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The Journal of Immunology, 2001, 167: 6421-6430.
Copyright © 2001 by The American Association of Immunologists

Functional and Structural Similarity of V{gamma}9V{delta}2 T Cells in Humans and Aotus Monkeys, a Primate Infection Model for Plasmodium falciparum Malaria1 ,2

Claudia A. Daubenberger3,*, Maxence Salomon*, William Vecino{dagger}, Beatrice Hübner*, Heike Troll{ddagger}, Raul Rodriques{dagger}, Manuel E. Patarroyo{dagger} and Gerd Pluschke*

* Department of Molecular Immunology, Swiss Tropical Institute, Basel, Switzerland; {dagger} Instituto de Inmunologia, Universidad Nacional de Colombia, Santafe de Bogota, Leticia Colombia; and {ddagger} Solvias Aktiengesellschaft, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{gamma}{delta} T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human {gamma}{delta} T cells carrying the V{gamma}9V{delta}2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating V{gamma}9V{delta}2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the V{gamma}9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human {gamma}{delta} T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of V{gamma}9V{delta}2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The precise role of human lymphocytes expressing the {gamma}{delta} TCR in immune responses is unclear. Typically, 2–5% of lymphocytes in the peripheral blood of adults express the {gamma}{delta} TCR. V{gamma}9V{delta}2 T cells constitute the majority of {gamma}{delta} T cells in peripheral blood, tonsils, and spleen (1, 2). Several observations have shown that {gamma}{delta} T cells dramatically increase in blood of patients infected with Mycobacterium tuberculosis (3), Brucella melitensis (4), Francisella tularensis (5), Listeria monocytogenes (6), Leishmania donovani (7), and Plasmodium falciparum (8). The expansion of V{gamma}9V{delta}2 T cells suggests that this subpopulation is part of the early immune response to infection. Furthermore, {gamma}{delta} T cells have been shown to be essential for protective immune responses in rodent models of malaria and tuberculosis (9, 10). {gamma}{delta} T cells exhibit features characteristic for the adaptive immune system, such as memory phenotype, junctionally diverse TCR, and the ability to undergo either anergy or expansion depending on the availability of costimulation (2). In contrast, {gamma}{delta} T cells can also be regarded as an element of the innate immune system, because some {gamma}{delta} T cell subpopulations exhibit limited TCR diversity and are rapidly stimulated in early phases of immune responses (2).

Isopentenyl pyrophosphate (IPP),4 a 246-Da molecule with a five-carbon isoprenyl chain and a pyrophosphate moiety, has been described as the first structurally identified ligand for human {gamma}{delta} T cells (11). Recently, 3-formyl-1-butyl pyrophosphate, a precursor in the synthesis pathway of IPP, was identified as the most likely natural ligand of V{gamma}9V{delta}2 T cells in parasitic and bacterial infections (12). No murine {gamma}{delta} T cell reactivity to these low molecular-mass Ags has yet been demonstrated. Therefore, no rodent model is presently available to establish experimentally the biological significance of responses against phosphorylated, nonpeptide ligands in humans.

Contemporary living primates are classified into New World monkeys (Platyrrhini) and Old World simians (Catarrhini). The Platyrrhini and Catarrhini radiated approximately 60 million years ago (13). Aotus monkeys, belonging to the Platyrrhini, has been shown to be susceptible to various infectious diseases also affecting man, such as malaria, bilharzia, leishmaniasis, and hepatitis A (14, 15, 16, 17). A. nancymaae belongs to the few species that are susceptible to the major human malaria parasites P. falciparum and Plasmodium vivax, and this animal model is currently used to evaluate new malaria blood stage vaccine candidates (18). Nonhuman primate models allow longitudinal surveys of certain cell subpopulations, investigation of specific tissues such as the bone marrow and spleen, and also to conduct adoptive cell transfer or depletion experiments (19).

