Comparative gene expression by WC1⁺ γδ and CD4⁺ αβ T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1⁺ γδ T cells

Abstract

The functions of γδ T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized αβ T cells. However, their conservation throughout evolution suggests that γδ T cells are biologically unique. In ruminants, γδ T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from αβ T cells. In fact, bovine WC1⁺ γδ T cells can act as APC for αβ T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1⁺ γδ and CD4⁺ αβ T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific αβ and γδ T cell clones express many of the same genes, γδ T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1⁺ γδ T cells when compared with CD4⁺ αβ T cells selected from peripheral blood.

INTRODUCTION

The functions of γδ T cells are still poorly defined, despite their discovery in 1986 [1]. Several of the well-characterized properties of αβ T cells are shared by γδ T cells, such as cytolytic effector cell function and expression of related perforins, granulysin, and Fas/Fas ligand (FasL), as well as secretion of T cell cytokines [2]. However, restricting the characterization of γδ T cells to examining those functions typically associated with αβ T cells potentially biases or limits a more complete understanding of γδ T cell biology.

Cattle have large numbers of circulating γδ T cells. In young ruminants, γδ T cells can comprise up to 70% of circulating leukocytes, with the percentage decreasing to 10–25% as animals approach maturity [3, 4]. The majority of γδ T cells in the circulation expresses a unique scavenger receptor, workshop cluster 1 (WC1), and does not express CD2, CD6, or CD8, whereas the majority of γδ T cells in the spleen and intestine expresses these CD molecules but lacks the WC1 molecule [5]. Although genes homologous to WC1 have been identified in humans and mice, they are not expressed in species other than ruminants and pigs [6, 7]. WC1⁺ γδ T cells in ruminants express inflammatory cytokines [8, 9] similar to the V_γ9V_δ2 subset in humans [10]. WC1⁺ γδ T cells also migrate to sites of infection, suggesting an inflammatory role [11], which was confirmed in global gene expression analyses on these cells [12, 13].

The high percentage of circulating WC1⁺ γδ T cells in ruminants suggests that these cells are important and distinct from αβ T cells. In addition, the decrease in percentage of WC1⁺ γδ T cells as animals reach adulthood indicates a role in immune defense prior to the development of a fully competent immune system [14]. In support of a potential role for γδ T cells in bridging innate and adaptive immunity, WC1⁺ γδ T cells can act as APC for αβ T cells, a result recently repeated with human γδ T cells [15, 16]. Previous functional gene expression studies have compared WC1⁺ and WC1⁻ γδ T cells [12, 13] and sorted polyclonal γδ and αβ T cell populations by serial analysis of gene expression (SAGE) [17]. The current study was designed to determine whether WC1⁺ γδ T cells and CD4⁺ αβ T cells, which respond to the same antigen, have different gene expression profiles and specifically, whether WC1⁺ γδ T cells differentially express genes associated with innate immune cell function. To this end, two independently derived sets of WC1⁺ γδ and CD4⁺ αβ T cell clones, which respond to major surface protein 2 (MSP2) of Anaplasma marginale, an intraerythrocytic rickettsial pathogen of cattle, were compared [8, 18]. T cell clones were used, as MSP2-responsive γδ T cells are rare in MSP2-immunized cattle, and γδ T cells enriched from PBMC did not respond to this antigen. Gene expression profiles of activated T cell clones were compared using an oligonucleotide microarray format, and the differential expression of differentially regulated immune function genes was confirmed by real-time RT-PCR and by protein or functional analyses, where possible. We demonstrate that although CD4⁺ αβ and WC1⁺ γδ T cells do express similar levels of many of the same genes, interesting differences between these two cell types were also revealed. It is important that WC1⁺ γδ T cells expressed higher levels of genes more commonly associated with macrophages rather than T lymphocytes. To determine if the myeloid cell-associated gene expression pattern was generally representative of circulating WC1⁺ γδ T cells, a similar comparison was performed with WC1⁺ γδ and CD4⁺ αβ T cells sorted from peripheral blood. Many of the differentially regulated genes expressed by the T cell clones were similarly expressed by polyclonal populations.

MATERIALS AND METHODS

T cell clones

WC1⁺ γδ T cell clone 61.1G11 was isolated from MSP2-immunized Holstein steer 98B61 as described previously [8, 18]. γδ T cell clone 61.3G3 as well as CD4⁺ αβ T cell clones 61.10.2D7 and 61.3H5 were isolated from the same animal (98B61) in different experiments by limiting dilution cloning as described [8, 18]. The cell lines and clones were stimulated weekly with T cell growth factor (TCGF), 5 μg/ml A. marginale MSP2 peptide P10 and irradiated PBMC as a source of APC. Peptide P10 (VAGAFARAVEGAEVIEVRAIGSTSVMLNAC) is a 30-amino acid peptide in the carboxy-terminal, conserved region of MSP2, which contains epitopes for CD4⁺ αβ T cells and WC1⁺ γδ T cell clone 61.1G11 [8, 18, 19]. After expansion of the clones, cell surface phenotypes were determined by flow cytometry, and antigen responsiveness was analyzed with proliferation assays. All T cell clones were cryopreserved at the time they were responding to antigen (see Table 2) in 10% DMSO in heat-inactivated FBS and were thawed and restimulated with antigen, TCGF, and APC 1 week prior to stimulation with Con A plus IL-2 when RNA was isolated for gene expression analysis. Animals were housed and maintained according to university guidelines.

Positively selected cell populations

WC1⁺ γδ T cells and CD4⁺ αβ T cells were positively selected using anti-WC1 mAb BAQ4A or anti-CD4 mAb CC8 (provided by Chris Howard, Institute for Animal Health, Compton, UK) using goat anti-mouse IgG-coated MACS MicroBeads following the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The cells were passed through two columns to increase purity, which was greater than 97%, as determined by flow cytometry (data not shown).

Cell surface phenotypic analysis

Differentiation markers on T cell clones were analyzed within 1 week of gene expression analysis by indirect immunofluorescence and flow cytometry as described previously [20]. The mAb used were specific for bovine CD2 (mAb MUC2A), CD3 (mAb MM1A), CD4 (mAb CACT 138A), CD8 (mAb CACT 80C and BAT 82B), the δ chain of the γδ TCR (mAb CACT 61A or GB21A), WC1 (mAb BAQ 4A), CD14 (mAb CAM 36A), and CD11b (mAb MM10A). These mAb were purchased from the Washington State University Monoclonal Antibody Center (Pullman). Following staining, cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry (FACSort, BD Biosciences, San Jose, CA). Viable cells were gated on scattering characteristics, and analysis was performed with FCS Express 2 (De Novo Software, Thornhill, Ontario, Canada).

Lymphocyte proliferation assays

The proliferative response of clone 61.1G11 to A. marginale, MSP2, and peptide P10 was described previously [8, 18, 19]. Proliferation assays for all clones were performed in duplicate wells of round-bottomed, 96-well plates for 4 days, 7 days prior to stimulation for gene expression analysis, and 7 days after the last stimulation with antigen and irradiated PBMC as a source of APC. Positively selected cell populations were assayed at a concentration of 2 × 10⁵ cells/ml for 7 days at the time of sorting and stimulation for gene expression analysis. For T cell clones 61.1G11 and 61.10.2D7, the proliferation assay was performed immediately prior to freezing cells, which were thawed and restimulated with TCGF, 10 μg/ml A. marginale Florida strain homogenate and irradiated PBMC as APC for 1 week prior to stimulation with Con A plus IL-2 (see below) and mRNA collection. Clones 61.3G3 and 61.3H5 were never frozen, and proliferation assays were performed 7 days before stimulation and mRNA collection. T cells (3×10⁴ cells/well) were cultured in duplicate wells in a total volume of 100 μl complete medium containing 2 × 10⁵ APC/well and antigen. Antigens consisted of 10 μg/ml A. marginale Florida strain homogenate, native, gel-purified MSP2, synthetic peptides of MSP2 including P10, or negative-control antigens uninfected RBC (URBC) membranes and MSP2 peptide P12 (VPYACAGIGGNFVSVVDGHINPKFAYRVKA) [19]. Protein concentrations in all antigen preparations were determined by the Bradford assay. To determine proliferation, cells were radiolabeled for the last 18 h of culture with 0.25 μCi [³H]thymidine (Dupont New England Nuclear, Boston, MA), and radiolabeled nucleic acids were harvested and counted with a Betaplate 1205 liquid scintillation counter (Wallac, Gaithersburg, MD). The proliferation assay was performed two times for each clone with similar results. Results are presented from a representative experiment as the stimulation index (SI), determined as the mean cpm of replicate cultures of cells plus antigen/the mean cpm of replicate cultures of cells plus medium. A SI of ≥3.0 was considered statistically significant [21].

