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(Journal of Leukocyte Biology. 2006;80:939-952.)
© 2006 by Society for Leukocyte Biology

Comparative gene expression by WC1+ {gamma}{delta} and CD4+ {alpha}ß T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1+ {gamma}{delta} T cells

Kevin K. Lahmers*, Jodi F. Hedges{dagger}, Mark A. Jutila{dagger}, Mingqi Deng{ddagger}, Mitchell S. Abrahamsen{ddagger} and Wendy C. Brown*,1

* Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington;
{dagger} Veterinary Molecular Biology Laboratory, Montana State University, Bozeman, Montana; and
{ddagger} Department of Veterinary Pathobiology, University of Minnesota, St. Paul, Minnesota

1 Correspondence: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. E-mail: wbrown{at}vetmed.wsu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The functions of {gamma}{delta} T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized {alpha}ß T cells. However, their conservation throughout evolution suggests that {gamma}{delta} T cells are biologically unique. In ruminants, {gamma}{delta} 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 {alpha}ß T cells. In fact, bovine WC1+ {gamma}{delta} T cells can act as APC for {alpha}ß 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+ {gamma}{delta} and CD4+ {alpha}ß 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 {alpha}ß and {gamma}{delta} T cell clones express many of the same genes, {gamma}{delta} 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+ {gamma}{delta} T cells when compared with CD4+ {alpha}ß T cells selected from peripheral blood.

Key Words: MSP2 • macrophage scavenger receptor 1 • macrophage mannose receptor • and galectin-3


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The functions of {gamma}{delta} T cells are still poorly defined, despite their discovery in 1986 [1 ]. Several of the well-characterized properties of {alpha}ß T cells are shared by {gamma}{delta} 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 {gamma}{delta} T cells to examining those functions typically associated with {alpha}ß T cells potentially biases or limits a more complete understanding of {gamma}{delta} T cell biology.

Cattle have large numbers of circulating {gamma}{delta} T cells. In young ruminants, {gamma}{delta} 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 {gamma}{delta} 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 {gamma}{delta} 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+ {gamma}{delta} T cells in ruminants express inflammatory cytokines [8 , 9 ] similar to the V{gamma}9V{delta}2 subset in humans [10 ]. WC1+ {gamma}{delta} 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+ {gamma}{delta} T cells in ruminants suggests that these cells are important and distinct from {alpha}ß T cells. In addition, the decrease in percentage of WC1+ {gamma}{delta} 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 {gamma}{delta} T cells in bridging innate and adaptive immunity, WC1+ {gamma}{delta} T cells can act as APC for {alpha}ß T cells, a result recently repeated with human {gamma}{delta} T cells [15 , 16 ]. Previous functional gene expression studies have compared WC1+ and WC1– {gamma}{delta} T cells [12 , 13 ] and sorted polyclonal {gamma}{delta} and {alpha}ß T cell populations by serial analysis of gene expression (SAGE) [17 ]. The current study was designed to determine whether WC1+ {gamma}{delta} T cells and CD4+ {alpha}ß T cells, which respond to the same antigen, have different gene expression profiles and specifically, whether WC1+ {gamma}{delta} T cells differentially express genes associated with innate immune cell function. To this end, two independently derived sets of WC1+ {gamma}{delta} and CD4+ {alpha}ß 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 {gamma}{delta} T cells are rare in MSP2-immunized cattle, and {gamma}{delta} 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+ {alpha}ß and WC1+ {gamma}{delta} 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+ {gamma}{delta} 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+ {gamma}{delta} T cells, a similar comparison was performed with WC1+ {gamma}{delta} and CD4+ {alpha}ß 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
T cell clones
WC1+ {gamma}{delta} T cell clone 61.1G11 was isolated from MSP2-immunized Holstein steer 98B61 as described previously [8 , 18 ]. {gamma}{delta} T cell clone 61.3G3 as well as CD4+ {alpha}ß 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+ {alpha}ß T cells and WC1+ {gamma}{delta} 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.


