors (Mean ± SD). CD56 was expressed on 45.3% ± 16.1 of Vδ2+ cells from CA donors compared to 29.8% ± 18.3 from AA donors. The scatter-plots show individual values as well as mean ± standard deviation for each group of donors. P value is the result of an Unpaired t Test.

Next, we evaluated the proportional distribution of naïve (CD27+/CD45RA+), T central memory (CD27+/CD45RA−), T effector memory (CD27−/CD45RA−) and T effector memory RA (CD27−/CD45RA+) subsets among Vδ2+ T cells [38, 39]. For all of these values, there were no significant interactions between race and gender. Neither race, age nor gender were significant predictors of T effector memory cells. Only race was a significant predictor of the TemRA compartment (P = 0.033). Age was a significant predictor for the proportion of Naïve cells (P = 0.049). Race (P = 0.012), gender (P = 0.030) and age (P = 0.019) were significant predictors of the T central memory compartment (Table 3).

Table 3.

Distribution of Vd2 T cells among naïve/memory compartments

Naïve CM EM EMRA
CD27+/CD45RA+ CD27+/CD45RA− CD27−/CD45RA− CD27−/CD45RA+
CA (N=32) 15.12 46.50 25.11 13.26 Mean
17.64 20.32 16.72 16.63 St.Dev
10b 51c 25.4d 7.75e Median
AA (N=31) 10.31 60.52 20.85 8.30 Mean
11.90 20.75 15.53 13.98 St.Dev
7.69b 65c 16.4d 5.71e Median
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a

Subsets are defined as naïve, Central Memory (CM), Effector memory (EM) and Effector Memory RA+ (EMRA)

b

P>0.05

c

P=0.0036 without adjusting for age and gender

d

P>0.05

e

P=0.0155 without adjusting for age and gender

The value of Vγ2990–996 represents the proportion of Vγ2 chains in a single specimen, with mRNA coding region lengths between 990–996 nucleotides. This value closely approximates the proportion of Vγ2 chains containing the Vγ2-Jγ1.2 rearrangement [32] which is associated with responses to phosphoantigen stimulation [13]. The Vγ2990–996 was 0.70 ± 0.22 for CA donors with values ranging from 0.06 to 0.94 (Fig. 3). The Vγ2990–996 was 0.57 ± 0.25 for AA donors with values ranging from 0.06 to 0.9 (Fig. 3). Neither race, age nor gender were significant predictors for the Vγ2990–996. However, if only race was included in the model, it became a significant predictor (P = 0.046).

Figure 3.

Figure 3

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The fraction of Vγ2-Jγ1.2+ is lower for AA donors. RNA was collected from PBMC from Caucasian (n = 25) or African American (n = 26) donors. Following Vγ2 PCR amplification and runoff analysis, relative frequencies were averaged for each chain length. (A) Histogram plots show mean and standard deviation for every Vγ2 chain length, for each group of donors. (B) The fraction of chains with a length between 990–996 nucleotides (Jγ1.2) was on average 0.57 ± 0.25 among AA donors and 0.70 ± 0.22 among CA donors. The scatter plot shows for both groups individual Vγ2 990–996 values as well as mean ± St Dev. P value is result of a Wilcoxon Test.

Other markers of Vδ2+ T cells

Granzyme B (GrB) is another marker for cytotoxic T cells. We characterized Granzyme B production and tested Vδ2 lymphocytes for proliferation responses to phosphoantigen. These tests were only done on a subset of samples from the donor pool and were not included in the multivariate analysis. Among CA donors, 57.1% ± 16.9% of Vδ2 cells expressed GrB, compared to 42.6% ± 20.4 for AA donors (P = 0.012) (Fig. 4). The difference was in line with our finding from the multivariate analysis that only race was a significant predictor of the TemRA compartment where GrB expression is most frequent [40].

Figure 4.

Figure 4

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Lower Granzyme B expression in Vδ2 cells from AA compared with CA donors. The frequencies of Granzyme B+ Vδ2 cells were 42.6% ± 20.4% for AA donors and 57.1% ± 16.2% for CA donors. The scatter-plots show individual values as well as mean ± standard deviation for each group of donors. P value is the result of an Unpaired t Test.