Cell stimulation and RNA collection

To obtain RNA for microarray and real-time RT-PCR analyses, T cell clones were obtained 7 days after the last stimulation with antigen and APC and were washed three times. Cells were cultured with 5 μg/ml Con A and 5 U/ml recombinant human IL-2 (Boehringer Mannheim, Indianapolis, IN) at 4 × 10⁶ cells/well in 24-well plates in the absence of APC. RNA was collected after 6 h using the TRIzol reagent (Gibco BRL, Gaithersburg, MD) as described by the manufacturer. RNA levels were determined by spectrophotometry and in oligonucleotide arrays, RNA quality was assessed using an Agilent 2100 bioanalyzer (Palo Alto, CA). Clones 61.1G11 and 61.10.2D7 were stimulated identically and used for the oligonucleotide array and real-time RT-PCR analyses. Clones 61.3G3 and 61.3H5 as well as positively selected WC1⁺ and CD4⁺ sorted cells were stimulated similarly and used for real-time RT-PCR analyses.

Bovine oligonucleotide microarray

RNA extracted from WC1⁺ γδ T cell clone 61.1G11 and CD4⁺ αβ T cell clone 61.10.2D7 was used to probe the Affymetrix GeneChip® bovine genome array (Affymetrix, Santa Clara, CA), which represents ∼23,000 transcripts based on Unigene Build 57 (April 2004) and GenBank sequences. cDNA amplification and synthesis of biotin-labeled cRNA were performed with the one-cycle, target-labeling protocol with 1.8 μg total RNA as described in the GeneChip® Expression Analysis Technical Manual (March 2004). Hybridization was performed with 15 μg cRNA. Washing and staining were performed in the GeneChip® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChip® Scanner 3000. GeneChip® operating software (GCOS v.1.1, Affymetrix) [22, 23] was used for data collection and analysis with scaling to an arbitrary target intensity of 500, and further analysis was done using Microsoft Excel. The cutoff for inclusion in the differentially regulated gene category was a 2.0-fold difference between αβ and γδ T cell RNA expression levels. Complete microarray data are available online, Gene Expression Omnibus Accession Numbers GSM52151 and GSM52152.

Real-time RT-PCR analysis of selected genes

In separate experiments, the relative expression levels of selected genes of interest were compared in γδ T cell clone 61.1G11 and αβ T cell clone 61.10.2D7 or γδ T cell clone 61.3H5 and αβ T cell clone 61.3G3 by using real-time RT-PCR. RNA (1 μg) from each clone was reverse-transcribed using the iQ iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s specifications. Relative amounts of mRNA were analyzed by using the relative quantitation method with a reference standard curve of RNA isolated from unstimulated PBMC using the TRIzol method (User Bulletin #2, Applied Biosystems, Foster City, CA). The gene symbols, forward and reverse primers, product lengths, and GenBank accession numbers of the sequences used to design the primers are listed in Table 1. PCR was performed using SYBR green I (Bio-Rad) in 25 μl reactions. Primers were used at a final concentration of 100 nM. Reactions were performed with a melt temperature of 95°C for 10 s and extension at 58°C for 60 s with the exception of MSR1, which had an extension temperature of 56.4°C. A melt curve was performed for each reaction to confirm uniformity of the amplicons. The standard curve was generated using duplicate samples of tenfold serial dilutions of cDNA from unstimulated PBMC, and unknown samples were run in triplicate. cDNA for the γδ clone and the αβ clone was generated without RT and used to rule out genomic DNA contamination. After conversion to relative concentrations using the standard curve, each value was normalized to 18S rRNA values, and ratios were determined.

Cytokine and NO determinations

T cell clones were harvested as before, 7 days after culture with antigen plus irradiated APC, and were subsequently cultured in 24-well plates at 1.3 × 10⁶ cells per ml with Con A plus IL-2 in the absence of APC. Supernatants collected after 24 h from αβ T cell clone 61.10.2D7 and γδ T cell clone 61.1G11 as well as identically stimulated, positively selected CD4⁺ αβ T cells and WC1⁺ γδ T cells were used to measure IFN-γ, TNF-α, IL-10, and NO. All assays were statistically analyzed with one-tailed Student’s t-test.

The levels of IFN-γ in supernatants diluted 1:4–1:100 were determined by ELISA (Bovigam, CSL Ltd., Parkville, Victoria, Australia) and compared with a standard curve obtained with a supernatant from a Mycobacterium bovis-purified protein derivative-specific T cell clone, which contained 440 U IFN-γ/ml (previously determined by the neutralization of vesicular stomatitis virus) [25]. In the assay, 1.7 ng corresponds to ∼1 unit IFN-γ [26].

The TNF-α capture ELISA was done as described previously [27, 28]. Briefly, Immulon II ELISA plates (Dynax Technologies, Chantilly, VA) were coated with anti-bovine TNF-α mAb 1D11-10 [provided by Veterinary Infectious Disease Organization (VIDO), Saskatoon, Saskatchewan, Canada]. Bound TNF-α was detected by incubation with a rabbit anti-TNF-α serum (VIDO) and subsequently, with biotinylated goat-anti rabbit IgG (heavy and light chains; Zymed Laboratories, San Francisco, CA), streptavidin-alkaline phosphatase (Gibco BRL), and the substrate p-nitrophenyl phosphate (AP-Yellow BioFX Laboratories, Owings Mills, MD). Samples were analyzed by comparison with a standard curve prepared using recombinant bovine TNF-α (VIDO), diluted to 0.02–10.0 ng/ml.

The IL-10 capture ELISA was done as described previously [24] with the following modifications. Black 96-well microplates (Porvair, Shepperton, UK) were incubated overnight at 4°C with capture mAb CC318 at 6 μg/ml in coating buffer. Following blocking with PBS containing Tween 20 (PBST), containing 1% BSA, 100 μl cell culture supernatants were added to each well and incubated for 1 h. Following six washes with PBST, 100 μl 2 μg/ml biotin-labeled secondary mAb CC320 was added to each well and incubated for an additional 1 h. The plates were washed six times with PBST, and 100 μl of the Super Signal ELISA Femto maximum sensitivity substrate (Pierce Biotechnology, Inc., Rockford, IL) was added, and the plates were evaluated within 5 min. The relative light unit value was read on a Betaplate 1205 liquid scintillation counter and luminometer (Wallac). The amount of IL-10 in culture supernatants was determined by comparison with a standard curve using recombinant bovine IL-10, where 1 unit IL-10 is the reciprocal dilution of recombinant IL-10 expressed in COS-7 cell supernatants, which inhibited IFN-γ production by M. bovis-specific T cells stimulated with a purified protein derivative by 50% [24]. The results are expressed as units/ml IL-10.

Nitrite (NO₂⁻) present in culture supernatants was measured in a Griess assay [28, 29]. Briefly, culture supernatants were transferred (50 μl/well) to 96-well, flat-bottomed plates, and 50 μl/well each 1% (w/v) sulfanilamide (Sigma Chemical Co., St. Louis, MO) in 2.5% H₃PO₄ and subsequently, 0.1% (w/v) naphthylethylenediamine dihydrochloride (Sigma Chemical Co.) in 2.5% H₃PO₄ was added to the supernatants, and the absorbance at 540 nm was compared with an NaNO₂ standard curve. Results are presented as the mean μM concentration of NO₂⁻ in triplicate cultures ± 1 sd.

RESULTS

Response of clones to antigen

The two pairs of T cell clones, each comprised of one CD4⁺ αβ T cell clone and one WC1⁺ γδ T cell clone, were selected for this study, as they responded by proliferation to A. marginale MSP2, and one pair additionally responded to MSP2 peptide P10. WC1⁺ γδ T cell clone 61.1G11 was previously shown to proliferate in response to A. marginale MSP2 and to a conserved region peptide, P10, of this protein [8, 18]. αβ T cell clones 61.10.2D7 and 61.3H5 and γδ T cell clone 61.3G3 were more recently isolated from animal 98B61 in a similar manner. Proliferative responses of T cell clones to A. marginale, MSP2, and peptide P10 are shown in Table 2. Clones 61.3H5 and 61.3G3 responded to A. marginale and MSP2 but not peptide P10, whereas γδ T cell clone 61.1G11 and αβ clone 61.10.2D7 responded to all three antigens. In a separate assay, CD4⁺ αβ T cell clone 61.10.2D7 was tested with MSP2 and had a SI of 58.9 compared with a SI of 0.7 to negative-control URBC antigen. Positively selected CD4⁺ T cells from MSP2-immunized animals responded to A. marginale and MSP2, as demonstrated previously in other studies [30, 31], whereas the WC1⁺ T cells did not proliferate to antigen (data not shown).

