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The Journal of Immunology, 2001, 166: 2296-2302.
Copyright © 2001 by The American Association of Immunologists

V{gamma}2 TCR Repertoire Overlap in Different Anatomical Compartments of Healthy, Unrelated Rhesus Macaques1

Alex V. MacDougall*, Patrick Enders2,{dagger}, Glen Hatfield2,{dagger}, David C. Pauza2,{dagger} and Eva Rakasz3,*

* Wisconsin Regional Primate Research Center; and {dagger} Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53715


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{gamma}{delta} T cells show preferential homing that is characterized by biased TCR repertoire at different anatomical locations. The processes that regulate this compartmentalization are largely unknown. A model that allows repeated multiple sample procurement under different conditions and enables with relatively straightforward extrapolation to a human situation will facilitate our understanding. The peripheral blood V{gamma}2 T cell population is the best-characterized human {gamma}{delta} T cell subset. To determine its diversity at multiple immunocompartments matching blood, colon, and vagina samples from rhesus macaques were investigated. Four joining segments used in V{gamma}2-J{gamma} transcripts were identified, including one segment with no human counterpart. Like in humans, the rhesus peripheral blood V{gamma}2 TCR repertoire was limited and contained common sequences that were shared by genetically heterogeneous animals. Furthermore, this subset comprised several phylogenetically conserved V{gamma}2 complementarity-determining region 3 (CDR3) motifs between rhesus and humans. Common sequences were also found within the colon and vagina of the same animal, and within the peripheral blood and intestine of different unrelated animals. These results validate rhesus macaques as a useful model for {gamma}{delta} TCR repertoire and homing studies. Moreover, they provide evidence that the concept of limited but overlapping V{gamma} TCR repertoire between unrelated individuals can be extended including the mucosa of the digestive and reproductive tract.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The V{delta}2/V{gamma}2+ T cell subset of peripheral blood is one of the largest circulating T cell populations in humans. This population represents only a minor subset in neonates, but by adulthood it is ~4–8% of the circulating T cells, and ~70–90% of circulating {gamma}{delta} T cells (1, 2). The expansion of this cell group is assumed to be driven by Ag-specific processes (3, 4). Their elevated cell numbers during numerous bacterial infections, e.g., Mycobacterium tuberculosis (5, 6, 7), Listeria (8, 9), Plasmodium sp. (10, 11), Toxoplasma (12), or Brucella melitensis (13) suggest an important but yet unclarified role in the immune response against these microorganisms. V{delta}2/V{gamma}2+ T cells form a minor population in the mucosa (14, 15). Mechanisms that control the translocation and retention of V{delta}2/V{gamma}2+ T cells at a particular tissue compartment are largely unknown, but are of major significance in studies that aim to manipulate these cells.

The Ag recognition of {gamma}{delta} T cells is not MHC restricted (16, 17, 18), and it does not require Ag processing via the known Ag processing pathways (19, 20, 21). {gamma}{delta} TCRs have complementarity-determining region 3 (CDR3)4 length diversity that are similar to Ig chains. Like the light chain of Igs compared with the heavy ones, {gamma} TCR chains have more limited CDR3 length diversity than do {delta} TCR chains (22, 23, 24). The repertoire of peripheral blood V{gamma}2 TCR chain is limited by the prevalent use of V{gamma}2-J{gamma}1.2 recombination (25), and by the high frequency of canonical CDR3 sequence irrespective of age, sex, or genetic background (26). V{gamma}2+ T cells expressing germline-encoded CDR3 sequence seem to be of extrathymic origin because they appear in the prethymic fetal liver in a relatively high abundance (27). These cells are functional because they respond to mycobacterial Ags by proliferation and cytokine secretion. Furthermore, they are maintained through adulthood as well (26). A subset of peripheral blood V{delta}2/V{gamma}2+ T cells are stimulated by small alkylphosphate and/or alkylamine molecules that derive from microorganisms, host cells, or the metabolism of nutrients (28, 29, 30, 31, 32). Efforts to determine the significance of this subset have been hindered by the lack of a well-established in vivo model system that includes similar cells. {gamma}{delta} T cells that recognize alkylphosphate or alkylamine ligands have not been found in rodents. However, rhesus macaques do have V{delta}2/V{gamma}2+ T cells that respond to these same molecules. In adult animals the V{gamma}2+ and V{delta}2+ T cell subsets represent ~0.7 and 0.4% of the lymphocyte population, respectively (our unpublished observations, Ref. 33). Although the relative proportion of the V{delta}2/V{gamma}2+ T cell subset in the peripheral blood of rhesus monkeys remains to be established, an ~2-fold expansion of both the V{gamma}2+ (34) and V{delta}2+ subset (our unpublished observation) as a function of age suggests that factors shaping the human V{gamma}2+ TCR repertoire might also operate in captive macaques. Previously we described that rhesus monkeys’ V{gamma}2 CDR3 length diversity is extremely limited and similar to the profile of human beings. We also observed that this repertoire restriction showed a tissue-specific pattern between peripheral blood and sigmoid colon (34).