The proliferation response to phosphoantigen, represented by the stimulation index, is a measure of functionality for Vδ2 cells in human PBMC. Stimulation index is defined as: (absolute number of Vδ2 cells after phosphoantigen + IL2) ÷ (absolute number of Vδ2+ cells after IL2). The mean stimulation index for CA donors was 33.5 ± 31.0, with values ranging from 5.5 to 97 (data not shown). For AA donors, the mean stimulation index was 35.2 ± 46.4 with a range from 1.7 to 160.3 (data not shown). There was no significant difference in the mean values between the two groups. Our results indicate that Vδ2 cells from AA or CA donors have similar capacities for responding to phosphoantigen stimulation.

Phosphoantigen stimulation also increased the proportion of CD56+ Vδ2 T cells. After phosphoantigen stimulation, the CD56+ subset was 67.7% ± 13.9% for CA and 50.2% ± 20.7% for AA donors (p < 0.05, data not shown). The proportional increases in CD56+ Vδ2 cells after phosphoantigen stimulation were similar for AA (1.7 fold) and CA donors (1.5 fold), consistent with the stimulation index data.

Characteristics of the Vγ2 chain repertoire

We used molecular cloning to evaluate the Vγ2 chain repertoire in multiple donors. Plasmid libraries were constructed from 8 AA and 6 CA donors; sequences were obtained for the V-J regions. Sequence data identified J segments used in each chain and defined the non-templated nucleotides in each CDR3 region. Within the library of sequences for each donor, we identified sequences present more than once (repeated). Amino acid sequences (clonotypes) that were present in more than one donor (identical at all positions within the V, N and J regions) were defined as public clonotypes.

Public clonotypes represent the most commonly selected Vγ2 sequences. The profile of public clonotypes can reveal similarities or differences in the Vγ2 T cell repertoire that in turn, reflect the selection and amplification mechanisms.

For 8 AA donors, 29 public clonotypes were identified (Table 3); 16 sequences were common to 2 donors (55%), 5 were common to 3 donors (17%), 2 were common to 4 donors (7%), 3 were common to 5 donors (10%), 2 were found in 6 different donors and 1 clonotype was found in all 8 donors. The public clonotype found in all donors (CDR3 region sequence of CALWEVQELG, called the canonical sequence) represents the germline configuration of Vγ2 and Jγ1.2 segments without deletion/addition of non-templated nucleotides during DNA recombination. For this collection of public clonotypes, 21 of 29 (72%) were also found in the list of public clonotypes for CA donors (Table 4).

Table 4.