We present in this work data demonstrating that A. nancymaae constitutes an animal model to investigate the role of V{gamma}9V{delta}2 T cells stimulated by nonpeptide ligands during immune responses against infectious diseases. This is based on T cell in vitro stimulation analyses with IPP and the comparison of TRGV-J and TRDV-D-J rearrangements of A. nancymaae and human IPP-stimulated {gamma}{delta} T cells. Furthermore, our results provide additional evidence that the CDR3 region of the V{gamma}9V{delta}2 TCR might be critically involved in the recognition of IPP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

A. nancymaae were caught in the Colombian Amazon Area close to Leticia, Colombia, and kept in the primate research facility of the Instituto de Inmunologia (Santafe de Bogota, Leticia, Colombia). The animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996).

Proliferation assays

Spleen cells derived from spleenectomized A. nancymaae and PBMC of human volunteers were isolated by Ficoll-Hypaque (Amersham Pharmacia Biotech, Dübendorf, Switzerland) density gradient centrifugation and were used either after cryopreservation in liquid nitrogen or freshly for proliferation assays. Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 2 mM L-glutamine, 1 mM Na-pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM nonessential amino acids (culture medium). A total of 4–5 x 105/well of freshly isolated human PBMC or thawed A. nancymaae spleen cells were cultivated in the presence or absence of 100 µM IPP in 96-well flat-bottom plates in the presence of 100 U/ml of recombinant human (rh)IL-2. [3H]Thymidine (1 µCi/well; Amersham Pharmacia Biotech), was added to the cultures for the last 16 h before they were harvested by an automated harvesting device (Inotech, Wohlen, Switzerland) and assayed for [3H]thymidine incorporation by liquid scintillation counting with an LKB-Wallac counter (LKB Instruments, Sweden). Data are expressed as {Delta} cpm (mean cpm of triplicate cultures in the presence of IPP and rhIL-2 - mean cpm of triplicate cultures in the absence of IPP plus rhIL-2).

Analysis of in vitro T cell subset expansion

Spleen cells of A. nancymaae were diluted to 1 x 106 cells/ml in culture medium and cultivated for the indicated time periods with culture medium in 48-well plates (Nunc, Roskilde, Denmark) in the presence of 100 U/ml rhIL-2, PHA plus 100 U/ml rhIL-2 and 100 µM IPP plus 100 U/ml rhIL-2. Cells were recovered from the wells and stained for flow cytometry before and after in vitro cultivation with a series of mAb specific for defined human surface receptors. Briefly, cells were resuspended in HBSS containing 1% BSA and 0.01% NaN3 (FACS buffer) in a concentration of 5 x 106 cells/ml and 100 µl was dispensed in every FACS tube. After centrifugation, the supernatant was discarded and the cells were mixed with 10 µl of Ab specific for human T cell surface Ags which were diluted 1/20 in FACS buffer. After incubation at 4°C for 30 min, the cells were washed once with FACS buffer, resuspended in 100 µl of appropriately diluted goat anti-mouse FITC-conjugated Ab (DAKO, Glostrup, Denmark) and incubated for another 30 min on ice. After washings, the cells were resuspended in 100 µl of FACS buffer. When using mAb labeled directly with FITC, cells were incubated for 30 min on ice and then washed and resuspended in FACS buffer. FITC-labeled isotype controls, unstained cells, and cells incubated with secondary reagent only were included as controls. Fluorescence was measured on a FACScan (BD Biosciences, Mountain View, CA). Cells were gated using forward and side scatter parameters for dead cell exclusion. In each sample, 10,000 events were measured and data were analyzed using CellQuest (BD Biosciences) to determine the frequencies and mean fluorescence intensities. The mAb used in this study included anti-pan {gamma}{delta} TCR (clone B1.1, BD PharMingen, San Diego, CA; clone 11F2, BD Biosciences; clone IMMU 520, Immunotech, Luminy, France), anti-{alpha}{beta} TCR (clone WT31, BD Biosciences), anti-V{gamma}9 (clone IMMU 360, Immunotech), anti-CD3 (clone SK7, BD Biosciences; clone SP34, BD PharMingen), anti-CD4 (clone OKT4, American Type Culture Collection, Manassas, VA, CRL-8002; clone MT310, DAKO A/S; clone RFT-4, Southern Biotechnology Associates, Birmingham, AL), anti-CD8 (clone SFCI21Thy2D3, Beckman Coulter, Fullerton, CA; clone OKT8, ATCC CRL-8014; clone DK25, DAKO A/S), anti-CD25 (clone M-A251, Southern Biotechnology), anti-CD69 (clone FN50, Southern Biotechnology), and anti-CD80 (clone BB1, Southern Biotechnology).