Oligonucleotide array

Cells were harvested 7 days after culture with antigen and irradiated APC and then stimulated with IL-2 plus Con A for mRNA collection without APC to avoid potential contamination with RNA from APC. Furthermore, analysis of the T cell clones for CD3 and CD14 expression by flow cytometry after 7 days of culture and washing demonstrated that all cells were CD3⁺ and CD14⁻, indicating there were no residual myeloid cells in the cultures. These cells appropriately expressed CD4 or the γδ TCR (Fig. 1). Comparison of gene expression in γδ and αβ T cells was first done with Con A/IL-2-stimulated γδ T cell clone 61.1G11 and αβ T cell clone 61.10.2D7 using the bovine oligonucleotide array. Transcripts (1343) were expressed at twofold or higher levels in the γδ T cell clone, and 606 transcripts were expressed at twofold or higher levels in the αβ T cell clone. Immune function-associated genes with increased expression in γδ or αβ T cell clones are listed in Table 3. As expected, γ and δ TCR mRNA was expressed at higher levels in the γδ T cell clone, 61.1G11. Conversely, αβ T cell clone 61.10.2D7 had higher levels of CD2, CD4, and α and β TCR mRNA. Two γ-chain TCR transcripts were expressed at higher levels in the αβ T cell clone, which is not unexpected, as the majority of αβ T cells has rearranged but not functional γ- and δ-chain genes. Genes not typically associated with T lymphocytes such as MSR1, MMR, TLR4, and chemokines CCL2, CCL8, CXCL1, CXCL2, and CXCL6 were up-regulated in the γδ T cell clone. The XCL1 chemokine, which is typically associated with CD8⁺ T cells, NK cells, mast cells, and γδ T cells, was highly expressed by the WC1⁺ subset of γδ T cells. In addition, CD68 and CD11b were expressed at higher levels in the γδ T cell clone. The oligonucleotide array results thus suggested that the γδ T cell clone had a phenotype which shared characteristics of myeloid cells.

Real-time RT-PCR analysis of genes expressed by γδ T cell clone 61.1G11 and αβ T cell clone 61.10.2D7

Thirty-eight genes were selected for real-time RT-PCR analysis to confirm differential expression by γδ and αβ T cell clones. The relative gene quantitation method using a standard curve was used to compare the differences in transcript levels in RNA isolated from clones 61.1G11 and 61.10.2D7. The differences predicted by the microarray and the actual differences in transcript levels identified by real-time RT-PCR are listed in Table 4. The expression patterns detected by the oligonucleotide array were confirmed for 31 of the 38 transcripts when analyzed by real-time RT-PCR. As observed with the oligonucleotide array, chemokines CCL2, CCL8, CXCL1, CXCL2, CXCL6, and XCL1 were expressed at higher levels in the γδ T cell clone. Similarly, as predicted from the microarray data, genes typically associated with αβ T cells such as RANTES (CCL5) and MIP-1α (CCL3) were expressed at similar levels in αβ and γδ T cells. Real-time RT-PCR analysis also confirmed up-regulated expression in γδ T cells of mRNA encoding TLR4, MSR1, MMR, and CD11b, a component of Mac-1, which are surface molecules typically associated with macrophages.

Protein expression and functional assays of γδ T cell clone 61.1G11 and αβ T cell clone 61.10.2D7

To assure that the increased transcript expression identified with the oligonucleotide array and by real-time RT-PCR analysis correlated with protein expression, protein levels were measured when possible based on availability of reagents. Cytokines TNF-α, IFN-γ, and IL-10 were compared using ELISAs, and NO production was analyzed using the Griess reaction, which measures nitrite in the supernatant (Table 5). The relatively large production of IFN-γ by clone 61.10.2D7 is typical of bovine CD4⁺ T cell lines and clones specific for MSP2 [19]. The amounts of cytokine or nitrite measured in supernatants corresponded with mRNA expression quantified by real-time RT-PCR. Furthermore, flow cytometric analysis showed significant expression of CD11b on γδ T cells but not αβ T cells, which again confirmed array and real-time PCR data (Fig. 2). Flow cytometry also showed that CD2, CD4, and CD6 were expressed on the αβ T cell clones but not on the γδ T cell clones (ref. [8] and data not shown), as predicted by the oligonucleotide array.

Real-time RT-PCR analysis of γδ T cell clone 61.3G3, αβ T cell clone 61.3H5, and WC1⁺ and CD4⁺ sorted populations

To determine if gene expression profiles obtained for clones 61.1G11 and 61.10.2D7 were unique to these cells or more generally representative of MSP2-responsive CD4⁺ αβ T cell and WC1⁺ γδ T cells, additional, real-time RT-PCR analysis was performed on another set of clones, γδ T cell clone 61.3G3 and αβ T cell clone 61.3H5. The clones selected for this comparison responded to A. marginale MSP2 but did not respond to peptide P10, unlike the previous set of clones (Table 2), indicating that the two pairs of clones are of different origins. Many of the comparisons of gene expression for the second pair of T cell clones were similar to those of the previous comparison (Table 6). Specifically, the majority of genes analyzed with the additional set of T cell clones had similar patterns of expression to those obtained by previous real-time PCR analysis of T cell clones. It is important that the γδ T cell clones expressed relatively high levels of CCL2, CXCL1, CXCL2, CXCL6 MSR1, MMR, galectin-3, CD68, and CD11b, genes that are generally associated with myeloid cells, as well as the γδ T cell-associated chemokine XCL1.

To determine whether the results obtained with cloned T cells specific for A. marginale MSP2 were representative of polyclonal cell populations, positively selected WC1⁺ T cells and CD4⁺ T cells were compared. This experiment demonstrated similar patterns of gene expression observed when comparing αβ and γδ T cell clones. When compared with clones 61.1G11 and 61.10.2D7, 16 of 20 genes analyzed had the same patterns of expression, and when compared with clones 61.3G3 and 61.3H5, 12 of the 20 genes had similar patterns of expression. Once again, genes typically associated with myeloid cells were expressed at much higher levels in positively selected γδ T cells than in αβ T cells. These genes included CCL2, CCL8, CXCL1, CXCL2, CXCL6, galectin-3, TLR4, CD68, and CD11b. The XCL1 chemokine was also up-regulated. These data suggest that the results obtained with T cell clones are representative of T cell populations selected from peripheral blood, although the majority of T cells in the population is not specific for MSP2.

DISCUSSION

We have postulated that WC1⁺ γδ T cells may express higher levels of genes typically associated with cells of the innate immune response rather than CD4⁺ αβ T cells. Comparison of gene expression in clonal populations of γδ and αβ T cells from the same individual, which respond to the same antigen, enabled us to determine whether γδ T cells and αβ T cells have differentially expressed immune response genes, by controlling for differences in antigen responsiveness. Our results show that the WC1⁺ subset of γδ T cells expresses higher levels of genes encoding chemokines and surface receptors, which are more typically expressed by myeloid cells, and therefore suggest that although γδ and αβ T cells may have some redundant functions, γδ T cells may function in a unique way during an inflammatory response.

Two primary hypotheses have been proposed to explain the preservation of γδ and αβ T cells throughout evolution. The first suggests that γδ T cells have effector functions similar to those of αβ T cells but as a result of their different mechanism(s) of antigen recognition, respond to atypical types of antigens. In fact, it has been demonstrated recently that murine γδ T cells interact with their ligand by a germ-line-encoded, single side-chain of the D_γ region [32, 33], which differs from antigen recognition by αβ T cells through complementary-determining region 3 (CDR3). The bovine WC1⁺ γδ and CD4⁺ αβ T cell clones used in this study also recognize antigen differently. Whereas CD4⁺ T cells are MHC class II-restricted, the γδ T cell clones are not [8]. In addition, sequence analysis suggested that γδ T cell clones do not recognize antigen through CDR3 [8]. The second hypothesis proposes that γδ and αβ T cells are functionally different. In support of this, γδ T cells have been shown to be uniquely involved in epidermal tissue repair and protection of normal airway function [34–36]. However, these hypotheses are not mutually exclusive. The αβ and γδ T cell clones analyzed in our study recognize MSP2 but have markedly different responses to the same stimulus, which strongly argues that WC1⁺ γδ T cells are functionally different from CD4⁺ αβ T cells, although many immune function genes are similarly expressed.

