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Originally published online as doi:10.1189/jlb.0703326 on October 2, 2003

Published online before print October 2, 2003
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(Journal of Leukocyte Biology. 2004;75:68-75.)
© 2004 by Society for Leukocyte Biology

Subset-specific, uniform activation among V{gamma}6/V{delta}1+ {gamma}{delta} T cells elicited by inflammation

Christina L. Roark*,1, M. Kemal Aydintug*,1, Julie Lewis{dagger}, Xiang Yin*, Michael Lahn*, Youn-Soo Hahn*, Willi K. Born*, Robert E. Tigelaar{dagger} and Rebecca L. O’Brien*,2

* Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, Colorado; and
{dagger} Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut

2Correspondence: Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: obrienr{at}njc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The V{gamma}6/V{delta}1+ cells, the second murine {gamma}{delta} T cell subset to arise in the thymus, express a nearly invariant T cell receptor (TCR), colonize select tissues, and expand preferentially in other tissues during inflammation. These cells are thought to help in regulating the inflammatory response. Until now, V{gamma}6/V{delta}1+ cells have only been detectable indirectly, by expression of V{gamma}6-encoding mRNA. Here, we report that 17D1, a monoclonal antibody, which detects the related epidermis-associated V{gamma}5/V{delta}1+ TCR, will also bind the V{gamma}6/V{delta}1+ cells if their TCR is first complexed to an anti-C{delta} antibody. Features of this special condition for recognition suggest the possibility that an alternate structure exists for the V{gamma}6/V{delta}1 TCR, which is stabilized upon binding to the anti-C{delta} antibody. Using the 17D1 antibody as means to track this {gamma}{delta} T cell subset by flow cytometry, we discovered that the response of V{gamma}6/V{delta}1+ cells during inflammation often far exceeds that of other subsets and that the responding V{gamma}6/V{delta}1+ cells display a strikingly uniform activation/memory phenotype compared with other {gamma}{delta} T cell subsets.

Key Words: T cells • T cell receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {gamma}{delta} T lymphocytes comprise a type of T cell in mice and humans whose function appears to be separate from that of other lymphocytes (reviewed in ref. [1 ]). The {gamma}{delta} T cells bear cell-surface T cell receptors (TCRs) related to but distinct from those carried by {alpha}ß T cells and often respond to autologous ligands rather than foreign antigens. Several lines of evidence suggest that {gamma}{delta} T cells act to regulate adaptive- and innate-immune responses, particularly during inflammation. Controversies over the role they play may be explained by findings suggesting that {gamma}{delta} T cell subsets have different functions [1 ].

The {gamma}{delta} T cells differ from the {alpha}ß T cells in their tendency to limit rather than to maximize the potential diversity of their TCRs. This is often seen as restricted pairing of certain V{gamma} and V{delta} chains, but in mice, the limitation goes so far that two subsets of {gamma}{delta} T cells arise whose TCRs are virtually invariant. These "canonical" TCRs are highly conserved among rodents [2 ], and they arise in the fetal thymus as a result of controlled gene rearrangements [3 , 4 ]. One of these {gamma}{delta} T cell populations, the V{gamma}6/V{delta}1 subset (GV2S1/DV101S1), is generated only for a short period of time in the late fetal stages [3 ]. It represents the second {gamma}{delta} T cell subset to arise in the thymus, preceded by the closely related V{gamma}5/V{delta}1 subset (GV1S1/DV101S1), whose TCR is also generally invariant.

The V{gamma}6/V{delta}1 subset has been reported to predominate among the T cells normally present in certain epithelial sites, in particular, the female reproductive tract [5 ] and the lung [6 ]. A preferential expansion of V{gamma}6/V{delta}1+ cells has also been noted in a variety of experimental systems that induce an inflammatory response. These include Listeria infection of the liver [7 ] and kidney [8 ], autoimmune and infection-induced orchitis [9 , 10 ], experimental allergic encephalomyelitis [11 ], drug-induced kidney damage [12 ], and Escherichia coli intraperitoneal infection [13 ]. The subset also expands in the uterine lining during pregnancy [14 ]. Despite their responses, the functional role played by V{gamma}6/V{delta}1+ cells during inflammation is not known; there is some evidence that they play a down-regulatory role [8 9 10 ], but some groups disputed this [15 , 16 ].