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  		Table 2. Proliferative Responses of T Cell Clones to A. marginale Antigens

 
Positively selected cell populations
WC1+ {gamma}{delta} T cells and CD4+ {alpha}ß 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 {delta} chain of the {gamma}{delta} 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 x 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 (3x104 cells/well) were cultured in duplicate wells in a total volume of 100 µl complete medium containing 2 x 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 x 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+ {gamma}{delta} T cell clone 61.1G11 and CD4+ {alpha}ß 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 {alpha}ß and {gamma}{delta} 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 {gamma}{delta} T cell clone 61.1G11 and {alpha}ß T cell clone 61.10.2D7 or {gamma}{delta} T cell clone 61.3H5 and {alpha}ß 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 {gamma}{delta} clone and the {alpha}ß 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.


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  		Table 1. Primer Sequence for Real-Time RT-PCR

 
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 x 106 cells per ml with Con A plus IL-2 in the absence of APC. Supernatants collected after 24 h from {alpha}ß T cell clone 61.10.2D7 and {gamma}{delta} T cell clone 61.1G11 as well as identically stimulated, positively selected CD4+ {alpha}ß T cells and WC1+ {gamma}{delta} T cells were used to measure IFN-{gamma}, TNF-{alpha}, IL-10, and NO. All assays were statistically analyzed with one-tailed Student’s t-test.

The levels of IFN-{gamma} 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-{gamma}/ml (previously determined by the neutralization of vesicular stomatitis virus) [25 ]. In the assay, 1.7 ng corresponds to ~1 unit IFN-{gamma} [26 ].

The TNF-{alpha} capture ELISA was done as described previously [27 , 28 ]. Briefly, Immulon II ELISA plates (Dynax Technologies, Chantilly, VA) were coated with anti-bovine TNF-{alpha} mAb 1D11-10 [provided by Veterinary Infectious Disease Organization (VIDO), Saskatoon, Saskatchewan, Canada]. Bound TNF-{alpha} was detected by incubation with a rabbit anti-TNF-{alpha} 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-{alpha} (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-{gamma} 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Response of clones to antigen
The two pairs of T cell clones, each comprised of one CD4+ {alpha}ß T cell clone and one WC1+ {gamma}{delta} 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+ {gamma}{delta} 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 ]. {alpha}ß T cell clones 61.10.2D7 and 61.3H5 and {gamma}{delta} 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 {gamma}{delta} T cell clone 61.1G11 and {alpha}ß clone 61.10.2D7 responded to all three antigens. In a separate assay, CD4+ {alpha}ß 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 {gamma}{delta} TCR (Fig. 1 ). Comparison of gene expression in {gamma}{delta} and {alpha}ß T cells was first done with Con A/IL-2-stimulated {gamma}{delta} T cell clone 61.1G11 and {alpha}ß T cell clone 61.10.2D7 using the bovine oligonucleotide array. Transcripts (1343) were expressed at twofold or higher levels in the {gamma}{delta} T cell clone, and 606 transcripts were expressed at twofold or higher levels in the {alpha}ß T cell clone. Immune function-associated genes with increased expression in {gamma}{delta} or {alpha}ß T cell clones are listed in Table 3 . As expected, {gamma} and {delta} TCR mRNA was expressed at higher levels in the {gamma}{delta} T cell clone, 61.1G11. Conversely, {alpha}ß T cell clone 61.10.2D7 had higher levels of CD2, CD4, and {alpha} and ß TCR mRNA. Two {gamma}-chain TCR transcripts were expressed at higher levels in the {alpha}ß T cell clone, which is not unexpected, as the majority of {alpha}ß T cells has rearranged but not functional {gamma}- and {delta}-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 {gamma}{delta} T cell clone. The XCL1 chemokine, which is typically associated with CD8+ T cells, NK cells, mast cells, and {gamma}{delta} T cells, was highly expressed by the WC1+ subset of {gamma}{delta} T cells. In addition, CD68 and CD11b were expressed at higher levels in the {gamma}{delta} T cell clone. The oligonucleotide array results thus suggested that the {gamma}{delta} T cell clone had a phenotype which shared characteristics of myeloid cells.