In these investigations we determined the V{gamma}2 TCR CDR3 repertoire diversity of rhesus macaques in different anatomical compartments at the nucleic acid level. We provide evidence for overlap of the V{gamma}2 TCR repertoire between distinct mucosal compartments within a single animal. In addition, we present data that support the concept of a restricted and shared V{gamma}2 TCR repertoire between distinct animals of heterogeneous genetic background. Our results provide an initial framework for future studies aimed at determining the homing mechanisms of {gamma}{delta} T cells.


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

Rhesus macaques (Macaca mulatta) were housed at the Wisconsin Regional Primate Research Center according to the National Institutes of Health "Guide to the Care and Use of Laboratory Animals". The animal cohort used in the primer extension length profile analysis included nine clinically healthy animals aged between 4 and 14 years, among them six females and three males. In the CDR3 sequencing analysis, animals 1 and 2 were 4 years old, and animal 3 was 9 years old. Venous blood collection and vaginal and sigmoid biopsy procedures were conducted under anesthesia using 20 mg/kg ketamine hydrochloride and 0.03 mg/kg buprenorphine. Further pain management was accomplished with 0.03 mg/kg buprenorphine administered i.m. at 3, 24, and 32 h after the procedure. PBMC were isolated by density gradient separation on Ficoll (Sigma, St. Louis, MO). Cells were washed with PBS and stored as dry pellets at -80°C. Cells (3 x 106) were processed for mRNA isolation. One vaginal punchbiopsy specimen (~16-mm3 size) and four to five colon biopsy specimens (~1–2 mm3 each) were obtained and stored in RNALater solution (Ambion, Austin, TX) at -20°C until mRNA isolation.

mRNA isolation and RT-PCR

PolyA RNA was isolated with the MicroFastTrack Kit (Invitrogen, San Diego, CA) according to the manufacturer recommendation. cDNA synthesis was accomplished in 20 µl volume using the Reverse Transcription System (Promega, Madison, WI). PCR was performed in a Perkin-Elmer (Applied Biosystems, Foster City, CA) 9600 thermal cycler with the following thermal cycling profile: denaturation of cDNA at 94°C for 3 min, hot start at 65°C, denaturation 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 90 s, with a final extension step (72°C for 10 min) when 40 cycles were completed. The reaction buffer consisted of 50 mM KCl, 20 mM Tris-HCl pH 8.3, 2 mM MgCl2, 100 µg/ml BSA, 1 mM of each primer, 0.2 mM of each dNTP, and 1.25 U of Taq polymerase. PCR products were visualized on 1.5% agarose gels. Oligodeoxynucleotide primers (Genosys; Sigma) were designed on the basis of highly conserved sequences between human, chimpanzee, bovine, pig, and mouse. The PCR products contained a segment of the TCR variable region, spanning the CDR3 region and including the total length of the J segment, and a small part of the constant region first exon. The constant segment specific primer amplified both the C{gamma}1 and C{gamma}2 genes (32). Oligonucleotide primer sequences were as follows: V{gamma}2: AGA CCT GGT GAA GTC ATA C; C{gamma}1&2: GTT GCT CTT CTT TTC TTG CC.

Primer extension product length determination

Five cycles of primer extension (94°C for 45 s, 60°C for 45 s, 72°C for 45 s) were performed with V{gamma}2 TCR PCR amplificates as templates using a 6-FAM-labeled primer that was specific for {gamma} constant regions (35). The reaction products were analyzed by capillary electrophoresis on a 310 Genetic Analyzer (Perkin-Elmer), and fragment lengths were determined using GeneScan 2.1 software and internal standards (Tamra 500; Perkin-Elmer). The areas under each peak (sequences with identical CDR3 lengths) were represented as a fraction of the total area of all peaks combined. The 6-FAM-labeled primer extension products contained a segment of the TCR variable region and spanned the CDR3 region, including the total length of the J segment with 40 bases of the constant domain first exon. The 6-FAM-labeled oligonucleotide primer used for primer extension reaction was: AAT AGT GGG CTT GGG GGA AAC.