Of particular interest is the higher level of expression of genes in WC1⁺ γδ T cells, which are typically associated with myeloid cells, detected in clone 61.1G11 by oligonucleotide array and confirmed by real-time RT-PCR for this clone, clone 61.3G3, and a polyclonal population of WC1⁺ γδ T cells. These genes include chemokines and surface receptors CCL2, CXCL1, CXCL2, CXCL6, MSR1, MMR, galectin-3, CD68, and CD11b. The up-regulated expression of these genes in γδ T cells is not likely explained by contaminating APC, as flow cytometric analysis of the clones within 1 week of RNA extraction demonstrated their purity. Furthermore, as the T cell clones were cultured in an identical manner, any residual APC in the cultures should be the same for αβ and γδ T clones. Similarly, populations of T cells sorted immediately prior to stimulation were also highly pure and free of contaminating APC. Of particular interest is the consistent finding that genes commonly associated with myeloid cells were expressed at higher levels by γδ T cells in the three comparative experiments. Additional myeloid-associated genes expressed at higher levels by γδ T cells in two of the three comparative studies were TLR4 and CCL8.

CCL chemokines are chemoattractants for monocytes, NK cells, and activated T cells, and the CXCL chemokines attract granulocytes and naïve T cells. CCL2, CCL8, CXCL1, CXCL2, and CXCL6 are typically produced by macrophages, keratinocytes, endothelial cells, and stromal cells [37]. Conversely, XCLI, also known as lymphotactin, is the only member of the C group of chemokines and is typically produced by activated CD8⁺ T cells, NK cells, mast cells, and γδ T cells. CD68 is macrosialin, a sialoprotein typically associated with macrophages. MSR1 is associated with binding cell-wall components of Gram-positive and Gram-negative bacteria and endotoxin. MMR binds the bacterial carbohydrate, mannose, and is typically found on the surface of macrophages and endothelial cells. Galectin-3, also known as Mac-2, binds galactose and is abundant on macrophages. TLR4 binds LPS and is also found on phagocytic cells. CD11b is a component of Mac-1, which binds to ICAM-1, complement component 3, and fibrinogen, and is normally distributed on myeloid and NK cells. Many of these receptors are important in recognition of pathogen-associated molecular patterns, including LPS, and several have been shown to be functional in γδ T cells [38]. The expression by WC1⁺ γδ T cells of these chemokines and surface receptors is consistent with a role for these cells in recruiting inflammatory cells during infection.

Previous studies have compared gene expression in CD8⁺ WC1⁻ and CD8⁻ WC1⁺ γδ T cell subsets of cattle using sorted cell populations [12, 13, 17]. SAGE and microarray analysis suggested that γδ T cells express a myeloid phenotype [13] in that transcripts for MSR1, CD68, and TNF-α were expressed at higher levels in WC1⁺ CD8⁻ γδ T cells when compared with CD8⁺ WC1⁻ γδ T cells. These genes were also regulated differentially in our comparison of WC1⁺ γδ T cells with CD4⁺ αβ T cells and support the concept that WC1⁺ γδ T cells share properties of myeloid cells. The demonstration that bovine γδ T cells, including cultured WC1⁺ γδ T cells, present antigen to αβ T cells [15] is also consistent with WC1⁺ γδ T cells sharing functions associated with APC of myeloid origin and supports their role in bridging innate and adaptive immunity. More recently, professional APC function was documented for human γδ T cells [16], lending further support to the concept that γδ T cells have evolved to function in some ways like myeloid cells.

In addition to the real-time RT-PCR confirmation of the oligonucleotide array data, protein determinations were done for proteins which we could measure: IFN-γ, TNF-α, and IL-10. When comparing monoclonal T cells, cytokine assays confirmed the oligonucleotide array and PCR results. The inversely correlated production of IFN-γ and TNF-α may be explained by differences in transcriptional regulation of their genes and because IFN-γ is differently regulated in γδ T cells and αβ T cells [39, 40]. Although nitrite levels were relatively low compared with levels produced by activated bovine macrophages [28, 29], elevated nitrite in supernatants from clone 61.1G11 versus 61.10.2D7 paralleled the transcript levels detected in the oligonucleotide array and with real-time RT-PCR. This is significant, as it not only indicates protein production but also that the NO synthase produced is functional, as demonstrated by nitrite production. It has recently been shown that human γδ T cells make NO [41], and others have demonstrated NO production by murine CD4⁺ T cells [42], indicating that the low levels of NO production by the bovine T cell clones are not unusual.

The role of γδ T cells in the immune response A. marginale, a pathogen that infects erythrocytes, is not known. It is interesting that calves under 6 months of age, which have high numbers of WC1⁺ γδ T cells in peripheral blood, are resistant to Anaplasma infection [43, 44]. It is possible that γδ T cells, which are so prevalent at a young age, play a role in age-associated resistance to anaplasmosis and other hemoparasitic diseases [45].

In summary, this study is the first to compare γδ and αβ T cell clones that respond to the same bacterial antigen. These cells have some functional overlap, but there are striking differences between the two populations. The γδ T cell clones express higher levels of surface receptors and many chemokines not typically associated with αβ T cells, which are usually associated with myeloid or stromal cells. These findings are supported by results of other studies, which demonstrate antigen-presenting capabilities by γδ T cells and the direct response of γδ T cells to LPS [38]. Taken together, these findings strongly support the concept that γδ T cells have cytolytic and cytokine secretory properties commonly associated with αβ T lymphocytes but also have potential to recruit cells via chemokines and perform some functions of professional APC, thereby allowing these “jacks of all trades” to help initiate an adaptive immune response and still respond to antigen. Previous studies also suggest that WC1⁺ γδ T cell clones used in this study recognize and respond to antigen differently than CD4⁺ αβ T cell clones specific for the same protein [8]. Further experiments are needed to analyze the functional relevance of the myeloid-associated gene products expressed at higher levels in the γδ T cells. In addition, the mechanisms of antigen recognition by these γδ T cells are not defined and warrant further studies.