Public clonotypes for Caucasian Donors

Clonotype ND001 ND006 ND008 ND018 ND019 ND020
CALW----DP----QELGKKIKVFGPGTKLIIT 2 1
CALW----GQ-----ELGKKIKVFGPGTKLIIT 1 1
CALW----V-----QELGKKIKVFGPGTKLIIT 1 1 1
CALWE---A------ELGKKIKVFGPGTKLIIT 38 2 1 3
CALWE---AG-----ELGKKIKVFGPGTKLIIT 1 1
CALWE---AK-----ELGKKIKVFGPGTKLIIT 6 2
CALWE---AP----QELGKKIKVFGPGTKLIIT 2 1
CALWE---A-----QELGKKIKVFGPGTKLIIT 2 8 3 7 7 7
CALWE---AQ------LGKKIKVFGPGTKLIIT 1 1
CALWE---AS----QELGKKIKVFGPGTKLIIT 1 1
CALWE---D-----QELGKKIKVFGPGTKLIIT 2 2 2 1
CALWE---DV-----ELGKKIKVFGPGTKLIIT 1 1
CALWE---E------ELGKKIKVFGPGTKLIIT 1 1 1 1
CALWE---EK-----ELGKKIKVFGPGTKLIIT 1 2
CALWE----------ELGKKIKVFGPGTKLIIT 3 2
CALWE---E-----QELGKKIKVFGPGTKLIIT 2 2
CALWE---G-----QELGKKIKVFGPGTKLIIT 2 1 1 2
CALWE---I-----QELGKKIKVFGPGTKLIIT 1 4
CALWE---K------ELGKKIKVFGPGTKLIIT 2 3
CALWE---L-----QELGKKIKVFGPGTKLIIT 1 3 2
CALWE---MQ-----ELGKKIKVFGPGTKLIIT 1 9
CALWE---NQ-----ELGKKIKVFGPGTKLIIT 2 2
CALWE---P-----QELGKKIKVFGPGTKLIIT 3 1 2
CALWE---PQ----QELGKKIKVFGPGTKLIIT 5 1
CALWE---------QELGKKIKVFGPGTKLIIT 1 4 6
CALWE---RL----QELGKKIKVFGPGTKLIIT 1 1
CALWE---R-----QELGKKIKVFGPGTKLIIT 1 1
CALWE---S-----QELGKKIKVFGPGTKLIIT 2 1 1
CALWE---T-----QELGKKIKVFGPGTKLIIT 4 3 2
CALWE---VD-----ELGKKIKVFGPGTKLIIT 23 1
CALWEV--E------ELGKKIKVFGPGTKLIIT 1 2 6 2
CALWEV---------ELGKKIKVFGPGTKLIIT 5 4 2 2
CALWEV--G------ELGKKIKVFGPGTKLIIT 1 4 1 2 1
CALWEV--H------ELGKKIKVFGPGTKLIIT 5 1
CALWEV--HQ------LGKKIKVFGPGTKLIIT 1 1
CALWEV--K------ELGKKIKVFGPGTKLIIT 1 1 1 1
CALWEV--LE-----ELGKKIKVFGPGTKLIIT 2 2
CALWEV--L------ELGKKIKVFGPGTKLIIT 9 1 4
CALWEV--P------ELGKKIKVFGPGTKLIIT 1 3 3 2
CALWEV--P-----QELGKKIKVFGPGTKLIIT 1 1
CALWEV--QD-----ELGKKIKVFGPGTKLIIT 1 1
CALWEV--------QELGKKIKVFGPGTKLIIT 5 20 25 13 7 19
CALWEV--QK------LGKKIKVFGPGTKLIIT 1 1 1
CALWEV--Q-----QELGKKIKVFGPGTKLIIT 1 1
CALWEV--R------ELGKKIKVFGPGTKLIIT 2 4 25 2 1 5
CALWEV--RG-----ELGKKIKVFGPGTKLIIT 1 1
CALWEV--RK------LGKKIKVFGPGTKLIIT 3 1
CALWEV--R-----QELGKKIKVFGPGTKLIIT 1 2
CALWEV--SG------LGKKIKVFGPGTKLIIT 1 1
CALWEV--T------ELGKKIKVFGPGTKLIIT 1 2 1
CALWEV--WK------LGKKIKVFGPGTKLIIT 1 1
Total # of public Jg1.2 77 84 105 43 66 79
Total # of Jg1.2 196 158 163 75 84 154
Fraction of public Jg1.2 (%) 39.29 53.16 64.42 57.33 78.57 51.30
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Among CA donors we identified 51 public clonotypes. 30 public clonotypes (59%) were found in 2 donors, 9 clonotypes were in 3 donors (18%), 8 were found in 4 donors (16%), 1 was found in 5 donors and 3 were found in all 6 donors. All of the clonotypes found in at least 5 of the CA donors (including the canonical sequence) were also present in AA donors; only 3 of the common clonotypes (in at least 4 of 6 CA donors) were not on the list of public clonotypes for AA donors.

The pattern, abundance and distribution of public Vγ2 chain clonotypes were similar among AA and CA donor groups. The larger number of public sequences found in CA donors probably reflects the larger number of total Vγ2-Jγ1.2 sequences obtained for each sample. Generally, public clonotypes unique to either group were less abundant (fewer repetitions) and present in fewer donors within the group. The most common clonotypes were present in both groups. Overall, the Vγ2 chain repertoire in AA and CA donors suggests strongly that selection and amplification mechanisms are similar in these groups, resulting in substantial overlap in the public clonotypes.

DISCUSSION

Human γδ T cells are derived from double negative thymic precursors, like αβ T cells, but γδ T cells develop along a separate pathway that does not involve MHC-restriction and does not generate lineages that are marked by expression of cell surface CD4 or CD8 glycoproteins. Most importantly, there is no known negative selection step during γδ T cell ontogeny; cells capable of recognizing self-antigens are released to the periphery.

In the absence of MHC restriction and thymic negative-selection, extrathymic selection and amplification drives repertoire maturation [18]. One product of extrathymic selection is a repertoire dominated by cells expressing the Vγ2Vδ2 receptor [8–10] and mostly including the Vγ2-Jγ1.2 rearrangement [11, 12]. The common presence of phosphoantigens and the strong responses of Vγ2-Jγ1.2 Vδ2 cells are believed to be the force behind extrathymic selection and amplification of the Vγ2Vδ2 T cell subset. However, the mechanisms controlling steady state levels of Vγ2Vδ2 T cells have not been explored. Unlike circulating αβ T cells, the Vδ2 subset is mostly composed of antigen-experienced, positively selected memory cells with potent effector functions including chemokine or cytokine release [41–43] and cytotoxicity [44]. Naïve cells are rare in this population [45].