Amplification and sequencing of TRDV and TRGV gene segments

For the amplification of TRGV-J-C and TRDV-D-J-C rearrangements, total RNA was extracted from spleen cells using the RNeasy extraction kit (Qiagen, Valencia, CA). Single-stranded cDNA was synthesized using oligo(dT) primer (Life Technologies, Rockville, MD) in combination with Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s recommendation. For the analysis of the TRDV-D-J-C rearrangements expressed by unstimulated cells, primers V{delta}2 (5'-GCAGGAGTCATGTCAGCCAT) and C{delta} (5'-GACAAGCGACATTTGTTCCA-3') were used. Similarly, for the analysis of TRGV-J-C rearrangements, primers V{gamma}9 (5'-ATCAACGCTGGCAGTCC-3') and C{gamma} (5'-AAGGAAGAAAAATAGTGGGC-3') were used. For the amplification of TRGV-J-C rearrangements from in vitro cultivated cells, primers V{gamma}9 (5'-ATCAACGCTGGCAGTCC-3') and P23 (5'-GGGGGAAATGTCTGCCCAAGG-3') and for the TRDV-D-J-C transcripts primers P77 (5'-TTTTTCATGACAAAAACGGATGG-3') and P78 (5'-TCCTTCACCAGACAAGCGAC-3') were used. PCR were run with the following profile: 1 min at 95°C, 30 s at 50°C, and 30 s at 72°C. PCR products were purified using a PCR product purification kit (Qiagen) according to manufacturer’s protocol and cloned into the pGEM 5 T vector (Promega, Madison, WI). Plasmid dsDNA was isolated using the plasmid purification kit (Qiagen). Plasmid inserts were sequenced in both directions using the ABI Prism 310 Genetic Analyzer (PerkinElmer, Foster City, CA). Sequence data were collected using the ABI Prism data collection software and processed using the Sequence Navigator program (PerkinElmer). Sequences were analyzed using software coming from the server of IMGT (http://imgt.cines.fr:8104; initiator and coordinator, M.-P. Lefranc, Montpellier, France), the international ImMunoGeneTics database (20). The nomenclature of Aotus TRGV, TRDV, TRGJ, TRDJ, and TRDD gene segments follows the international ImMunoGeneTics database (http://imgt.cnusc.fr:8140) (20).

Phylogenetic analysis

The deduced amino acid sequences of TRGV9 segments of human, Pan troglodytes, and A. nancymaae were aligned using the CLUSTAL W computer program (http://bioweb.pasteur.fr). The phylogenetic analysis is based on the comparison of framework and CDR1 and CDR2 regions of sequences available in GenBank. Phylogenetic analysis was performed using the PHYLIP 3.572 package available under http://bioweb.pasteur.fr. A neighbor-joining phylogenetic tree (21) was constructed from genetic distance values (22). The reliability of the tree was examined by the bootstrap test (23).

Detection of IFN-{gamma} by real-time quantitative RT-PCR and ELISA

Cells were harvested after the indicated time periods of stimulation, washed in HBSS, and frozen at -80°C. Total RNA was prepared in one batch using the RNeasy extraction kit (Qiagen). The RNA was eluted with water and stored at -80°C. For cDNA synthesis, ~700 ng total RNA was transcribed with cDNA transcription reagents (PerkinElmer) with the use of random hexamers. Measurement of gene expression was performed using the ABI Prism 7700 Sequence Detection System (PerkinElmer) and the predeveloped assay reagents for measurement of human IFN-{gamma} (PerkinElmer; part number 4308250S). Every reaction was performed in triplicate and reported as the average. Amplification of 18S RNA was used as an internal control template for the normalization of the amplification. Real-time monitoring of fluorescent emission from cleavage of sequence-specific probes by the nuclease activity of Taq polymerase allowed definition of the threshold cycle during the exponential phase of amplification.