Studies on the role of the V{gamma}6/V{delta}1+ subset have been limited by lack of an antibody that specifically recognizes this TCR. We report here that 17D1, a monoclonal antibody (mAb) initially thought to be idiotype-specific for the mouse V{gamma}5/V{delta}1 canonical TCR [17 ], in fact, also recognizes V{gamma}6/V{delta}1+ cells if their TCR is first complexed to an anti-C{delta} mAb. Using the 17D1 mAb as a staining reagent to detect V{gamma}6/V{delta}1+ cells, we directly examined the distribution of this subset, tracked its accumulation during Listeria-induced inflammation of the liver and spleen and in the draining lymph nodes of complete Freund’s adjuvant (CFA)-treated mice, and directly examined memory/activation markers expressed by responding V{gamma}6/V{delta}1+ cells. Although rare in most sites, the V{gamma}6/V{delta}1+ cells were found to increase to a much greater extent than other {gamma}{delta} T cell subsets during certain types of inflammation, an increase additionally remarkable for its associated acquisition of a nearly uniform pattern of activation/memory markers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Age and sex-matched C57Bl/10J and C57BL/6J mice bred in-house from Jackson stock (Jackson Laboratories, Bar Harbor, ME) were used in these experiments, at 5–12 weeks of age (or see, as noted, Table 2 ). Male DBA/1 mice, 8–10 weeks of age, were purchased from Jackson Laboratories and used in the CFA experiments (see below).


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  		Table 2. The Distribution of V{gamma}6/V{delta}1+ Cells in Various Tissues of Normal B10 Mice

 
Flow cytometry
One-, two-, and three-color analyses were performed as described previously [10 ], except that for some normal cell analysis, Fc{gamma} receptors were preblocked by a 10-min incubation in 50% normal mouse serum plus 50% 2.4G2 hybridoma supernatant or with purified 2.4G2 mAb at 5 µg/ml. The mAb used include GL3 (anti-C{delta}) [18 ], 403-A10 (anti-C{delta}) [19 ], UC7-13D5 (anti-C{delta}; eBioscience, San Diego, CA) [20 ], 2.11 (anti-V{gamma}1, GV5S1) [21 ], F536 (anti-V{gamma}5, GV1S1) [22 ], UC3 (anti-V{gamma}4, GV3S1) [23 ], GL1 (anti-V{gamma}7, GV4S1) [18 ], GL2 (anti-V{delta}4, DV104S1) [18 ], 17C (anti-V{delta}6.3, ADV7S1) [24 ], F45.152 (anti-V{delta}5, DV105S1) [25 ], 17D1 (anti-V{gamma}5/V{delta}1) [17 ], and KT3 (anti-CD3) [26 ]. Phycoerythrin (PE)-labeled mAb for TCR activation/memory markers (CD62L, CD44, and CD45RB) were purchased from BD Sciences PharMingen (San Diego, CA). Except as noted, all of these mAb were grown and purified in our laboratory by Protein A or G chromatography and were conjugated to biotin or fluorescein isothiocyanate (FITC) using standard methods. However, 17D1 [a rat immunogloulin (Ig)M antibody] lost all activity when purified in this way and was therefore instead prepared by ammonium sulfate precipitation from the supernatant of hybridoma cells cultured without fetal bovine serum in protein-free hybridoma medium II (Gibco-BRL, Grand Island, NY). A mAb cocktail was used in some experiments, composed of a mixture of biotinylated versions 2.11, F536, UC3, GL1, GL2, 17C, and F45.152. PE–streptavidin (Rockland, Gilbertsville, PA) or allophycocyanin–streptavidin (Caltag, Burlingame, CA) was used to detect biotinylated mAb. All staining steps were performed for 20–30 min at 4°C in the presence of sodium azide. For 17D1 staining of V{gamma}6/V{delta}1+ cells, cells were first incubated with anti-C{delta} antibody for 20–30 min, washed two times, and then incubated with biotinylated 17D1 for 20–30 min. Analysis was performed on data generated with a FACScan or FACSCalibur flow cytometer, using CellQuest or Flowjo software.