Figure 1
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  		Figure 1. Flow cytometric analysis of T cell clones; {gamma}{delta} T cell clones 61.1G11 and 61.3G3 are presented (a and b, respectively). {alpha}ß 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 {gamma}{delta} TCR is represented by the open black histograms.

 

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  		Table 3. Immune Function-Related Genes Differentially Regulated in the Oligonucleotide Array When Comparing {gamma}{delta}T Cell Clone 61.1G11 and {alpha}ßT Cell Clone 61.10.2D7

 
Real-time RT-PCR analysis of genes expressed by {gamma}{delta} T cell clone 61.1G11 and {alpha}ß T cell clone 61.10.2D7
Thirty-eight genes were selected for real-time RT-PCR analysis to confirm differential expression by {gamma}{delta} and {alpha}ß 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 {gamma}{delta} T cell clone. Similarly, as predicted from the microarray data, genes typically associated with {alpha}ß T cells such as RANTES (CCL5) and MIP-1{alpha} (CCL3) were expressed at similar levels in {alpha}ß and {gamma}{delta} T cells. Real-time RT-PCR analysis also confirmed up-regulated expression in {gamma}{delta} T cells of mRNA encoding TLR4, MSR1, MMR, and CD11b, a component of Mac-1, which are surface molecules typically associated with macrophages.


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  		Table 4. Real-Time RT-PCR Analysis of Selected Immune Function Genes When Comparing {gamma}{delta} T Cell Clone 61.1G11 with {alpha}ß T Cell Clone 61.10.2D7

 
Protein expression and functional assays of {gamma}{delta} T cell clone 61.1G11 and {alpha}ß 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-{alpha}, IFN-{gamma}, 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-{gamma} 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 {gamma}{delta} T cells but not {alpha}ß 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 {alpha}ß T cell clones but not on the {gamma}{delta} T cell clones (ref. [8 ] and data not shown), as predicted by the oligonucleotide array.


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  		Table 5. Comparison of Cytokine and NO Produced by {gamma}{delta} and {alpha}ß T Cells

 

Figure 2
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  		Figure 2. Flow cytometric analysis of {gamma}{delta} T cell clone 61.1G11 and {alpha}ß 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).

 
Real-time RT-PCR analysis of {gamma}{delta} T cell clone 61.3G3, {alpha}ß 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+ {alpha}ß T cell and WC1+ {gamma}{delta} T cells, additional, real-time RT-PCR analysis was performed on another set of clones, {gamma}{delta} T cell clone 61.3G3 and {alpha}ß 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 {gamma}{delta} 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 {gamma}{delta} T cell-associated chemokine XCL1.


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  		Table 6. Real-Time RT-PCR Analysis of Selected Immune Function Genes When Comparing {gamma}{delta} T Cell Clone 61.3G3 and {alpha}ß T Cell Clone 61.3H5 or WC1+ and CD4+ Cells Selected from PBMC

 
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 {alpha}ß and {gamma}{delta} 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 {gamma}{delta} T cells than in {alpha}ß 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have postulated that WC1+ {gamma}{delta} T cells may express higher levels of genes typically associated with cells of the innate immune response rather than CD4+ {alpha}ß T cells. Comparison of gene expression in clonal populations of {gamma}{delta} and {alpha}ß T cells from the same individual, which respond to the same antigen, enabled us to determine whether {gamma}{delta} T cells and {alpha}ß T cells have differentially expressed immune response genes, by controlling for differences in antigen responsiveness. Our results show that the WC1+ subset of {gamma}{delta} 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 {gamma}{delta} and {alpha}ß T cells may have some redundant functions, {gamma}{delta} T cells may function in a unique way during an inflammatory response.