IFN-{gamma} secreted into cell culture supernatant of the cultures analyzed with real-time quantitative PCR was determined by ELISA using the predeveloped ELISA system for human IFN-{gamma} (M 700A-E, M 701-B from Endogen, Woburn, MA; and RPN2787 from Amersham) following the manufacturer’s instructions. We attempted to measure IPP-induced IFN-{gamma} production by Aotus cells using these two ELISA systems. However, IFN-{gamma} could be readily detected in supernatants of human but not in Aotus cultures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aotus {gamma}{delta} T cells proliferate in vitro in response to IPP

Up to date, no reactivity of murine {gamma}{delta} T cells with phosphorylated nonpeptidic ligands has been demonstrated and hence a rodent model system is not available to establish the biological significance of such responses. Therefore, we investigated whether T cell subpopulations of A. nancymaae are stimulated by IPP, a representative of this group of ligands. Spleen cells were incubated in vitro with IPP in the presence of rhIL-2 or with rhIL-2 alone. In parallel, human PBMC of two donors were incubated under similar conditions. Proliferative responses were detectable after 48 h of stimulation, and the difference in the incorporation of radioactivity between stimulated and unstimulated cultures was comparable between Aotus and human cells (Fig. 1Go). No proliferation in the cultures could be observed in the absence of rhIL-2 (data not shown).



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  		FIGURE 1. Proliferative responses of Aotus spleen cells and human PBMC stimulated with IPP plus rhIL-2 are comparable. Spleen cells of two A. nancymaae ({diamondsuit} and {blacksquare}) and PBMC of two human volunteers ({blacktriangleup} and •) were stimulated with 100 µM IPP plus 100 U/ml rhIL-2 for the indicated time periods. For the last 16 h, [3H]thymidine was present. Shown are {Delta} cpm (mean cpm of triplicate cultures in the presence of IPP plus rhIL-2 - mean cpm of triplicate cultures in the absence of IPP). The results are representative for three independent experiments.

 
Based on these observations, we wanted to analyze the phenotype of the expanding cell populations. Therefore, we tested a series of mAb against human CD Ags and TCR for cross-reactivity with the corresponding Aotus cell surface receptors. Spleen cells of two animals were expanded in vitro for 14 days by stimulation with PHA and rhIL-2, stained, and analyzed by flow cytometry. The results are summarized in Table IGo. One of two analyzed anti-CD3 mAb, all three anti-CD4 mAb, and an anti-CD80 mAb exhibited cross-reactivity. Although all three pan-{gamma}{delta} TCR-specific mAb did not cross-react, a small fraction of Aotus T cells was stained with the anti-human V{gamma}9 mAb IMMU 360. None of the anti-CD8 mAb reacted with the Aotus cells.


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  		Table I. Cross-reactivity of mAb specific for human T cell surface Ags with A. nancymaae cells1

 
Aotus spleen cells and human PBMC were stimulated for 20 days with IPP plus rhIL-2 and then analyzed by flow cytometry with cross-reactive mAbs. In three independent experiments a significant and reproducible expansion of TCR V{gamma}9-expressing cells starting at 6–8% before stimulation and reaching 50–80% of the total cell count after 20 days of incubation with IPP in both human and monkey cultures was observed. After 20 days, the cells started to reduce their size and proliferation seized. One representative experiment is shown in Fig. 2Go. Obviously, the combination of IPP and rhIL-2 but not rhIL-2 alone is responsible for the expansion of V{gamma}9 cells, which is in accordance with results in humans.



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  		FIGURE 2. Stimulation of cultures of Aotus spleen cells with IPP plus rhIL-2 lead to expansion of V{gamma}9 T cells in cultures as demonstrated by flow cytometry. Aotus cells were cultivated with 100 µM IPP and 100 U/ml rhIL-2 and analyzed on days 0 (A), 7 (B), and 20 (C). Corresponding control cultures stimulated with rhIL-2 alone were analyzed on days 7 (D) and 20 (E), respectively. Cells were harvested, stained with FITC-labeled anti-V{gamma}9 mAb IMMU 360 and PE-labeled anti-CD3 mAb SP34, and analyzed by flow cytometry.