Listeria infection
Listeria monocytogenes EGD were freshly grown from frozen aliquots in tryptose phosphate broth (Difco Laboratories, Detroit, MI). C57BL/10 mice were inoculated with 2–3 x 103 L. monocytogenes EGD by a 0.2-cc intravenous (i.v.) injection (tail vein), killed 5 days after inoculation, and spleen and liver T cells were prepared as described previously [27 ].

CFA-elicited inflammation
DBA/1 mice were immunized with 100 µl of a 1:1 emulsion of phosphate-buffered saline (PBS)/CFA intradermally (i.d.) at the base of the tail on days 0 and 21. Inguinal and popliteal lymph nodes were removed on day 42 for analysis. Cells were isolated and passed over nylon–wool columns to enrich for T cells and were stained as described above.

Generation of soluble {gamma}{delta} TCRs
Baculovirus constructs containing the desired V{gamma} and V{delta} genes were produced by polymerase chain reaction amplification of appropriate cDNAs using methods similar to those described previously [28 ]. Constructs were transfected into Sf9 insect cells, and the recombinant baculoviruses were propagated in High Five insect cells for protein production using standard methods (BD Sciences PharMingen). The soluble {gamma}{delta} TCRs were purified from culture supernatant using sepharose-affinity columns prepared with cyanogen bromide-activated CL-4B beads (Sigma Chemical Co., St. Louis, MO) coupled to anti-C{delta} (GL3; M. K. Aydintug et al., manuscript in preparation).

MAb-binding inhibition assay
We determined the ability of the soluble TCR preparation to bind to the anti-C{delta} mAb GL3, which recognizes {gamma}{delta} TCRs in native form only, using a staining inhibition assay. Here, a titered amount (0.25–2.0 mg) of each soluble TCR was mixed with 40 ng GL3 mAb, and after incubating 1 h, the mixture was tested for its ability to stain a hybridoma bearing a {gamma}{delta} TCR. Binding of the GL3 mAb was detected using a FITC-labeled anti-hamster Ig secondary antibody (Jackson ImmunoResearch, West Grove, PA). The reduction in staining with soluble TCR-treated GL3 as compared with untreated GL3 constitutes a relative measure of the amount of intact, correctly folded, soluble TCR present in individual preparations.

Soluble TCR enzyme-linked immunosorbent assay (ELISA)
A capture ELISA was performed using the anti-C{delta} mAb GL3, using a standard ELISA protocol [29 ]. Briefly, 96-well ELISA plates were coated overnight with GL3 mAb, postcoated with gelatin, and soluble TCRs then captured on the wells from serially diluted samples. Biotinylated 17D1 was used detect the bound, soluble TCRs, together with horseradish peroxide-conjugated streptavidin and colorimetric substrate (tetramethyl benzidine; Zymed, San Francisco, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The 17D1 mAb can detect V{gamma}6/V{delta}1+ cells
The mAb 17D1 was initially considered to be specific for the V{gamma}5/V{delta}1 canonical TCR. Later, it was found that 17D1 could also detect V{gamma}1+ cells co-expressing a V{delta}1 chain, a combination rare or absent in normal mice but present among skin {gamma}{delta} T cells of V{gamma}5 gene-disrupted mice [17 ]. The V{delta}1 chain of these cells matched the canonical sequence for the V{gamma}5/V{delta}1 subset, whereas the V{gamma}1 chain had a highly constrained but not invariant junctional sequence. Thus, another V{gamma} in conjunction with this particular V{delta}1 chain was with certain restrictions able to provide a structure similar enough to the V{gamma}5/V{delta}1 canonical TCR to be recognized by 17D1. The V{gamma}6/V{delta}1 and V{gamma}5/V{delta}1 canonical TCRs are likewise similar; both express a V{delta} chain of identical sequence, and whereas they express different V{gamma}s, their {gamma} junctions are identical [5 , 30 ]. We found that if first complexed to an anti-C{delta} mAb, V{gamma}6/V{delta}1+ cells could also be detected with 17D1 (Fig. 1A ). No staining with 17D1 was evident with T cell hybridomas expressing unrelated {gamma}{delta} TCRs, including a hybridoma expressing the canonical V{gamma}6 together with a different V{delta} (see BNT-18.8.8, Table 1A ). Although the canonical V{gamma}6/V{delta}1 TCR was not absolutely required, one noncanonical version evidently failed to stain with 17D1 (Fig. 1B and Table 1B ). Pretreatment with anti-C{delta} mAb was necessary for detectable 17D1 staining of V{gamma}6/V{delta}1+ but not V{gamma}5/V{delta}1+ cell lines; treatment of V{gamma}6/V{delta}1+ cells with anti-C{delta} after 17D1 resulted in anti-C{delta} staining only (not shown). Three anti-C{delta} mAb, derived independently (GL3, 403-A10, and UC7-13D5), were effective in conferring 17D1 staining (two shown in Fig. 1C ), although an anti-CD3 mAb was not effective (Fig. 1C) .