Cloning and sequencing

RT-PCR products were isolated from a 1% agarose gel, purified by filter through a PCR cleanup spin column (Wizard PCR preps DNA Purification System; Promega), and ligated with a pCRII-TOPO vector (Invitrogen) for 5 min at room temperature. A portion of the ligation was used to transform DH10 cells according to the manufacturer recommendations (Invitrogen). Transformants were plated on Luria-Bertani agar with ampicillin prespread with 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (40 mg/ml in dimethylformamide) and isopropyl-thio-{beta}-D-galactopyranoside (5 mM in H2O). Lac- colonies were processed by standard alkaline lysis. Pellets containing plasmid DNA were washed with 70% ETOH. Plasmids were sequenced using C{gamma}182 primer and the BigDye fluorescent sequencing kit (Applied Biosystems). Sequencing products were analyzed with ABI 377XL automated fluorescence sequencer (Applied Biosystems). Sequence data were analyzed with MacVector 6.5 software (Oxford Molecular Group, Hunt Valley, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High degree of homology between rhesus and human V{gamma}2 and J{gamma} TCR segments

Our initial approach was to determine the extent of homology of V{gamma}2 and J{gamma} TCR segments between M. mulatta and Homo sapiens. Germline sequences of TCR chain gene segments of rhesus macaques have not been published so far. Having used cDNA samples, our analysis is limited to consensus sequences derived from data of five unrelated animals. For the same reason, we defined the position of nucleic and amino acid replacements on the basis of corresponding human data (36, 37, 38, 39). We sequenced the V{gamma}2 TCR chain segment that is adjacent to the 3' end of CDR2 hypervariable regions stretching from amino acid residue at position 81 to the carboxyl-terminal of the chain segment. At this region four of the detected eight mutations were nonsynonymous. At the amino acid level, these exchanges resulted in a glycine to serine at the 84th residue, an isoleucine to leucine at the 95th residue, and a glutamic acid to lysine replacement at the 97th residue (Fig. 1Go). The extent of homology was >93% at nucleic acid and 93% at amino acid levels.



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  		FIGURE 1. Comparison of nucleotide and amino acid sequences of V{gamma}2 and J{gamma} TCR segments. Homology was determined on the basis of cDNA sequences. We provided consensus sequences representing J{gamma} TCR segments truncated at the 5' end.

 
In humans, five different J{gamma} sequences have been identified and designated as J{gamma}1.1, J{gamma}1.2, J{gamma}1.3, J{gamma}2.1, and J{gamma}2.3 (37, 38, 39). Having sequenced >500 clones, we encountered four different {gamma}-joining (J{gamma}) segments. On the basis of sequence homology, we designated them as J{gamma}1.1, J{gamma}rh, J{gamma}1.2, and J{gamma}2.3. We did not identify sequences that would match the human J{gamma}1.3 or J{gamma}2.1 gene segments. The region showing the highest homology to the human J{gamma}1.1 sequence had only one, nonsynonymous mutation, which changed the phenylalanine at the fourth residue to an isoleucine (Fig. 1Go). The degree of homology was 97% at nucleic acid, and 93% at amino acid level. Additionally, we observed an unknown sequence that was designated J{gamma}rh. J{gamma}rh showed the highest homology to the J{gamma}1.1 segment, and five nonsynonymous mutations caused changes of glycine to tryptophan at the second residue, phenylalanine to isoleucine at the fourth residue, isoleucine to lysine at the sixth residue, and lysine to arginine at the 12th residue (Fig. 1Go). The overall extent of homology between J{gamma}rh and human J{gamma}1.1 was >93% at the nucleic acid, and 75% at the amino acid level. Whether this domain represents an allotypic variant or a distinct joining gene segment will require further studies.