 
Sequence analysis of TRGV and TRDV gene segments of A. nancymaae

After having established that V{gamma}9 T cells of Aotus are stimulated by IPP, we characterized the diversity of the {gamma}{delta} TCR repertoire at the nucleotide sequence level. TGV-J and TRDV-D-J transcripts from both freshly isolated Aotus spleen cells and IPP-stimulated cultures were reverse transcribed and amplified by PCR. The resulting PCR products were cloned and sequenced. The comparative analyses of Aotus cDNA sequences and human germline TRDV, TRDD, TRDJ, TRGV, and TRGJ sequences were performed using IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr).

The Aotus equivalent of the human TRDV2 sequence displayed 84% identity at the amino acid level with the human homolog. Aotus homologs of the human TRGV9 and TRGV2 gene segments were 85 and 62% identical at the amino acid level with their human counterparts, respectively. Both synonymous and nonsynonymous base differences with respect to the closest human counterparts were found throughout all regions of the TRGV and TRDV genes (Fig. 3Go). Two alleles of the Aotus TRGV2 homolog differing in one synonymous and nonsynonymous base change were both found several times in different transcripts (Fig. 3Go).




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  		FIGURE 3. Alignment of nucleotide sequences and deduced amino acid sequences of TRDV and TRGV gene segments of Aotus with their closest human counterpart. Identities among sequences are shown as dashes with respect to the Aotus sequences. The numbering system is in accordance with the ImMunoGeneTics database system (http://imgt.cnuc.fr:8140). Framework region (FR) 1, FR 2, and FR 3 are located at positions 1–26, 39–55, and 66–104, respectively. CDR 1, CDR 2, and germline CDR 3 are located at positions 27–38, 56–65, and 105–115 (20 ).

 
A phylogenetic tree constructed with deduced amino acid sequences of TRGV gene segments derived from human, P. troglodytes, and A. nancymaae is shown in Fig. 4Go. The TRGV9 sequences form a separate clade from the other TRGV gene segments and the Aotus TRGV2 alleles are grouped together with the human TRGV2 homolog (Fig. 4Go). Therefore, the interspecies divergence between the TRGV9 and TRGV2 genes of the three primates compared is smaller than the intraspecies divergence of the different TRGVgene segments building the TRGV repertoire.



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  		FIGURE 4. Phylogenetic unrooted bootstrap tree of deduced amino acid sequences of TRGV segments of human (H.s.), P. troglodytes (P.t.), and A. nancymaae (A.s.). The FR, CDR 1, and CDR 2 regions of primate TRGV sequences available in GenBank were used for tree construction. Sequences were aligned using the CLUSTAL W computer program and the phylogenetic tree was constructed using the PHYLIP 3.572 package. A genetic distance matrix was constructed using the Dayhoff PAM matrix. The reliability of the neighbor-joining phylogenetic tree was tested using bootstrap values. The GenBank accession numbers for the human and P. troglodytes TRGV gene segments included are M13429, M13430, X15272, X13355, M13434, X07205, and X61069, respectively.

 
Identification of TRDJ and TRGJ gene segments in A. nancymaae

Two TRGJ and three TRDJ gene segments were identified from the TRGV-J and TRDV-D-J transcripts (Fig. 5Go). Somatic additions of non-germline-encoded nucleotides and exonuclease trimming at the TRGV-J and TRDV-D-J junctions make it impossible to determine exactly the 5' ends of the germline-encoded TRGJ and TRDJ sequences in the transcripts. All identified Aotus TRGJ and TRDJ segments have human counterparts, and the level of identity at the amino acid level ranges from 80 to 100% (Fig. 5Go). The vast majority of both the TRGV-J and TRDV-D-J rearrangements sequenced were in frame. Screening of the TRDV-D-J rearrangements for homologs to human TRDD segments yielded evidence for the existence of an Aotus equivalent for the human TRDD3 segment (data not shown).



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  		FIGURE 5. Alignment of nucleotide sequences and deduced amino acid sequences of TRDJ and TRGJ gene segments of Aotus with their closest human counterpart. Identities among sequences are shown as dashes with respect to the human sequences. Synonymous and nonsynonymous exchanges are shown in italics and bold, respectively.