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  		Figure 1. The 17D1 mAb can detect hybridomas bearing a V{gamma}6/V{delta}1 TCR. (A) 17D1-staining profiles of representative cell lines bearing a V{gamma}5/V{delta}1, V{gamma}6/V{delta}1, or V{gamma}1/V{delta}6.3 TCR. Cells were incubated with biotinylated 17D1 only or with biotinylated 17D1 after prestaining with FITC-conjugated GL3 (anti-C{delta}). Staining with GL3–FITC plus biotinylated F536 (anti-V{gamma}5) is also shown as a control. (B) 90BPL-2, a V{gamma}6/V{delta}1 hybridoma expressing a canonical and a noncanonical V{delta}1 chain (see Table 1B ), stains poorly with 17D1 as compared with TCR level-matched hybridoma 123BLT-27, bearing the canonical V{gamma}6/V{delta}1 TCR only. Cells stained with biotinylated 17D1 were prestained with GL3–FITC. (C) Staining of 90BPL-3.27, a V{gamma}6/V{delta}1+ canonical hybridoma, with 17D1, following various pretreatments including anti-C{delta} mAb GL3 and UC7-13D5 (denoted UC7) and anti-CD3 mAb KT3. (D) Binding of 17D1 to purified TCRs in an ELISA assay. The V{gamma}5/V{delta}1 canonical TCR is 5:1, 6:1 is the V{gamma}6/V{delta}1 canonical TCR, 4:5 is a V{gamma}4/V{delta}5+ TCR originally derived from the T22-reactive hybridoma line KN6, and {alpha}ß represents the negative control, an {alpha}ß TCR-derived from the ovalbumin-reactive hybridoma DO-11.10. O.D., Optical density. (E) Antibody-inhibition assay, showing the ability of the two soluble TCR preparations to block the anti-C{delta} mAb GL3. Blocking is measured by the decrease in the GL3 available to bind to a {gamma}{delta} TCR+ hybridoma (70BET49); bound GL3 was detected with a FITC-labeled, anti-hamster IgG secondary antibody. MFI, Mean fluorescence intensity.

 

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  		Table 1A. 17D1 Staining Profiles for Hybridoma Cells Bearing a Variety of {gamma}{delta} TCRs

 

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  		Table 1B. V{gamma} and V{delta} Junctional Sequences that Permit, or Fail to Permit, 17D1 Binding after Pretreatment with Anti-C{delta}

 
17D1 has been confirmed by immunoprecipitation to recognize the TCR of V{gamma}5/V{delta}1+ cells [17 ]. To verify that it binds the TCR as well on V{gamma}6/V{delta}1+ cells, we tested whether 17D1 is able to specifically detect the V{gamma}6/V{delta}1 TCR in an ELISA. Using soluble recombinant {gamma}{delta} TCRs generated in vitro and captured on assay plates with anti-C{delta} mAb, we found that 17D1 bound to the canonical V{gamma}6/V{delta}1 and V{gamma}5/V{delta}1 TCRs, although it bound the V{gamma}5/V{delta}1 TCR better (Fig. 1D) , apparently as a result of an affinity difference, as these two TCR preparations were of nearly identical quality (Fig. 1E) . In contrast, an unrelated, soluble {gamma}{delta} TCR and a soluble {alpha}ß TCR showed no 17D1 binding.