We observed the greatest variation in the J{gamma}1.2 segment. All four amino acid replacements were localized to the NH2 terminus of the protein segment (Fig. 1Go). The extent of homology was 90% at nucleic acid and 76% at protein level. Surprisingly, we found no mutation at all in the J{gamma}2.3 sequences. Altogether these data point out several differences and confirm the anticipated high homology of {gamma} TCR chain gene segments between rhesus monkeys and human beings.

Limited diversity of amino acid sequences coded by V{gamma}2-J{gamma}1.2 CDR3 regions in peripheral blood; shared sequences between different unrelated animals

Previously we established that the diversity of V{gamma}2 CDR3 lengths in rhesus macaque peripheral blood is extremely restricted (34). To understand the molecular basis for this restriction we sequenced samples from three adult unrelated animals. The V{gamma}2-J{gamma}1.2 junction region was preferentially expressed in all three animals (Fig. 2Go), and V{gamma}2-J{gamma}2.3 junctions were detected less frequently. J{gamma}1.1 and J{gamma}rh joining regions were underrepresented in all three animals. Almost all of the V{gamma}2-J{gamma}1.2, ~30% of the V{gamma}2-J{gamma}2.3, and 20% of the V{gamma}2-J{gamma}rh recombinations were in frame. More than 70% of the J{gamma}1.2-containing segments had six-amino-acid-long CDR3 regions. The CDR3 lengths encompassed by V{gamma}2-J{gamma}2.3 junctions were more diverse. We often found transcripts shared either by all three, or just two of the animals or we detected clones with identical amino acid sequences. The amino acid motif WEVQQF was abundant, being represented by clones with four distinct coding sequences. The CDR3 regions WEVQGQF, WEGQQF, WEVKQF, WESQQF, and WEVGQF were found in two or all three of the monkeys, sometimes with identical transcripts. In the case of V{gamma}2-J{gamma}2.3 recombination we found no shared nucleic or amino acid sequences. Overall, repeated encounters with common V{gamma}2-J{gamma}1.2, but not V{gamma}2-J{gamma}2.3 motifs in three unrelated adult animals provide evidence for a strong, permanent selection for these receptor sequences that was independent of genetic background.




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  		FIGURE 2. Restricted CDR3 diversity in the peripheral blood V{gamma}2 TCR repertoire of rhesus macaques. In frame junctions are labeled +, out of frame junctions are labeled -. Number of clones is the frequency at which a particular sequence was found among cDNA clones.

 
Increased proportion of V{gamma}2-J{gamma}2.3 transcripts in the sigmoid colon; shared motifs between different animals

To assess the extent of V{gamma}2 TCR diversity in mucosal compartments, we sequenced cDNA samples derived from sigmoid colon biopsies. V{gamma}2-C{gamma} transcripts from peripheral blood samples were characterized by a dominant 284-base-long primer extension product length (Fig. 3GoA). (The size of this product is determined by our choice of primer sequences and is used here for identification only.) In contrast, V{gamma}2-C{gamma} transcripts from intestinal samples cannot be characterized by one predominant length, but usually they contain products of shorter lengths (Fig. 3GoB). Sequencing revealed that the majority of transcripts are V{gamma}2-J{gamma}2.3 recombinations. However, a significant number of the V{gamma}2-J{gamma}2.3 recombinations produced out of frame sequences, and we even detected a higher number of out of frame V{gamma}2-J{gamma}1.2 recombinations (Fig. 4Go). Next we determined whether the functional transcripts still display a similar length pattern in the blood and sigmoid colon. We found an enrichment for shorter functional transcripts in the intestine, the pattern of which was primarily due to an increased usage of the shorter J{gamma}2.3 joining segment (Fig. 5Go).



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  		FIGURE 3. Primer extension product length diversity in V{gamma}2 TCR family from peripheral blood and sigmoid colon. Scattergrams show the relative amount of each peak of primer extension product length analysis. Each symbol represents an individual monkey, altogether including nine animals. A, Peripheral blood. B, Sigmoid colon.

 



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  		FIGURE 4. Restricted CDR3 diversity in V{gamma}2 TCR repertoire of rhesus macaque sigmoid colon. In frame junctions are labeled +, out of frame junctions are labeled -. Number of clones is the frequency at which a particular sequence was detected. Sequences labeled with bold letters are found in multiple samples from the same donor. Sequences labeled with italic bold letters are motifs shared between different animals.