Table 1

Primer Sequence for Real-Time RT-PCR

Gene symbol	Forward (5′–3′)	Reverse (5′–3′)	Product size	GenBank accession no.
18S RNA	CTGAGAAGACGGTCGAACTTGACT	TCCGTTAATGATCCTTCCGCAGGT	90 bp	AY779626
CCL2	AGTCACCAGCAGCAAGTGTCCTAA	TTCTTGGGTTGTGGAGTGAGTGCT	165 bp	NM174006.2
CCL3	TCAAGCCTGGTGTCATCTTCCAGA	ATGTATTCCTGGACCCAGTCCTCA	82 bp	AY050252
CCL5	AACGCTTTGGAGTTGAGCTAGGGT	TGCTTAGGACAAGAGCGAGAAGCA	82 bp	NM175827.2
CCL8	TGCATGTAGGATGGTGAGGTCCTT	AGCACACATCCACTTACAGGAGCA	94 bp	NM174007.1
CCR5	ACATGCTGGTTGTCCTCATCCTGA	AGCCCAGAATGGGATGGTGATGAT	119 bp	NM_001011672
CD11a	TGAACTCCATGAGGGTGAGGATCA	AGTGAAACTGTGCCAAGCACCTAC	102 bp	BI681282
CD11b	AGGAAATCTCGACAGGAGCTCGAT	TCTGCGTACCCTCAATCGCAAAGA	139 bp	AJ535320
CD11c	TGTCAACCTGAGCTTCTTAGGGCT	TTGCTAACGTGCGTCGATAGGACA	158 bp	BM366605
CD14	TCTAGCGCCGTTCAGTGTATGGTT	TCCCTTGAGAAACTGTTCCAGGCT	81 bp	NM_174008.1
CD18	AACTGGCAGAAAGCAACATCCAGC	TCCACCACGTTCCTGGAATCTTCA	133 bp	NM175781
CD68	TGATGAGAGGCAGCAAGATGGACT	GGGCCATAGCTTCAGTTGCAGAAA	164 bp	CK774183
CXCL1	ACATCCAGAGCGTGAAGGTGACAA	TGAGACACACTTCCTGACCAGTCT	93 bp	NM175700.1
CXCL2	AGAAGCTCTTGGATGGCTGTTCCA	AGATGGCCTTAGGAGGTGGTGATT	91 bp	NM174299.2
CXCL6	TTGTGAGAGAGCTGCGTTGTGTGT	TCCCTTCCATTCTTCAAGGTGGCT	139 bp	NM174300.2
CXCL8	GGTGCAGAAGGTTGTGCAGGTATT	ACACCAGACCCACACAGAACATGA	176 bp	NM_173925.2
CXCR4	ACCTGTGGCTAGTGGTGTTTCAGT	TTTGGAGTGGGACAGCTTGGAGAT	113 bp	NM174301.2
FasL	CCAGCCAAAGGCATACAGCATCAT	ACCAGGGCAATTCCATAGGTGTCT	160 bp	AB035802.1
Galectin-3	TCGCATTGGGCTTTACTGTACCCA	TCCGGACCACTGAATGTGCCTTAT	97 bp	CK849687
GzmA	ATGACTCAGTCTTTGCTGATCGGG	AGAAGCATCTTGCAGCCCTCTGAA	121 bp	CK950789.1
Granulysin	ACGCTGTGGTTCTTGTCGGAGAAT	ACAGACCTTTCAGCAGCCTCATCT	128 bp	CB454206
ICAM-1	TGAAGAACTCAACCTGAGCTGCCT	TCGACTGCACTGTGAACATGACCT	146 bp	NM_174348.2
ICAM-3	TGAAGAACTCAACCTGAGCTGCCT	ATTGGTGCCATGGTCCTCTCTTCT	169 bp	NM_174349.1
IFN-αR1	TCCACATGGTATGAGGTTGAGCCA	AGCTTGAACGATCCATAGCCCACA	157 bp	NM_174552.2
IFN-γ	GAATGGCAGCTCTGAGAAACTGGA	CGGCCTCGAAAGAGATTCTGACTT	171 bp	M29867
IL-1α	CCACTTCGTGAGGACCAGATGAAT	TCTTCAGAATCTTCCCACTGGCTG	124 bp	M37210
IL-1β	TCCATGGGAGATGGAAACATCCAG	GACGTTTCGAAGATGACAGGCTCT	194 bp	NM_174093
IL-10a	GGTGATGCCACAGGCTGAG	AGCTTCTCCCCCAGTGAGTTC	69 bp	NM_174088.1
IL-18	TTCTGCTCTCCAATGCTTTCAGCG	TCTGCAGCCATCTTTATGCCTGTG	156 bp	NM_174091.2
IRF1	ATTGTAGTGGACAGCGCAGACAGA	ATTGTAGTGGACAGCGCAGACAGA	141 bp	CK960990
Integrin β3	TCCCACTTGCTGGTGTTTACCACT	TCCATGGTGGTGGAGGCAGAATAA	137 bp	CK838238
MMR	GTGTCACCTGAGGTTATATGTACTTGAC	AGAAGCAACCAGCTAAGTGTCCTG	127 bp	BF440340
MSR1	TCTGTGAAGTTCGATGCTCGCTCA	TTTCAGGAGCTGAGCTGCCACTAT	174 bp	NM_174113.1
Perforin	TCCTTTCCAGTTTGGCACAGAGGT	TGGACCACGGTCTCCTGAAATTCT	178 bp	CK771825
Selenoprotein P	TTCAGGTCTTCATCACCACCACCA	GCAACAGCAGCTACTCAAAGCAGA	199 bp	AB032826.1
TLR2	TGTGAAGAGCGAGTGGTGCAAGTA	TCAATGGGCTCCAGCAGAATCAGA	102 bp	NM_174197.2
TLR4	ACCTGTATTCAAGGTCTGGCTGGT	TTGCACAGTCCCTCCAGGAAAGAT	113 bp	NM_174198.3
TNF-α	CATTGCAGTCTCCTACCAGACCAA	TCCTCCCTGGTAGATGGGTTCATA	121 bp	Z14137
XCL1	AATCTGTGCTGATCCTCAAGCTGC	AAAGTGCTGGAAGGTCACTACCCA	147 bp	NM175716.2

Primer sequences were based on a previously published, real-time PCR protocol using TaqMan chemistry [24]. GzmA, Granzyme A; IRF1, IFN response factor 1; MMR, macrophage mannose receptor; MSR1, macrophage scavenger receptor 1.

Table 2

Proliferative Responses of T Cell Clones to A. marginale Antigens

Antigen	Proliferation (SI)
	αβ T cell clones		γδ T cell clones
	61.10.2D7	61.3H5	61.1G11	61.3G3
A. marginale	52.4a	6.3	101.4	10.7
MSP2	NDb	5.5	6.8	11.4
URBC	1.3	0.7	1.7	0.7
P10	192.4	0.7	9.8	1.4
P12	0.4	1.4	1.1	2.4

^a

T cell clones (3×10⁴ cells/well) were cultured in duplicate wells for 3 days with 2 × 10⁵ irradiated, autologous PBMC as a source of APC and antigen, radiolabeled overnight, and harvested. Results are presented as the SI, calculated as the mean cpm of cells cultured with 10 μg/ml antigen/mean cpm of cells cultured with medium. Statistically significant results are in bold.

^b

Not determined in the assay performed 1 week prior to Con A and IL-2 stimulation. In a previous experiment, the SI of this clone to MSP2 compared with medium was 58.9.

Figure 1

Flow cytometric analysis of T cell clones; γδ T cell clones 61.1G11 and 61.3G3 are presented (a and b, respectively). αβ T cell clones 61.10.2D7 and 61.3H5 are presented (c and d, respectively). Control staining without primary antibody is represented by the solid silver in the histograms. Staining for CD4 is represented by the open gray histogram, and staining for the γδ TCR is represented by the open black histograms.

Table 3

Immune Function-Related Genes Differentially Regulated in the Oligonucleotide Array When Comparing γδT Cell Clone 61.1G11 and αβT Cell Clone 61.10.2D7