The TCR requirements for 17D1 binding can thus be assessed from hybridomas staining with 17D1. Although V{delta}1 expression seems crucial, 17D1 has no strict requirement for a particular D{delta} reading frame or even J{delta} (Table 1B) . However, the {delta} junctional length may be important, as the one nonbinding V{delta}1 identified also has a longer junction than do any of those that bind. Although all V{gamma}6+ cell lines we tested in this study had the canonical V{gamma}6 sequence, neither V{gamma}6 nor the canonical {gamma} junction could be required, as the V{gamma}1/V{delta}1+ cells that arise in V{gamma}5 knockout mice can bind 17D1 even without anti-C{delta} pretreatment, although length and/or sequence restrictions for these V{gamma}1 junctions probably exist [17 ].

V{gamma}6/V{delta}1+ cells are rare in most normal tissues
We first tested the ability of 17D1 to stain normal lymphocytes using cells from V{gamma}6/V{delta}1 transgenic mice, whose T cells predominantly express the V{gamma}6/V{delta}1 TCR [31 ]. After anti-C{delta} pretreatment, 17D1 was able to stain most of the splenic, transgenic {gamma}{delta} T cells in these mice (Fig. 2A ).



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  		Figure 2. 17D1 also stains normal V{gamma}6/V{delta}1-bearing cells following preincubation with anti-C{delta}. (A) Nylon–wool-nonadherent V{gamma}6/V{delta}1 transgenic spleen cells were stained as indicated. Of the T cells in these mice that express an {alpha}ß TCR, many were found to weakly co-express a (presumably transgene-encoded) {gamma}{delta} TCR as well (left). When prestained with GL-3 FITC, virtually all of the {gamma}{delta} TCR+ cells also stain with biotinylated 17D1 (center). Consistent with the hybridoma staining, 17D1 cannot stain theV{gamma}6/V{delta}1+ transgenic T cells without anti-C{delta} pretreatment (right). Percentages shown are relative to total events. (B) 17D1 detects many {gamma}{delta} T cells in the livers of Listeria-infected mice. Five-week-old C57BL/6 mice were used in this experiment. Infected mice received 2 x 103 L. monocytogenes by i.v. injection 5 days earlier; sham-infected controls, instead, received an inoculum of the same volume containing nonpyrogenic saline only. Nylon–wool-purified liver T cells from individual mice were stained as indicated in each profile (percentages shown are relative to total C{delta}+ cells). (C) The expansion of V{gamma}6/V{delta}1+ {gamma}{delta} T cells in the spleens of Listeria-infected mice exceeds that of other {gamma}{delta} T cell subsets. Nylon–wool-purified T cells from spleens of the same mice analyzed in B, infected or sham-infected, were analyzed by two-color flow cytometry. The number of cells in each subset was calculated based on the percent of total events that each represented, multiplied by the number of total nylon–wool-nonadherent cells obtained from each spleen. Error bars represent the SD for each group of three individual spleens.