We used a multivariate model to test the impact of age, gender and race on Vδ2+ T cell levels. Age was negatively associated with Vδ2 T cell levels similarly to what was reported by Caccamo et al. [29]. In our study, age-related decline of Vδ2 cells were the same for CA and AA groups and there were no significant effects of gender. However, larger sample sizes might have revealed an impact of gender similar to that reported by Caccamo, et al. [29]. Race was the most important factor affecting Vδ2 cell levels.

The differences in Vδ2 cell numbers were consistent with a lower proportion of CD56+ cells, and lower granzyme B expression in the Vδ2 cell subset of AA donors. Differential granzyme expression is possibly related to a shift in the distribution of Vδ2 cells across different memory compartments. Granzyme B is expressed at higher levels in the terminally-differentiated TEMRA cell subset (CD27− CD45RA+) [38] which is lower (8.3% versus 13.26%) in AA donors. All of these differences might have been consistent with decreased strength of response to phosphoantigens due either to alterations in the signal transduction pathway, differences in antigen presentation or differences in TCR structure and repertoire. Within the limits of our data, we did not find substantial differences in the stimulation index for phosphoantigen in AA versus CA groups. Based on this finding, we felt it was unlikely that antigen presentation or signal transduction would differ among groups.

We also evaluated the complexity of Vγ2-Jγ1.2 chains, looking for the degree of clonality and the presence of public clonotypes that might reveal differences between our donor groups. In the Vγ2-Jγ1.2 sequences from 8 AA donors, we identified 29 clonotypes that were found in 2 or more donors. For CA donors, we identified 51 sequences present in 2 or more donors. The larger number of public clonotypes identified in the CA group is likely due to a larger yield of molecular clones with the Vγ2-Jγ1.2 sequence obtained for each sample. There was a substantial overlap between AA and CA public repertoire, with 72% of public Vγ2 chains from the AA group being identical to public chains in the CA group. The reverse was also true, with 41% of CA public chains also identified as public clonotypes in the AA group. Importantly, the common public chains (defined by their presence in most or all donors) were very similar in both groups. We believe that overlap among public clonotypes provides the strongest evidence for similar selection and amplification of Vγ2Vδ2 cells in both donor groups. Accordingly, we do not find substantial differences in TCR structure or repertoire selection that can account for the population differences in baseline values or phenotypic variation.

The existing models for Vγ2Vδ2 T cell repertoire maturation focus on selection and amplification driven by common phosphoantigens. One might extrapolate from these models, that the same forces shaping the repertoire also control the circulating levels of Vγ2Vδ2 T cells. Our results show that selection/amplification do not control the circulating cell levels. The differences in CD56 or Granzyme B expression, and the change in proportion of TemRA cells all point to differences in T cell homeostasis among the groups studied here. These data do not imply a functional defect in Vδ2 cells for the AA group. The key is that proliferation responses to phosphoantigen, which were similar among the groups, would rapidly mitigate any baseline differences. Our data do show group-specific differences in the distributions of memory cell populations that might be related to the control of cell numbers.

Our studies do not address the myriad of potential environmental factors that might influence T cell homeostasis. All donors in this study claimed to be in good health and all lived in and around the metropolitan area of Baltimore, Maryland. Beyond this level, we have no information on their prior exposure to environmental agents that might impact Vδ2 T cell levels. We have limited data on populations other than individuals living in Baltimore or Southern China. We have some information for adult women in Cameroon (n = 28) who had median Vγ2Vδ2 T cell levels intermediate between the AA and CA groups reported here, but these women were not age-matched to the groups in our study (mean age: 28 ± 5 years) and were all at the end of pregnancy. Nonetheless, we recognize that our results might be influenced by using only a local population. Larger studies are needed to determine whether the impact of race on Vγ2Vδ2 T cell levels can be generalized, including studies of additional racially or ethnically distinct populations.