 
Comparison of TRGV-J and TRDV-D-J rearrangements expressed in IPP-stimulated and unstimulated cultures

Human and Aotus cells were stimulated for 20 days with IPP in the presence of 100 U/ml rhIL-2. By that time, 50–80% of cultured cells expressed the TRGV9 receptor as demonstrated by FACS analysis (Fig. 2Go). To investigate the diversity of the complementarity-determining region 3 (CDR 3) of the TCR, sequences of TRGV9-J and TRDV2-D-J transcripts were analyzed. As depicted in Fig. 6GoB, the Aotus TRGV9 gene segment was joined in 20/21 transcripts to TRGJP and once to TRGJP2. In all 11 human sequences analyzed, TRGV9 was associated with TRGJP. The CDR3 regions of both the human and Aotus TRGV9-J rearrangements were diverse, probably due to exonuclease trimming and additions of non-germline-encoded nucleotides. A maximum of nine non-germline-encoded nucleotides was found in a single rearrangement. The N region was occupied in 6 of 20 Aotus transcripts and 2 of 11 human transcripts by an Arg residue encoded by three of the six different Arg codons available (Fig. 6Go, B and C).



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  		FIGURE 6. Alignment of the nucleotide sequences and deduced amino acid sequences of TRGV9-J transcripts of Aotus V{gamma}9 T cells before expansion with IPP (A), after stimulation with IPP (B), and of human V{gamma}9 T cells after expansion with IPP (C). Shown are the clones, and the rearrangement of the gene segments used.

 
For comparison, sequences derived from IPP-unstimulated Aotus {gamma}{delta} cells are shown in Fig. 6GoA. Two different TRGV gene segments dominated, namely TRGV9 and TRGV2. Although the TRGV9 gene was associated exclusively with TRGJP, TRGV2 was rearranged with TRGJP1 or TRGJ2 segments. In the N region of this group of 10 sequences Arg was present twice.

Sequences of TRDV2-D-J transcripts from IPP-stimulated and unstimulated cultures are shown in Fig. 7Go. TRDV2/TRDD3/TRDJ1 and TRDV2/TRDD3/TRDJ2 rearrangements were found. In the sequences derived from IPP-unstimulated cells, the same gene segments were found as in the IPP-stimulated cultures (data not shown). In transcripts from human IPP-stimulated cells, TRDJ1 and TRDD3 dominated (Fig. 7GoB). Despite their heterogeneity, almost all TRDV2/TRDD/TRDJ1 rearrangements carried a strongly hydrophobic amino acid (Val, Leu, or Ile) at a conserved position (codon 109) relative to the TRDV framework residues (Fig. 7Go). Usage of different nucleotide triplets indicates that these amino acids are encoded by non-germline-encoded nucleotides.



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  		FIGURE 7. Alignment of the nucleotide sequences and deduced amino acid sequences of TRDV2-D-J transcripts of Aotus and human {gamma}{delta} T cells after stimulation with IPP (A and B), respectively. Shown are the clones, and the rearrangement of the gene segments used.

 
Detection of IFN-{gamma} gene and protein expression in IPP-stimulated cultures

The next set of experiments was designed to analyze the kinetics of IPP-induced production of IFN-{gamma}. A real-time quantitative PCR method using the predeveloped assay system for human IFN-{gamma} was used. Within 8 h of stimulation IPP induced in the presence of rhIL-2 a >400-fold increase in IFN-{gamma} mRNA in human PBMC. Surprisingly, in cultures not supplemented with rhIL-2 a small but consistent peak of IFN-{gamma} was also present (Table IIGo). The predeveloped assay system turned out to be unsuitable for the amplification of Aotus IFN-{gamma} cDNA.