 
The actual prevalence of the V{gamma}6/V{delta}1 subset has been unclear as a result of the necessity to detect these cells by indirect means. We therefore next determined the percentage of V{gamma}6/V{delta}1+ {gamma}{delta} T cells in various tissues of normal mice by flow cytometry using labeled anti-C{delta} and 17D1 mAb (Table 2 ). For comparison, we also determined the number of V{gamma}6/V{delta}1 cells in most of these tissues by their lack of staining with a mAb cocktail, which detects most other {gamma}{delta} T cell types, at the same time or in other experiments. The V{gamma}6/V{delta}1+ subset was rare or absent among the {gamma}{delta} T cells of normal spleen, adult thymus, and blood. In contrast, as earlier studies predicted [5 , 6 , 14 ], the V{gamma}6/V{delta}1+ cells comprised a substantial fraction of the {gamma}{delta} T cells present in the fetal and newborn thymus, the newborn spleen, the lung, and the placenta/decidua. In general, the percentage of cells detected by 17D1 agreed well with the percentage calculated by staining with the cocktail. However, some discrepancy appeared in fetal and newborn thymuses, where 17D1 staining usually detected more cells than did the cocktail method, even when the percentage of V{gamma}5+ cells (determined concurrently) was first subtracted from the 17D1+ cells. This is probably because we often had difficulty in fully separating cells in these profiles that expressed low levels of V{gamma}5. We suggest that these low V{gamma}5+ cells in the developing thymus, which nonetheless appear to stain well with 17D1, actually coexpress V{gamma}5 and V{gamma}6 chains. Indeed, virtually all V{gamma}6+ cells also carry a rearranged V{gamma}5 gene, although it is not expressed in mature cells [32 ].

Although they represented only ~7% of {gamma}{delta} TCR+ cells in the C57BL/10 lung (Table 2) , in BALB/c mice, ~30% of the pulmonary {gamma}{delta} T cells were found by 17D1 staining to be V{gamma}6/V{delta}1+ (not shown), revealing an apparent strain difference in relative abundance of this subset. Despite their seeming preference for mucosal sites, V{gamma}6/V{delta}1+ cells were virtually undetectable among intraepithelial cells of the intestinal mucosa, although {gamma}{delta} T cells are comparatively abundant in this tissue. In the normal liver, although in most cases, the V{gamma}6/V{delta}1+ cells were rare, we occasionally observed outliers in which they represented 6–7% of the total {gamma}{delta} T cells (obvious outliers not included in the averages shown in Table 2 ). We suspect this might indicate that an otherwise undiscovered infection or wound is present in our presumed "normal" controls. Juvenile mice also had a higher average level of liver V{gamma}6/V{delta}1+ cells than was typical in full-grown adults (8 weeks +).

17D1 can be used to track the response of the V{gamma}6/V{delta}1+ cells elicited during inflammation
Indirect methods have shown that the V{gamma}6/V{delta}1 {gamma}{delta} T cell subset expands during inflammation, including L. monocytogenes infection [7 ]. Using 17D1, we discovered that in the liver and spleen of Listeria-infected mice, the response of the V{gamma}6/V{delta}1 subset substantially exceeds that of other subsets. Figure 2B shows flow cytometry profiles obtained from liver T cells of three sham-infected controls versus three individual Listeria-infected mice; younger mice, not yet fully grown (~5 weeks old), were used for this experiment to test whether the higher "background levels" of V{gamma}6/V{delta}1+ cells we noted in juveniles would affect the response. Whereas the uninfected controls indeed had higher percentages of 17D1+ cells than is typical for older mice, an increase in the V{gamma}6/V{delta}1+ subset was apparent in all three of the infected mice. In similar experiments (Table 2 and data not shown), we have verified that few if any of the 17D1+ cells present in naive or Listeria-infected spleens and livers instead represent V{gamma}5/V{delta}1+ cells, as they failed to stain with an anti-V{gamma}5 mAb; hence, virtually all of the 17D1+ cells shown in Figure 2B must represent V{gamma}6/V{delta}1+ cells. Using the percentages shown, we calculated that the actual number of liver V{gamma}6/V{delta}1+ cells in this experiment had increased an average of 24-fold at this point in the infection, whereas liver {gamma}{delta} T cells as a whole increased by an average of only 2.4-fold. In the spleens of the same mice, 17D1 staining revealed a somewhat lesser but again highly biased increase in the same subset, such that the infection-driven increase in V{gamma}6/V{delta}1+ cell numbers was 18-fold versus twofold or less for other subsets (Fig. 2C) . Moreover, Listeria-elicited increases in liver V{gamma}6/V{delta}1+ cells as high as 64-fold were noted in some experiments (not shown). As V{gamma}6/V{delta}1+ cells are quite rare in the blood, spleen, and lymph nodes of uninfected mice, it seems likely that these expansions are largely the result of proliferation rather than infiltration, although we have yet to test this directly.