 


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  		FIGURE 5. Primer extension product length diversity from in frame V{gamma}2-J{gamma} junctions of matching peripheral blood and sigmoid colon. A, Peripheral blood. B, Sigmoid colon. Histogram displays data derived from animal 1. Number of clones represents the frequency of individual clones.

 
The amino acid sequence WEVQQF encoded by the TGGGAGGTGCAACAGTTT sequence was abundant in the blood and was also found in the colon samples from all three animals. The possibility that this clone arose from contaminating blood cells cannot be excluded, although the lack of other sequences representing expanded clones in the blood makes contamination improbable. Additionally, we detected a V{gamma}2-J{gamma}2.3 transcript expressing the CDR3 motif WEVRYK in two animals; in one of them it was a repeated transcript, suggesting previous expansion of this clone.

To assess the reproducibility of our sampling method we simultaneously processed two pools of samples (biopsies taken at the same time) from the same animal. We observed significant overlap between the two sets of clones, irrespective of whether the recombinations were in frame or not (Fig. 4Go, animal 3/1 and 2 bold-faced sequences). We also compared the retrieved sequences with a set of clones that was derived from specimens taken 2 months previously. Again common motifs were detected with both in and out of frame recombinations (Fig. 4Go, animal 3/3 bold-faced sequences). Altogether, the excellent reproducibility of V{gamma}2 TCR chain repertoire in parallel samples argues against sampling errors. These results indicate that the sigmoid colon contains an oligoclonally expanded V{gamma}2 TCR+ cell subset, and the repertoire is shared among genetically distinct animals.

Common V{gamma}2-C{gamma} motifs between intestinal and vaginal samples of the same animal

To determine the diversity of V{gamma}2 TCR repertoire at another mucosal site, we analyzed vaginal samples from six animals. We found that the average primer extension product length diversity is very similar to the peripheral blood (Fig. 6Go). Sequence data derived from two animals showed no elevated frequency of V{gamma}2-J{gamma}2.3 transcripts (Fig. 7Go). As expected on the basis of published human data (40, 41, 42, 43), there was a significant overlap between the vaginal and matching blood V{gamma}2 TCR repertoires. Because the female reproductive tract is highly vascularized, additional experiments are needed to show whether this overlap can be attributed to contaminating blood cells. Remarkably, we found two in frame transcripts, one of them V{gamma}2-J{gamma}1.2 and the other V{gamma}2-J{gamma}2.3, which were present in both the vaginal and intestinal repertoires. These sequences were not encountered in the peripheral blood. V{gamma}2-J{gamma}2.3 transcript WEIKPSYYK was found repeatedly in both mucosal tissues, suggesting that it represents a T cell population that is common at both tissue sites.



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  		FIGURE 6. Primer extension product length diversity in V{gamma}2 TCR family from vagina. Scattergram shows the relative amount of each peak of primer extension product length analysis. Each symbol represents an individual monkey, altogether including six animals.

 


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  		FIGURE 7. Restricted CDR3 diversity in V{gamma}2 TCR repertoire from rhesus macaques vagina. In frame junctions are labeled +, out of frame junctions are labeled -. Number of clones is the frequency at which a particular sequence was found among cDNA clones. Sequences labeled with italic bold letters are motifs shared between colon and vagina of the same donor.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our objective was to assess the diversity of V{gamma}2 TCR repertoire at different anatomical compartments in rhesus macaques to establish an in vivo model for V{gamma}2 T cell repertoire studies. We observed that the CDR3 length diversity for the V{gamma}2 T cell subset in peripheral blood is similarly limited in humans and rhesus macaques (25, 26, 34). In these studies we show that this similarity relies on extensive structural homology within the peripheral blood V{gamma}2 TCR repertoire for M. mulatta and H. sapiens. Rhesus monkeys have four counterparts of the known human J{gamma} segments (38, 39) but, like in humans, the V{gamma}2-J{gamma}1.2 junctions predominate. The overwhelming majority of these junctions have CDR3 length of 6 ± 1 amino acids in both humans and macaques. In humans the canonical V{gamma}2-J{gamma}1.2 junction encodes the amino acid sequence WEVQEL. Although rhesus monkeys do not express this particular sequence, a CDR3 region was identified that encoded the motif WEVQQF and was frequently encountered at multiple anatomical locations in all of the investigated animals. On the basis of consensus sequences of rhesus V{gamma}2-J{gamma}1.2 recombinations and sequence homology between human and rhesus, we postulate that this sequence might be a canonical sequence in macaques. A common CDR3 motif WEVXEL was observed in 30–50% of human peripheral blood V{gamma}2-J{gamma}1.2 junctions (26). In rhesus the WEVXQF motif was observed at similar frequencies (20.8–44.7% of the blood V{gamma}2-J{gamma}1.2 junctions). Finally, several motifs that included the carboxyl-terminal of V{gamma}2 segment and amino acids encoded by palindrome/N-nucleotides were found in both humans and rhesus monkeys (WEVQ, WESQ, WEAQ, WEVG). These phylogenetically conserved sequences suggest strong selection for the structures represented by them. The mechanism of selection remains to be established; however, it equally well could be attributed to the presence of ubiquitous Ags (44), a diversifying machinery of limited capacity (45), or a selection pressure for certain {gamma}-chain conformations that may or may not have direct contact with the Ag (46).