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  		Table II. Real-time quantitative PCR analysis of human PBMC of two donors stimulated with IPP for the expression of IFN-{gamma} mRNA1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human V{gamma}9V{delta}2 T cells recognize small, nonpeptidic microbial metabolites containing critical phosphate residues in a TCR-dependent way (24). These phosphorylated ligands are broadly distributed in living organisms, and IPP was described as the first structurally identified natural ligand for human {gamma}{delta} cells (11). IPP is widespread in organisms, from bacteria to fungi and higher eukaryotes, and is an essential precursor in the synthesis of isoprenoids (25). IPP has been identified as highly selective stimulator of human V{gamma}9V{delta}2 T cells as determined by staining of in vitro expanded cells and by screening large panels of human {gamma}{delta} T cell clones (24, 26). The functional role of {gamma}{delta} T cells in host defense against infections is difficult to study in humans due to limitations in supply with well-defined clinical specimens. Lack of rodent {gamma}{delta} T cell populations responding to low m.w. phosphorylated ligands excludes the use of established rodent infection models in this context. Therefore, we used IPP as a representative of non-protein T cell ligands to address the question of whether {gamma}{delta} T cells of A. nancymaae could be activated similarly to human cells. In spleen cells of A. nancymaae IPP induced a significant proliferative response detectable after 48 h. This response was comparable to that of human PBMC. Previous investigators have reported that in the absence of CD4+ T cells the combination of IPP plus rhIL-2 induces high levels of {gamma}{delta} T cell expansion, indicating that the secretion of IL-2 is one of the key helper functions provided by CD4+ T cells for {gamma}{delta} T cell expansion (27, 28). Aotus cells did not proliferate in response to IPP in the absence of rhIL-2 (data not shown), suggesting that Aotus {gamma}{delta} T cells also depend on the provision of IL-2 by other cell subsets.

Transfection experiments with {gamma} and {delta} TCR genes indicate that the {gamma}{delta} TCR is involved directly in the recognition of phosphorylated metabolites (29, 30). Transfectants expressing a TRGV9-J1 rather than the predominant TRGV9-JP chain did not respond to IPP, suggesting that the TRGJ segments crucially determine the fine specificity of the V{gamma}9V{delta}2 TCR. Furthermore, transfection experiments of a V{delta}1 chain replacing the V{delta}2 chain of a IPP-reactive TCR showed that the V{delta} chain is also involved in the recognition of IPP (29).

IPP-responsive Aotus T cell lines were stainable with the cross-reactive anti-human V{gamma}9 mAb IMMU 360. Nucleotide sequence analyses of the TRGV-J and TRDV-D-J rearrangements of IPP-stimulated Aotus cells showed that the V{gamma}9 and V{delta}2 TCR chains in Aotus and humans are similar with respect to 1) TRJ and TRD ne segment usage, 2) a high degree of sequence identity of the TRGV, TRGJ, TRDV, TRDJ, and TRDD gene segments used, and 3) the level of diversity of the CDR3 regions. The gene segments predominantly used by the IPP-responsive Aotus {gamma}{delta} T cells have a sequence identity to their human counterparts at the amino acid level of 84 and 85% for TRDV2 and TRGV9 respectively, 94% for TRDJ2, and TRGJP, and 100% for TRDJ1. Similar to the results in humans, the CDR3 region of IPP-stimulated cells is highly diverse. In the junctional region of the Aotus {gamma}-chain, non-germline-encoded Arg residues were found at higher frequency, indicating a positive selection for this amino acid. Furthermore, in the CDR3 region of the {delta}-chains the hydrophobic amino acids Leu, Val, or Ile located at a distinct position relative to the TRDV2 framework dominated in both species. This motif has been described previously in a series of human V{gamma}9V{delta}2 T cell clones and might result from Ag-driven peripheral selection (31).

At present it is unclear how phosphorylated compounds are presented to {gamma}{delta} T cells. The stimulation of freshly isolated human V{gamma}9V{delta}2 T cells requires the presence of APC (27), and the Ag might be presented by a novel extracellular pathway not requiring Ag uptake or processing (32). Our finding that highly conserved TCR sequences are shared between human and Aotus cells responding to IPP indicates that the trimolecular interaction between IPP, the potential presenting element, and the TCR has conserved structural features. This might suggest that an evolutionary conserved, nonpolymorphic IPP-binding molecule exists. Experiments with human V{gamma}9V{delta}2 T cells stimulated with APC derived from different Old and New World monkey species and vice versa could provide some insight into the evolutionary conservation of the processing events and presenting elements required for stimulation with non-protein-phosphorylated ligands.