V{gamma}6/V{delta}1+ cells elicited by treatment with CFA reveal activation/memory marker differences as compared with other responding {gamma}{delta} T cell subsets
Using the 17D1 mAb, we have also recently noted that an infiltration of V{gamma}6/V{delta}1+ cells is often evident in the draining lymph nodes of mice treated previously with CFA (Fig. 3 ; for unknown reasons, this infiltration varied in individual mice from nearly undetectable to predominant among the {gamma}{delta} T cells). When analyzed by flow cytometry, we found that the V{gamma}6/V{delta}1+ and V{gamma}1+ cells in the lymph nodes of mice showing a strong response of V{gamma}6/V{delta}1+ cells were almost diametrically opposed to one another with regard to expression of activation/memory markers. Whereas virtually all of the V{gamma}6/V{delta}1+ cells from the Freund’s adjuvant-treated mice show an activated/memory phenotype—CD62L low, CD44 high, and CD45RB low—only a minor subset of the V{gamma}1+ cells from the same mice fits this paradigm, as the majority is instead CD62L high, CD44 low, and CD45RB high (Fig. 3A) . In contrast, most of the V{gamma}6/V{delta}1+ and V{gamma}1+ {gamma}{delta} T cells isolated from untreated mice displayed a naïve activation profile, as expected (Fig. 3B) . By analogy with {alpha}ß T cells [33 ], this observation suggests that the expansion of the V{gamma}6/V{delta}1 {gamma}{delta} T cell subset in Freund’s adjuvant-treated mice is driven by TCR-mediated activation. Although the {gamma}{delta} T cells during the long-term inflammation evoked by CFA often expand to a greater extent in the draining lymph nodes than they do in the livers of mice infected briefly with Listeria, the increase of the V{gamma}6/V{delta}1+ subset again greatly exceeds that of other subsets, including the V{gamma}1+ cells (450-fold vs. tenfold, respectively, in the example shown in Fig. 3 ).



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  		Figure 3. The V{gamma}6/V{delta}1 subset, present in the draining lymph nodes of CFA-treated mice but not the V{gamma}1 subset, shows a highly activated phenotype. (A) Flow cytometry scatter profiles identifying the V{gamma}1 and V{gamma}6/V{delta}1 subsets among T cells derived from the draining (inguinal and popliteal) lymph nodes of a representative DBA/1 mouse previously treated by i.d. injection at the base of the tail with a 50% emulsion of CFA in PBS. Percentages of V{gamma}6/V{delta}1+ (GL3/17D1+) and V{gamma}1+ (GL3/2.11+) {gamma}{delta} T cells, as a fraction of all C{delta}+ cells, are indicated. Virtually no cells were detected in these samples that costained with GL3 and an anti-V{gamma}5 mAb (F536), indicating that the GL3/17D1+ cells detected are indeed V{gamma}6/V{delta}1+ (not shown). The V{gamma}6/V{delta}1+ and V{gamma}1+ subsets were further analyzed individually for cell-surface markers whose expression levels are dependent on an activated or memory phenotype by three-color flow cytometric analysis. Cells double-positive for anti-C{delta} and 17D1 (V{gamma}6/V{delta}1) or 2.11 (V{gamma}1) were used to set gates (upper right quadrants), and the activation/memory markers expressed in each gate were then determined. The percentages shown in the histograms indicate the relative frequency in each subset of cells bearing the memory-associated (e.g., high or low) level of the marker shown. (B) Cells from the same lymph nodes isolated from naive mice, prepared and analyzed as in A.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the demonstration of its preferential response during inflammation induced in diverse ways, the functional role of the V{gamma}6/V{delta}1+ subset has not yet been directly examined. The effects of {gamma}{delta} T cells in general during the responses studied in some models suggest an anti-inflammatory role for this subset, but lack of a mAb recognizing this subset has made these cells challenging to study. Our finding that 17D1 can be used as a reagent to specifically detect the V{gamma}6/V{delta}1 subset now clears the way for these experiments.