Gene ID	Gene name	Description	Accession	Ratioa
Genes with higher transcript levels in γδ T cell clone 61.1G11
TCR-associated genes
Bt.28004.2.S1_a_at	T cell receptor δ chain, V, D, and J regions, BTDV2	T cell receptor	D13656.1	675
Bt.28004.2.A1_a_at	T cell receptor δ chain, V, D, and J regions, BTDV2	T cell receptor	D13656.1	12.1
Bt.28007.1.A1_s_at	T cell receptor δ chain, V, D, and J regions, BTDV6	T cell receptor	CK969206	90.5
Bt.28732.1.S1_at	T cell receptor δ chain, a part of V, D, J, and C regions	T cell receptor	D90419.1	776
Bt.28732.1.S1_s_at	T cell receptor δ chain, a part of V, D, J, and C regions	T cell receptor	D90419.1	256
Bt.29691.1.S1_at	T cell receptor δ chain variable region, BVd1.16	T cell receptor	U73383.1	2.6
Bt.29689.1.S1_s_at	T cell receptor δ chain variable region, BVd1.21	T cell receptor	CK834180	256
Bt.29695.1.A1_s_at	T cell receptor γ chain variable region BVG3.1	T cell receptor	BE588480	7.0
Bt.29695.1.A1_at	T cell receptor γ chain variable region BVG3.1	T cell receptor	BE588480	6.1
Bt.29695.1.S1_at	T cell receptor γ chain variable region BVG3.1	T cell receptor	U73186.1	17.1
Bt.29694.1.S1_at	T cell receptor γ chain variable region BVG3.2	T cell receptor	U73187.1	17.1
Bt.29693.1.S1_at	T cell receptor γ chain variable region BVG7	T cell receptor	CK836176	73.5
Bt.29693.1.A1_at	T cell receptor γ chain variable region BVG7	T cell receptor	CK836176	3.7
Surface molecules and receptors
Bt.7027.1.S1_at	MHC class I-like family A1 (MHCLA1)	MHC class I	AF317556.1	3.3
Bt.1007.1.S1_at	MHC class II DM β-chain	MHC class II	CK773078	2.1
Bt.2227.1.S1_at	HLA class II histocompatibility antigen, DZ α chain	MHC class II	BI849171	5.7
Bt.4482.1.S1_at	Macrophage scavenger receptor type 1	Binding and phagocytosis of bacteria	NM_174113.1	90.5
Bt.4482.2.S1_a_at	Macrophage scavenger receptor type 1	Binding and phagocytosis of bacteria	X51689.1	3.7
Bt.4482.2.S1_at	Macrophage scavenger receptor type 1	Binding and phagocytosis of bacteria	X51689.1	2.3
Bt.20836.1.S1_at	Macrophage mannose receptor precursor	Binds high mannose structures of pathogens	CK946016	18.4
Bt.23089.1.S1_at	Macrophage mannose receptor precursor	Binds high mannose structures of pathogens	BF440340	8.6
Bt.6850.1.S1_a_at	CD3 δ	Component of CD3	X53269.1	2.1
Bt.2573.1.S1_at	CD9	Mediates platelet aggregation and activation	NM_173900.2	7.5
Bt.248.1.S1_at	CD38	NAD glycohydrolase, augments B cell proliferation	NM_175798.2	4.9
Bt.13496.2.S1_a	CD63	Lysosomal membrane protein	CK849512	3.5
Bt.2334.1.S1_at	Macrophage antigen CD68	Macrosialin, macrophage-specific marker	CK774183	8.6
Bt.20940.1.S1_at	T cell activation antigen (CD96 antigen)	Expressed on activated T cells	CK770291	2.8
Bt.4285.1.S1_at	CD14	Myeloid membrane protein (macrophage-specific)	NM_174008.1	8.0
Bt.9030.1.S1_at	Toll-like receptor 4 (TLR4)	Responds to LPS in conjunction with CD14	NM_174198.3	3.7
Bt.8140.1.S1_at	Fc fragment of IgE, high-affinity I	High-affinity receptor for IgE	NM_174537.1	4.9
Bt.284.1.S1_at	Fc fragment of IgG, high-affinity Ia (CD64)	High-affinity receptor for IgG	NM_174538.2	2.1
Bt.28015.1.S1_at	Fc-γ 2 receptor	Inhibits stimulation	Z37506.1	4.0
Bt.4110.1.S1_at	Histamine H1 receptor	Histamine H1 receptor	NM_174083.2	5.7
Bt.3843.1.S1_at	Immunoglobulin J chain	Component of immunoglobulin	NM_175773.1	9.2
Bt.13039.1.S1_at	Integrin, β 3 (CD61)	Associates with glycoprotein IIb/IIIa or CD51	CK838238	3.3
Bt.21219.1.S1_at	Integrin α-E chain (CD103)	Associates with β7 and binds E-cadherin in the gut	CK980975	2.6
Bt.9483.1.S1_at	Integrin α-D precursor (CD11d)	Binds CD50; expressed on leukocytes	BM364873	2.3
Bt.27978.1.S1_at	Leukocyte integrin α-M chain (CD11b)	Binds ICAM-1, complement 3, fibrinogen (Mac1)	AJ535320.1	6.5
Bt.1416.1.S1_at	Galectin-3	Galactose-specific lectin abundant on macrophages (Mac2)	CK849687	5.3
Bt.11241.1.S1_at	E-cadherin	A ligand for integrin α-E/β7	CK948176	5.3
Bt.5372.1.S1_at	Intercellular adhesion molecule 1 (CD54)	Binds LFA-1, Mac1	NM_174348.2	2.5
Bt.4293.1.S1_at	Intercellular adhesion molecule 3	T cell molecule that binds CD209 on dendritic cells	NM_174349.1	3.0
Bt.10077.1.S3_at	Interferon responsive factor 1	Regulates response to IFN-γ	CK960990	4.0
Bt.10077.1.S2_at	Interferon responsive factor 1	Regulates response to IFN-γ	CK950685	3.0
Bt.5768.2.S1_at	Interferon regulatory factor 7	Role in virus-activated transcription of IFN-α genes	BF774513	2.0
Bt.4557.1.S1_at	Interferon, α; receptor	Receptor for IFN-α	NM_174552.2	2.8
Bt.23148.1.S1_at	Interleukin 11 receptor, α	Receptor for IL-11	CK946408	2.3
Bt.7213.1.S1_at	Interleukin 12 receptor, β2	Receptor for IL-12 associated with IFN production	NM_174645.2	2.6
Bt.13851.1.S1_at	Interleukin-13 receptor α-1 chain precursor	Receptor for IL-13	CK966449	4.6
Bt.11174.1.S1_at	KIR2DL1 Killer cell immunoglobulin-like receptor	Inhibits NK cell cytotoxicity on binding MHC I (CD158a)	NM_174740.2	9.2
Bt.24545.1.S1_at	Natural killer cell receptor group D variant 2	Activating member of the NKG2 receptors	CK952080	2.6
Bt.28588.2.A1_a_at	Natural killer receptor 2B4 (CD244)	Modulates other receptor-ligand interactions to activate	CK971483	2.6
Secreted products
Bt.8144.1.S1_at	Chemokine (C motif) ligand 1 (XCL1)	Lymphocyte trafficking and development (Lymphoactin)	NM_175716.2	6.5
Bt.2408.1.S1_s_at	Chemokine (C-C motif) ligand 2 (CCL2)	Monocyte-chemoattractant protein-1	NM_174006.2	64.0
Bt.2408.1.S1_at	Chemokine (C-C motif) ligand 2 (CCL2)	Monocyte-chemoattractant protein-1	NM_174006.2	26.0
Bt.154.1.S1_at	Chemokine (C-C motif) ligand 8 (CCL8)	Monocyte-chemoattractant protein-2	NM_174007.1	2.3
Bt.611.1.S2_at	Chemokine (C-X-C motif) ligand 1 (CXCL1)	Neutrophil chemoattractant (GRO-α)	NM_175700.1	5.3
Bt.611.1.S1_at	Chemokine (C-X-C motif) ligand 1 (CXCL1)	Neutrophil chemoattractant (GRO-α)	U95813.1	7.5
Bt.611.1.S1_x_at	Chemokine (C-X-C motif) ligand 1 (CXCL1)	Neutrophil chemoattractant (GRO-α)	U95813.1	7.0
Bt.610.1.A1_at	Chemokine (C-X-C motif) ligand 2 (CXCL2)	Neutrophil chemoattractant (GRO-β)	NM_174299.2	3.7
Bt.23093.1.S1_at	Chemokine (C-X-C motif) ligand 3 (CXCL3)	Neutrophil chemoattractant (GRO-γ)	CF929989	5.7
Bt.7165.1.S1_at	Chemokine (C-X-C motif) ligand 6 (CXCL6)	Neutrophil chemoattractant (GCP-2)	NM_174300.2	2.3
Bt.8957.1.S1_at	Chemokine (C-X-C motif) receptor 4 (CXCR4)	Receptor for SDF-1 chemokine	NM_174301.2	3.3
Bt.191.1.S2_at	Interleukin 1, α	T cell and macrophage activation	NM_174092.1	2.3
Bt.191.1.S1_at	Interleukin-1, α	T cell and macrophage activation	M36182.1	2.5
Bt.4856.1.S2_at	Interleukin 1, β	T cell and macrophage activation	NM_174093.1	3.0
Bt.4856.1.S1_at	Interleukin 1, β	T cell and macrophage activation	M35589.1	2.6
Bt.423.1.S1_at	Interleukin 4	B cell activation	NM_173921.2	3.0
Bt.5.1.S1_at	Interleukin 5	Eosinophil growth, differentiation	NM_173922.1	10.6
Bt.12751.1.S1_at	Interleukin 7	Growth of pre-B cells and pre-T cells	NM_173924.2	2.0
Bt.155.1.S1_at	Interleukin 8 (CXCL8)	Lymphocyte-derived neutrophil-activating factor	NM_173925.2	2.0
Bt.4723.1.S1_at	Interleukin 10	Potent suppressant of macrophage function	NM_174088.1	2.8
Bt.12928.1.S1_at	Interleukin 13	B cell growth, inhibits macrophage inflammatory cytokines	NM_174089.1	22.6
Bt.19162.1.A1_at	Interleukin 13	B cell growth, inhibits macrophage inflammatory cytokines	CB456261	18.4
Bt.234.1.S1_at	Interleukin 18	Induces IFN-γ production; favors TH1	NM_174091.2	2.8
Bt.26527.1.S1_a_at	Interleukin-16 precursor	Chemoattractant for CD4 T cells, monocytes, eosinophils	CK848236	3.0
Bt.12756.1.S1_at	Tumor necrosis factor-α	Local inflammation, endothelial activation	NM_173966.1	3.3
Bt.9286.1.S1_at	LPS-induced TNF-α factor	Local inflammation, endothelial activation	CK980342	2.0