It is unusual to have persistent, memory T cell populations focused on a single antigen. The example of Vγ2Vδ2 T cells is similar to human invariant NKT cells that recognize CD1-restricted galactosylceramide molecules [46] and all express the identical Vα24 chain of the T cell receptor [47]. The γδ T cell repertoire does include a substantial proportion of identical Vγ2 clonotypes but in contrast to iNKT cells, the population is clonally diverse and there is a broad range for the strength of response to phosphoantigen [48]. These features, clonal diversity but redundant phosphoantigen recognition, arise from the chronic, positive selection mechanism first proposed by Brenner's group [18]. However, this mechanism does not account for differences in baseline levels that we report here. Human Vδ2 cell populations have the interesting feature of very few naïve cells and a stable repertoire [49] dedicated mainly to phosphoantigen recognition. Thus, studies on Vδ2 cells may provide insight into the control of T cell memory that may be relevant to natural and acquired immunity to pathogens in distinct population groups.

Table 5.

Public clonotypes for African-American Donors

Clonotypes LR33 EG41 YL43 MP068 ND023 ND030 ND037 ND045
CALWE---AK-----ELGKKIKVFGPGTKLIIT 1 1
CALWE---AL----QELGKKIKVFGPGTKLIIT 1 1
CALWE---AP----QELGKKIKVFGPGTKLIIT 5 1
CALWE---A-----QELGKKIKVFGPGTKLIIT 3 2 5 1 5
CALWE---AQ------LGKKIKVFGPGTKLIIT 1 1
CALWE---AS----QELGKKIKVFGPGTKLIIT 2 1
CALWE---D-----QELGKKIKVFGPGTKLIIT 5 1
CALWE---E------ELGKKIKVFGPGTKLIIT 2 3 1
CALWE---G-----QELGKKIKVFGPGTKLIIT 1 1 3 1 1 1
CALWE---P-----QELGKKIKVFGPGTKLIIT 2 1 1 6
CALWE---------QELGKKIKVFGPGTKLIIT 2 1
CALWE---S-----QELGKKIKVFGPGTKLIIT 2 1
CALWE---T-----QELGKKIKVFGPGTKLIIT 1 1 1
CALWEV--A------ELGKKIKVFGPGTKLIIT 1 3
CALWEV--G------ELGKKIKVFGPGTKLIIT 1 6 4 1 2
CALWEV--GG------LGKKIKVFGPGTKLIIT 1 30
CALWEV--H------ELGKKIKVFGPGTKLIIT 1 61 2
CALWEV--HG-----ELGKKIKVFGPGTKLIIT 5 2
CALWEV--K------ELGKKIKVFGPGTKLIIT 2 2
CALWEV--LE-----ELGKKIKVFGPGTKLIIT 1 1 1
CALWEV--L------ELGKKIKVFGPGTKLIIT 2 1 1 1 2
CALWEV--M------ELGKKIKVFGPGTKLIIT 1 2
CALWEV--N------ELGKKIKVFGPGTKLIIT 1 2
CALWEV--P------ELGKKIKVFGPGTKLIIT 2 2 2 1
CALWEV--------QELGKKIKVFGPGTKLIIT 5 6 9 11 18 73 4 15
CALWEV--QK-----ELGKKIKVFGPGTKLIIT 1 1 1
CALWEV--Q-----QELGKKIKVFGPGTKLIIT 1 1
CALWEV--R------ELGKKIKVFGPGTKLIIT 4 9 1 1 1 9
CALWEV--RG-----ELGKKIKVFGPGTKLIIT 1 1
Total # of public Jg1.2 25 21 32 14 48 109 80 59
Total # of Jg1.2 61 80 57 32 115 124 111 122
Fraction of public Jg1.2 (%) 41.0 26.3 56.1 43.8 41.7 87.9 72.1 48.4
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Public Jγ1.2 public clonotypes among CA (Table 3) and AA (Table 4) donors

A total of 51 public Vγ2-Jγ1.2 clonotypes were found for CA donors (Table 3) and 29 public Vγ2-Jγ1.2 clonotypes were found for AA donors (Table 4). The number of occurrences in each donor is reported for every clonotype and the fraction of Jγ1.2 sequences coding public clonotypes is calculated as: total number of Jγ1.2 sequences % total number of Jγ1.2 sequences coding public clonotypes. Highlighted clonotypes are present as public in both groups of donors. The clonotype in bold represents a germline encoded sequence, and it is present in all donors.

Acknowledgments

We are grateful to colleagues and study volunteers who made this work possible and especially to Drs. Maria Salvato, David Riedel, Mohammed Sajadi and Robert Redfield for critical comments. We are also grateful to Shannon Berg and Rebecca Boyce who helped to identify volunteers and obtain clinical material. The research was supported by PHS grant AI077394 (C.D.P.).

Footnotes

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