Why V{gamma}6/V{delta}1+ cells must be pretreated with anti-C{delta} to detect 17D1 binding is not clear, but at least two possibilities exist. First, the anti-C{delta} mAb may aggregate the TCRs on the cells, increasing avidity so that the 17D1 mAb, an IgM whose affinity for the V{gamma}6/V{delta}1 TCR may be low, now shows enhanced binding of the V{gamma}6/V{delta}1 TCR. Second, the structure of the V{gamma}6/V{delta}1 TCR might be altered in some way when bound to an anti-C{delta} mAb, such that the 17D1 epitope is revealed or stabilized. Our ELISA (Fig. 1D) did in fact indicate a higher affinity of 17D1 for V{gamma}5/V{delta}1 than for V{gamma}6/V{delta}1. However, pretreating V{gamma}6/V{delta}1+ cells with an anti-CD3 mAb, which should be equally able to aggregate TCRs, did not allow 17D1 to bind, whereas pretreatment with any of three different anti-C{delta} mAb did. Moreover, when V{gamma}5/V{delta}1+ cells are prestained with anti-C{delta}, they do not show any enhancement in 17D1 binding (e.g., see Fig. 1A ), as one might expect if aggregation were a factor. It thus appears likely that the anti-C{delta} mAb brings about or stabilizes a structural change in the V{gamma}6/V{delta}1 TCR. The ability of 17D1 to bind it might therefore reflect an unusual property of this TCR—of being able to assume two distinct structural forms—although determining whether this is indeed so and whether it is functionally important will require further study.

Using 17D1, we found in this study that V{gamma}6/V{delta}1+ cells, which expand in response to CFA, differ markedly from the V{gamma}1+ cells expanding in the same lymph node, in that the former appear to be almost uniformly activated or in a memory state. Others who have characterized {gamma}{delta} T cells for the same memory/activation markers examined here have reported that {gamma}{delta} T cells follow the paradigm set by {alpha}ß T cells for expression of these molecules [34 , 35 ] and moreover, that {gamma}{delta} T cells, which display the putative activated/memory phenotype, also behave like activated/memory {alpha}ß T cells in being longer-lived and having a higher turnover rate [35 ]. This suggests that the nearly uniform activated/memory phenotype of V{gamma}6/V{delta}1 cells, which respond robustly during inflammation, has likely been triggered by stimulation through the TCR, whereas the weaker responses of other subsets, which result in only a small increase in cells having an activated phenotype, may involve the TCR less frequently or even not at all. In several instances, {gamma}{delta} T cells have been found to carry innate receptors such as those expressed on macrophages [36 37 38 39 40 ]; these may, by themselves, be sufficient to stimulate infiltration and perhaps some degree of proliferation by {gamma}{delta} T cells [41 ]. The use of the 17D1 mAb to identify V{gamma}6/V{delta}1+ cells will likely serve as a valuable addition to further experiments aimed at determining what is needed to bring about {gamma}{delta} T cell subset responses.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the NIH (R01AI44920, T32AI07405, RO1AI40611, and RO1HL65410) and from the National Jewish Janet S. Lewald and R. W. Gitzen Jr./C. P. Gitzen Fellowships, respectively, to C. L. R. and M. K. A. C. L. R. was also supported by an Investigator Award from the Arthritis Foundation. We thank Bill Townend, Josh Loomis, and Shirley Sobus (National Jewish Medical and Research Center, Denver, CO) for flow cytometry advice and John Kappler for making available a control baculovirus encoding the soluble {alpha}ß TCR derived from the hybridoma DO-11.10.


   FOOTNOTES
 
1 These authors contributed equally to this manuscript. Back

Received July 14, 2003; revised August 28, 2003; accepted September 8, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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