Bt.29672.1.S1_at	Granzyme A precursor (gzmA gene)	Induces perforin-mediated apoptosis	CK950789	2.5
Bt.146.1.S1_at	Neutrophil β-defensin 4	Suppress cytokine production and NK cell activity	AF014107.1	16.0
Bt.13125.1.S1	Neutrophil β-defensin 4	Suppress cytokine production and NK cell activity	BM106372	6.1
Bt.21431.2.S1_at	Lymphotoxin β	Stimulates lymph node development	CB428714	3.7
Bt.21431.3.S1_at	Lymphotoxin β	Stimulates lymph node development	BE668004	2.3
Bt.21431.1.S1_at	Lymphotoxin β	Stimulates lymph node development	CK948133	2.0
Bt.27067.1.A1_at	Lymphotoxin α precursor	Stimulates killing, endothelial activation	CK774112	9.9
Bt.24345.1.S1_at	Complement component 2 precursor	Cleaves C3 and C5	CK772312	9.2
Bt.24345.2.S1_at	Complement component 2 precursor	Cleaves C3 and C5	BE487614	4.9
Bt.8803.1.A1_at	Complement C1r component precursor	Cleaves C1s to active protease	CK950026	4.3
Cytoplasmic molecules
Bt.142.1.S1_at	Neutrophil cytosolic factor 1	Part of NADPH oxidase; produces superoxide	NM_174119.2	13.0
Bt.143.1.S1_at	Neutrophil cytosolic factor 2	Part of NADPH oxidase; produces superoxide	NM_174120.2	12.1
Bt.7625.1.S1_a_at	Neutrophil cytosolic factor 4	Part of NADPH oxidase; produces superoxide	CK775962	2.5
Bt.23126.2.S1_at	Nitric oxide synthase 2A	Inducible nitric oxide synthase	AW654110	8.0
Bt.23126.1.S1_at	Nitric oxide synthase 2A	Inducible nitric oxide synthase	CK832507	4.3
Bt.8479.1.A1_at	NO synthase	Inducible nitric oxide synthase	BF040087	3.0
Bt.8947.1.S1_at	Legumain	Cysteine endoprotease that hydrolyzes asparaginyl bonds	NM_174101.2	3.7
Bt.15758.1.S1_at	Prostaglandin-endoperoxide synthase 2	Key enzyme in prostaglandin biosynthesis (COX2)	NM_174445.2	2.6
Bt.13273.1.S1_at	Suppressor of cytokine signaling 2	Regulator of cytokine signal transduction	NM_177523.2	2.5
Bt.22739.2.S1_at	Suppressor of cytokine signaling 4	Regulator of cytokine signal transduction	CB422658	2.0
Bt.23278.1.S1_at	Allograft inflammatory factor 1	Augments production of IL-6, IL-10, and IL-12	NM_173985.2	6.1
Bt.12304.1.S1_at	Interferon-stimulated protein, 15 kDa	Induces B cell proliferation and T cell cytokine production	NM_174366.1	4.6
Bt.9308.1.S1_at	Lymphocyte phosphatase-associated phosphoprotein	Cytoplasmic tyrosine kinase necessary for TCR signaling	BM252117	3.3
Bt.26983.1.S1_at	Interleukin-1β converting enzyme β isozyme	Cleaves and activates the inactive precursor of IL-1	CK776788	4.9
Bt.16623.1.A1_at	Nuclear factor of activated T cells 5, isoform c	Up-regulates members of the TNF family	CB422127	2.6
Bt.24855.2.S1_at	Tumor necrosis factor ligand superfamily member 13B	Involved in B cell proliferation	BE753440	2.5
Bt.18959.1.A1_at	Lymphocyte activation gene-3 (LAG-3/CD223)	Binds to MHC class II; may be involved in down-regulation	CB452278	2.6
Bt.10855.1.S1_at	Regulator of G-protein signaling 2 (RGS2)	Inhibits G-protein signaling including chemokine signals	CB169117	4.9
Bt.4781.1.S1_at	A disintegrin and metalloproteinase domain10 (ADAM10)	Cleaves L1 adhesion molecule to induce cell migration	NM_174496.2	2.1
Genes with higher transcript levels in αβ T cell clone 61.10.2D7
TCR-associated genes
Bt.28000.1.S1_at	T-cell receptor γ chain V and J regions	T cell receptor	BM362209	−11.3b
Bt.4289.1.S1_at	Bovine T cell receptor α chain, and constant regions	T cell receptor	CK960422	−6.0
Bt.29765.1.S1_at	T cell receptor β chain V-region	T cell receptor	CK836454	−337
Bt.1978.1.S1_a_at	T cell receptor, β cluster	T cell receptor	CB172009	−2.1
Bt.1978.1.S1_at	T cell receptor, β cluster	T cell receptor	CB172009	−2.6
Bt.1978.2.S1_x_at	T cell receptor, β cluster	T cell receptor	CK775366	−2.0
Bt.28013.1.S1_at	BTGT1 mRNA for T cell receptor γ chain	T cell receptor	BE752633	−27.9
Surface molecules and receptors
Bt.13087.1.S1_at	CD6	Binds CD166	AB042274.1	−4.3
Bt.48.1.S1_a_at	CD28	Binds CD80 and CD86	NM_181004.1	−24.3
Bt.48.3.S1_a_at	CD28 isoform 2	Binds CD80 and CD86	AY064487.1	−18.4
Bt.4497.1.S1_at	CD5 antigen (p56–62)	Expressed at high density on mature T cells	NM_173899.2	−27.9
Bt.29099.1.S1_at	T lymphocyte activation antigen CD80 precursor	Costimulator, Ligand for CD28 and CTLA4	CK955216	−2.1
Bt.24707.1.S1_at	CD86 mRNA	Costimulator, Ligand for CD28 and CTLA4	AY533858.1	−2.0
Bt.13097.1.S1_at	CD86	Costimulator, Ligand for CD28 and CTLA4	AJ291475.1	−2.5
Bt.3841.1.S1_at	CD83 antigen	Dendritic cell-associated molecule	CK773094	−8.0
Bt.3841.2.S1_at	CD83 antigen	Dendritic cell-associated molecule	BE755445	−9.9
Bt.10954.1.S1_at	T cell surface antigen CD2 precursor	Binds CD58 and activates T cells	CK774743	−68.6
Bt.10954.2.S1_a_at	T cell surface antigen CD2 precursor	Binds CD58 and activates T cells	AW658942	−73.5
Bt.10954.2.S1_at	T cell surface antigen CD2 precursor	Binds CD58 and activates T cells	AW658942	−14.9
Bt.27979.1.S1_at	CD4	Coreceptor for MHC class II	AJ535319.1	−9.9
Bt.79.1.S1_at	Cytotoxic T-lymphocyte-associated protein 4	Negative regulator of T cell activation	NM_174297.1	−4.3
Bt.11174.3.S1	KIR2DL1 Killer cell immunoglobulin-like receptor	Inhibits NK cell cytotoxicity on binding MHC I	AF490400.1	−4.9
Bt.22948.1.S1	KIR3DL1 Killer cell immunoglobulin-like receptor	Inhibits NK cell cytotoxicity on binding MHC I	NM_181451.1	−5.3
Bt.11174.2.S1	KIR2DL1 NK receptor KIR3DL1-like protein pseudogene	Inhibits NK cell cytotoxicity on binding MHC I	AY075103.1	−3.5
Bt.8547.1.S1_at	Signaling lymphocytic activation molecule (CD150)	Promotes T cell activation and IFN-γ production	NM_174184.2	−3.5
Bt.22692.1.A1_at	Interleukin-6 receptor precursor (CD126)	IL-6 receptor α	CK774531	−5.7
Bt.12301.1.S1_at	Prostaglandin E receptor 2	Decreased proliferation and cytokine release	NM_174588.2	−2.0
Bt.22749.1.A1_at	Prostaglandin F2 receptor negative regulator	Associates with CD9 and CD81	BP107094	−2.1
Bt.22634.1.S1_at	Prostaglandin F2-α receptor regulatory protein	Associates with CD9 and CD81	BP109430	−3.5
Bt.22634.2.A1_at	Prostaglandin F2-α receptor regulatory protein	Associates with CD9 and CD81	CB536196	−3.0
Bt.22634.3.S1_at	Prostaglandin F2 receptor negative regulator precursor	Associates with CD9 and CD81	CK946156	−2.5
Bt.352.2.S1_at	Prostaglandin F2α receptor isoform-α	Receptor for PGF2α	AB083784.1	−22.6
Bt.24531.1.S1_at	Interleukin-4 receptor α chain precursor (CD124)	IL-4 receptor	AV666299	−2.5
Bt.18375.1.S1_at	TNF ligand superfamily member 5 (CD40L)	Inducer of B cell proliferation and activation	CB443472	−97.0
Bt.49.1.S1_at	TNF ligand superfamily member 5 (CD40L)	Inducer of B cell proliferation and activation	NM_174624.2	−181
Bt.13148.1.S1_at	Vascular endothelial growth factor (VEGF)-receptor-1, fit-receptor protein	Receptor for VEGF with protein kinase activity	X94263.1	−3.3
Secreted products
Bt.523.1.S1_at	Interleukin 12 p40	Induces IFN-γ production	NM_174356.1	−13.0
Bt.538.1.S1_at	Interleukin 3	Synergistic action in early hematopoiesis	NM_173920.2	−42.2
Bt.3686.1.S1_at	Interleukin 6	T cell and B cell growth and differentiation	NM_173923.2	−3.3
Bt.4336.1.S1_at	Complement factor D precursor	Plasma serine protease cleaves B when bound to C3b	CK771764	−4.6
Bt.4138.2.S1_at	Vascular endothelial growth factor (VEGF)	Growth factor involved in angiogenesis	CB450531	−3.7
Bt.4138.1.S1_a_at	Vascular endothelial growth factor (VEGF)	Growth factor involved in angiogenesis	NM_174216.1	−4.0
Cytoplasmic molecules
Bt.5154.1.S1_at	Heat shock 70 kD protein 1	Inducible isoforms of Hsp70	NM_174550.1	−2.0
Bt.5154.1.S1_s	Heat shock 70 kD protein 1	Inducible isoforms of Hsp70	NM_174550.1	−2.0
Bt.12241.1.S1_at	Lymphocyte-specific G protein-coupled receptor EBI1	G-protein associated with CCR7	CK770049	−5.3