The diversity of V{gamma}2 TCR chain in the intestine is restricted, but different from peripheral blood, suggesting that selection mechanisms operating in blood are absent, or overruled in the intestine. There are no published examples of shared {gamma}{delta} TCR CDR3 sequences in adult human intestinal samples. However, common V{gamma}2 and V{delta}2 TCR CDR3 motifs have been observed between different fetal tissues (including gut and liver) of the same individual or between genetically distinct subjects (27, 47). Our results identify common V{gamma}2 TCR species in the colon of unrelated adult animals, suggesting a dominant and ubiquitous selection mechanism. Further studies are necessary to explore different possible explanations for this unexpected observation, e.g., shared sequences of V{gamma} but not V{delta} TCR might also exist in adult humans, or shared CDR3 motifs might be frequent in some mammals and appear in early human ontogeny, mainly as a reiteration of phylogeny.

In mice, the nonoverlapping {gamma} TCR chain variable gene segment usage of tissue resident {gamma}{delta} T cell populations (48, 49, 50, 51, 52, 53) and, in humans, the nonoverlapping CDR3 repertoire of {delta} TCR chains between intestine and blood (54, 55, 56) indicated strict compartmentalization of {gamma}{delta} T cell subsets. In human beings, {delta} TCR chain repertoires found at distant locations in the intestine (duodenum, colon) from a single individual contained common clones, suggesting the presence of ubiquitous Ags distributed throughout the digestive tract, or the original population of these sites by a small number of progenitor cells (56). Our data are the first to show that although the V{gamma}2 T cell repertoire of colon and vagina is mostly distinct, shared sequences can be detected. This observation might indicate the presence of common Ag(s) and/or a possible {gamma}{delta} T cell trafficking between the reproductive and digestive tract of healthy animals.

Processes controlling {gamma}{delta} T cell trafficking and tissue targeting remain largely unknown. Our results are consistent with studies that showed anatomical compartmentalization of {gamma}{delta} T cells. Within mucosal tissue sites, the {gamma}{delta} T cell populations are largely distinct, but the occasional appearance of common V{gamma}2 sequences suggests either cell trafficking between these sites or some overlap in the Ags that might be responsible for selecting and maintaining the local {gamma}{delta} T cell repertoire. The extensive similarity between V{gamma}2 TCR repertoire in rhesus and human shows that this animal model will be useful for elucidating mechanisms that control {gamma}{delta} cell biology.


   Acknowledgments
 
We thank Maria T. Zayas, Jody Helgeland, Leonard Acker, and Carol L. Emerson for their expert help with the endoscopy procedures, and the Animal Care Unit of Wisconsin Regional Primate Research Center for their extraordinary assistance. We also are grateful to Angelia Schoolmeister and Jacques L. Mitchen for their technical assistance. We thank Tracy Ruckwardt and Peter S. Evans for their invaluable help in primer extension product length analysis.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grant RR00167 (Regional Primate Center support) and AI 38491 (to C.D.P.). Back

2 Current address: Institute of Human Virology, Medical Biotechnology Center, 725 West Lombard Street, Baltimore, MD 21201. Back

3 Address correspondence and reprint requests to Dr. Eva Rakasz, University of Wisconsin-Madison, Wisconsin Regional Primate Research Center, 1220 Capitol Court, Madison, WI 53715. Back

4 Abbreviation used in this paper: CDR3, complementarity-determining region 3. Back

Received for publication August 17, 2000. Accepted for publication November 27, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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