Two primary hypotheses have been proposed to explain the preservation of {gamma}{delta} and {alpha}ß T cells throughout evolution. The first suggests that {gamma}{delta} T cells have effector functions similar to those of {alpha}ß 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 {gamma}{delta} T cells interact with their ligand by a germ-line-encoded, single side-chain of the D{gamma} region [32 , 33 ], which differs from antigen recognition by {alpha}ß T cells through complementary-determining region 3 (CDR3). The bovine WC1+ {gamma}{delta} and CD4+ {alpha}ß T cell clones used in this study also recognize antigen differently. Whereas CD4+ T cells are MHC class II-restricted, the {gamma}{delta} T cell clones are not [8 ]. In addition, sequence analysis suggested that {gamma}{delta} T cell clones do not recognize antigen through CDR3 [8 ]. The second hypothesis proposes that {gamma}{delta} and {alpha}ß T cells are functionally different. In support of this, {gamma}{delta} T cells have been shown to be uniquely involved in epidermal tissue repair and protection of normal airway function [34 35 36 ]. However, these hypotheses are not mutually exclusive. The {alpha}ß and {gamma}{delta} T cell clones analyzed in our study recognize MSP2 but have markedly different responses to the same stimulus, which strongly argues that WC1+ {gamma}{delta} T cells are functionally different from CD4+ {alpha}ß T cells, although many immune function genes are similarly expressed.

Of particular interest is the higher level of expression of genes in WC1+ {gamma}{delta} 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+ {gamma}{delta} 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 {gamma}{delta} 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 {alpha}ß and {gamma}{delta} 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 {gamma}{delta} T cells in the three comparative experiments. Additional myeloid-associated genes expressed at higher levels by {gamma}{delta} 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 {gamma}{delta} 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 {gamma}{delta} T cells [38 ]. The expression by WC1+ {gamma}{delta} 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+ {gamma}{delta} T cell subsets of cattle using sorted cell populations [12 , 13 , 17 ]. SAGE and microarray analysis suggested that {gamma}{delta} T cells express a myeloid phenotype [13 ] in that transcripts for MSR1, CD68, and TNF-{alpha} were expressed at higher levels in WC1+ CD8– {gamma}{delta} T cells when compared with CD8+ WC1– {gamma}{delta} T cells. These genes were also regulated differentially in our comparison of WC1+ {gamma}{delta} T cells with CD4+ {alpha}ß T cells and support the concept that WC1+ {gamma}{delta} T cells share properties of myeloid cells. The demonstration that bovine {gamma}{delta} T cells, including cultured WC1+ {gamma}{delta} T cells, present antigen to {alpha}ß T cells [15 ] is also consistent with WC1+ {gamma}{delta} 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 {gamma}{delta} T cells [16 ], lending further support to the concept that {gamma}{delta} 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-{gamma}, TNF-{alpha}, and IL-10. When comparing monoclonal T cells, cytokine assays confirmed the oligonucleotide array and PCR results. The inversely correlated production of IFN-{gamma} and TNF-{alpha} may be explained by differences in transcriptional regulation of their genes and because IFN-{gamma} is differently regulated in {gamma}{delta} T cells and {alpha}ß 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 {gamma}{delta} 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 {gamma}{delta} 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+ {gamma}{delta} T cells in peripheral blood, are resistant to Anaplasma infection [43 , 44 ]. It is possible that {gamma}{delta} 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 {gamma}{delta} and {alpha}ß 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 {gamma}{delta} T cell clones express higher levels of surface receptors and many chemokines not typically associated with {alpha}ß 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 {gamma}{delta} T cells and the direct response of {gamma}{delta} T cells to LPS [38 ]. Taken together, these findings strongly support the concept that {gamma}{delta} T cells have cytolytic and cytokine secretory properties commonly associated with {alpha}ß 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+ {gamma}{delta} T cell clones used in this study recognize and respond to antigen differently than CD4+ {alpha}ß 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 {gamma}{delta} T cells. In addition, the mechanisms of antigen recognition by these {gamma}{delta} T cells are not defined and warrant further studies.


   ACKNOWLEDGEMENTS
 
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-{alpha} ELISA.

Received May 24, 2006; revised June 15, 2006; accepted June 19, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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