^a

Gene transcript ratios are expressed as spot intensity for γδ T cell clone/spot intensity for αβ T cell clone.

^b

When gene expression levels are higher in αβ T cells, the ratios are expressed as αβ:γδ T cells and as negative values. Mac1, Membrane-activated complex-1; GRO-α, growth-related oncogene-α; GCP-2, granulocyte chemotactic protein-2; SDF-1, stromal-derived factor-1; COX2, cyclooxygenase-2.

Table 4

Real-Time RT-PCR Analysis of Selected Immune Function Genes When Comparing γδ T Cell Clone 61.1G11 with αβ T Cell Clone 61.10.2D7

Gene	Microarray prediction	Normalized γδ real-time PCRa	Normalized αβ real-time PCR	Ratiob ± sd
CCL2	64/26c	3,464	35.4	97.6	12.1
CCL3	1.0	1,948	3,052	−1.6d	0.2
CCL5	1.2	61	80	−1.3	0.2
CCL8	2.3	115	37	3.1	0.8
CCR5	1.0	17.0	37.7	−2.3	0.3
CD11a	1.2	3.00	6.84	−2.3	0.3
CD11b	6.5	0.36	0.04	9.1	1.4
CD11c	1.0	16.2	9.02	1.8	0.2
CD14	8.0	0.13	0.07	1.7	0.1
CD18	1.4	1.43	2.17	−1.5	0.1
CD68	8.6	1.08	0.14	4.8	1.1
CXCL1	7.5/7.0/5.3	54.2	18.1	3.0	0.4
CXCL2	3.7	88.8	16.8	5.3	0.3
CXCL6	2.3	253	51.9	4.9	0.5
CXCL8	2.0	3.27	1.87	1.8	0.1
CXCR4	3.3	1.51	0.82	1.8	0.7
FasL	1.9	17.6	15.3	1.2	0.2
Galectin-3	5.3	2.37	0.72	2.38	0.1
Granzyme A	2.5	579	295	2.0	0.1
Granulysin	1.3	9.3	7.97	1.2	0.1
ICAM-1	2.5	131	59.1	2.2	0.1
ICAM-3	3.0	2.79	1.16	2.4	0.1
IFN-α receptor 1	2.8	0.70	0.91	−1.3	0.5
IFN-γ	−1.3	295	1,657	−5.6	0.9
IL-1α	2.5/2.3	64.0	55.8	1.2	0.2
IL-1β	3.0/2.6	207	128	1.6	0.1
IL-10	2.8	30.1	5.96	5.1	1.3
IL-18	2.8	5.40	3.85	1.4	0.3
Interferon response factor 1	3.0/4.0	4.54	3.12	1.5	0.2
Integrin β3	3.3	3.79	1.65	2.3	1.1
MMR	18.4/8.6	1.18	0.07	10.9	2.6
MSR1	90.5/2.3/3.7	53.0	2.07	25.6	3.8
Perforin	1.0	38.2	50.5	−1.3	0.2
Selenoprotein P	239	0.22	0.10	2.3	0.6
TLR2	−2	0.47	1.58	−3.3	2.5
TLR4	3.7	12.1	4.83	2.5	0.3
TNF-α	3.3	19.28	4.75	4.1	0.3
XCL1	6.5	4,341	310	14.0	2.0

^a

Real-time RT-PCR value when compared with a standard curve for each gene and normalized to 18S ribosomal RNA for each sample.

^b

Ratio of normalized, real-time RT-PCR values of γδ T cell clone 61.1G11 to αβ T cell clone 61.10.2D7.

^c

When more than one prediction was made for gene expression by the oligonucleotide microarray, each is listed.

^d

When gene expression levels are higher in αβ T cells, the ratios are expressed as αβ:γδ T cells and as negative values.

Table 5

Comparison of Cytokine and NO Produced by γδ and αβ T Cells

Mediator	Levels of mediator in the supernatanta
	γδ T cell clone 61.1G11	αβ T cell clone 61.10.2D7
TNF-α	1.1 ± 0.03 ng/mlb	<80 pg/ml
IFN-γ	60.5 ± 29 ng/ml	483 ± 120 ng/ml
IL-10	1.42 ± 0.078 U/ml	<0.5 U/ml
Nitrite	1.6 ± 0 μM	0.71 ± 0 μM

^a

T cell clones were harvested 7 days after stimulation with TCGF, antigen, and APC, washed, and cultured for an additional 24 h without APC and with Con A and IL-2. Cytokine levels in cell supernatants were determined by ELISA, and nitrite was measured by the Griess assay. The limit of TNF-α detection was 80 pg/ml, and that of IL-10 was 0.5 U/ml. Results are presented as a mean ± 1 sd of duplicate cultures.

^b

When mediators were compared between clones, values in boldface type were shown to be significantly higher using a one-tailed Student’s t-test.

Figure 2

Flow cytometric analysis of γδ T cell clone 61.1G11 and αβ T cell clone 61.10.2D7 for surface expression of CD11b. Results are presented as histograms showing staining of clones 61.1G11 (a) and 61.10.2D7 (b) with mAb MM10A specific for CD11b (black line) and no primary antibody (silver area).

Table 6

Real-Time RT-PCR Analysis of Selected Immune Function Genes When Comparing γδ T Cell Clone 61.3G3 and αβ T Cell Clone 61.3H5 or WC1⁺ and CD4⁺ Cells Selected from PBMC

Gene	T cell clones				Selected PBMC
	Normalized γδ real-time PCRa value	Normalized αβ real-time PCR value	Ratiob ± sd		Normalized γδ real-time PCRa value	Normalized αβ real-time PCR value	Ratioc ± sd
CCL2d	647	2.40	269.6	45.8	3.37	0.35	9.6	2.4
CCL8	0.058	0.20	−3.4	0.2	0.054	0.0004	135	47.5
CD68	5.09	0.40	12.7	0.9	0.24	0.011	21.8	7.4
CD11b	0.93	0.024	38.7	6.2	0.05	0.007	7.1	2.4
CXCL1	150	1.28	117.2	8.9	35.6	0.49	72.7	18.8
CXCL2	892	5.11	174.5	14.2	332	3.22	103.1	24.9
CXCL6	996	11.7	85.1	11.0	94.3	1.06	89.0	18.7
CXCL8	3.67	0.06	61.2	2.4	1.05	0.019	55.3	12.6
Galectin-3	1.64	0.15	10.9	0.0	0.21	0.097	2.2	0.5
Granzyme A	63.9	639	−10.0	0.4	1.36	1.32	1.0	0.3
ICAM1	3.46	2.04	1.7	0.2	1.38	0.16	8.6	1.8
ICAM3	0.65	0.64	1.0	0.07	0.28	0.034	8.2	1.9
IFN-γ	1456	9253	−6.4e	0.7	235	0.23	1022	251
IL-10	4.70	5.58	−1.2	0.1	5.61	0.32	17.5	4.8
Integrin β3	47.7	26.0	1.8	0.2	57.0	22.9	2.5	0.6
MMR	0.22	0.04	5.5	1.4	0.076	0.013	5.8	1.2
MSR1	1.62	0.37	4.4	0.8	0.93	0.72	1.3	0.4
TLR4	7.53	5.57	1.4	0.1	3.45	1.00	3.5	0.8
TNF-α	27.2	13.0	2.1	0.1	13.5	0.75	18.0	4.6
XCL-1	22.7	0.004	5675	469.1	0.49	0.0047	104.2	26.0

^a

Real-time RT-PCR value when compared with a standard curve for each gene and normalized to 18S ribosomal RNA for each sample.

^b

Ratio of normalized, real-time RT-PCR values of γδ T cell clone 61.3G3 to αβ T cell clone 61.3H5.

^c

Ratio of normalized, real-time RT-PCR values of sorted WC1⁺PBMC to CD4⁺PBMC.

^d

Bolded values are those that have real-time PCR patterns of expression similar to clones 61.1G11 and 61.10.2D7.

^e

When gene expression levels are higher in αβ T cells, the ratios are expressed as αβ:γδ T cells and as negative values.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases, Grant K08 AI53594 and U.S. Department of Agriculture, National Research Initiative Competitive Grants Program, Grant 00-52100-9612. We are grateful to Kim Kegerreis, Daming Zhu, and Shelley Whidbee for excellent technical assistance, to Jayne Hope and Chris Howard for providing reagents for the IL-10 ELISA and mAb CC8, and to Dale Godson (VIDO) for providing reagents for the TNF-α ELISA.