It has been shown recently that the rapid stimulation of V{gamma}9V{delta}2 cells leads to a local release of IFN-{gamma} and TNF-{alpha}, facilitating the activation and regulation of other T cell subsets in an early stage of the immune response (24). Using real-time quantitative PCR, we found that IPP induces a massive IFN-{gamma} mRNA up-regulation which is detectable after only 4 h of incubation with IPP. The comparison of the IFN-{gamma} amino acid sequences of A. nancymaae and humans demonstrates an identity of 94%. Nevertheless, two predeveloped ELISA systems and a real-time quantitative PCR method for the detection of IFN-{gamma} have been tried unsuccessfully with cell culture supernatants and mRNA of Aotus spleen cells, respectively.

Six TRGV and four TRDV gene segments are used by human {gamma}{delta} T cells. Despite the organizational conservation of {gamma}{delta} TCR genes in different species, the interspecies and intraspecies divergence of TRGV genes is strikingly high (33). The phylogenetic analysis of TRGV sequences of primates demonstrate that human, chimpanzee, and Aotus TRGV9 sequences cluster together in a distinct clade separated from other representatives of the TRGV gene families. Hence the interspecies diversity of TRGV9 sequences is smaller than the intraspecies differences of other TRGV gene segments (34). The observation that TCR chain gene segments of nonhuman primates cluster in phylogenetic analyses according to family membership instead of species designations has been also described for TCR {alpha}- and {beta}-chain gene segments (35, 36, 37, 38, 39, 40). The high evolutionary conservation of TRGV9 sequences during more than 50 million years in conjunction with the conserved features of TCR rearrangements found in V{gamma}9V{delta}2 T cells responding to IPP suggests that these cells represent an evolutionary conserved immune surveillance system. This is supported by the finding that apart from {gamma}{delta} TCR gene sequences, effector functions displayed by {gamma}{delta} T cells derived from chimpanzees and humans are conserved in terms of lytic function specific for distinct target cells lines (41).

Levels of circulating human {gamma}{delta} T cells are enhanced during acute P. falciparum malaria, and a significant but transient increase in the number of {gamma}{delta} T cells is also observed in nonimmune P. vivax patients during clinical paroxysms (8, 42). The Rohmer pathway through which IPP can be synthesized is fully functional in P. falciparum (43) and merozoites of P. falciparum stimulate V{gamma}9 T cells (44). However, the mechanism of action of these stimulated {gamma}{delta} T cells and their precise physiological role is not understood. The functional and structural conservation of IPP-responsive {gamma}{delta} T cells between humans and A. nancymaae provides the opportunity to study the involvement of this T cell subpopulation in the host defense against P. falciparum, P. vivax, and probably other pathogens of medical importance for humans. Most vaccination strategies used so far depend on protein Ags mostly activating {alpha}{beta} T cells. However, functionally distinct lymphocyte subpopulations recognizing nonpeptide Ags are becoming better characterized (11, 26, 45) and the specific activation of these cells might be important for the future design of vaccine formulations (46). Our studies provide evidence that A. nancymaae might be a suitable animal model for evaluating the potential involvement of V{gamma}9 T cells in protective or pathogenic immune responses after vaccination studies targeting {gamma}{delta} T cells. This study extends our previous investigations characterizing the repertoire of TCRA, TCRB, and IGK chains, and the polymorphism of MHC-DRB, MHC-DQA1, and MHC-DQB molecules in A. nancymaae (35, 36, 47, 48, 49). Results of these studies demonstrate that the Aotus monkeys represent a nonhuman primate model with an immune system that has important structural and functional features in common with the human immune system.


   Acknowledgments
 
We thank Manuel Alfonso Patarroyo for conducting the spleenectomies of the animals. We are indebted to Clemens Ruch for continuous discussions during this project.


   Footnotes
 
1 Part of this project was supported by funds of the Swiss National Science Foundation (31-52068.97). The Instituto de Inmunologia is financed by the Colombian Ministry of Health and by Colciencias. Back

2 The sequence(s) presented in this article has been submitted to GenBank under accession number(s) AF333709–AF333732, AF336930–AF336946, and AF37841–AF378750. Back

3 Address correspondence and reprint requests to Dr. Claudia A. Daubenberger, Swiss Tropical Institute, Socinstrasse 57, CH 4002 Basel, Switzerland. E-mail address: claudia.daubenberger{at}unibas.ch Back

4 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; rh, recombinant human; FR, framework region; CDR, complementarity-determining region. Back

Received for publication June 20, 2001. Accepted for publication October 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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