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The Journal of Immunology, 2004, 172: 4167-4175.
Copyright © 2004 by The American Association of Immunologists

Detection of Cell Surface Ligands for the {gamma}{delta} TCR Using Soluble TCRs1

M. Kemal Aydintug2, Christina L. Roark2, Xiang Yin, J. M. Wands, Willi K. Born and Rebecca L. O’Brien3

Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206; and University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The natural ligands recognized by {gamma}{delta} TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific {gamma}{delta} T cell responses. Taking advantage of an established ligand for a particular {gamma}{delta} TCR, we demonstrated that a multimerized recombinant form of this {gamma}{delta} TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common {gamma}{delta} TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional {gamma}{delta} TCRs. One of these represents the mouse V{gamma}6/V{delta}1 invariant {gamma}{delta} TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related V{gamma}5/V{delta}1 invariant {gamma}{delta} TCR expressed by most epidermal T cells. The third is a V{gamma}1/V{delta}6.3 TCR, representative of a variable type frequently found on lymphoid {gamma}{delta} T cells. We found evidence that ligands for multiple {gamma}{delta} TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of {gamma}{delta} TCRs can be as tools to identify and characterize the natural ligands of {gamma}{delta} T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The {gamma}{delta} T lymphocytes constitute a relatively rare type of T cell in mice and humans whose function appears to be distinct from that of other lymphocytes, the {alpha}{beta} T cells and B cells (reviewed in Ref. 1). Despite elaborate mechanisms that {alpha}{beta} T and B cells undergo to prevent self-responsiveness, {gamma}{delta} T cells have frequently been reported to respond to autologous ligands (e.g.,2, 3, 4, 5, 6). Such studies suggest that some {gamma}{delta} T cells are in fact inherently biased toward the recognition of self, rather than foreign molecules, autologous ligands whose expression is induced by stress or inflammation. The functional role of {gamma}{delta} T cells also appears to differ from that of {alpha}{beta} T cells, and in both innate and adaptive immune responses, they may be more important for immunoregulation than for protection (7, 8, 9, 10, 11).

{gamma}{delta} T cells came to be regarded as subsets based on the type of TCR they express, because those bearing particular V{gamma}s and/or V{delta}s were found to be differentially distributed in certain tissues. As well, mounting evidence suggests that {gamma}{delta} T cell subsets also differ from one another in terms of function (6, 12, 13, 14, 15, 16, 17). Rather than maximizing the potential diversity of their Ag receptors like other lymphocytes, some {gamma}{delta} T cell subsets show diversity limitations. This finding is all the more surprising because the available V gene pool for the {gamma}{delta} TCR is considerably smaller than that for the {alpha}{beta} TCR or B cell Ag receptor of mice and humans. In some subsets, the diversity limitations mainly involve the restricted pairing of certain V{gamma} and V{delta} chains, but in rodents, they also give rise to two subsets whose TCRs are encoded by virtually invariant, canonical sequences (18, 19, 20). One of these subsets, bearing the V{gamma}5/V{delta}1 TCR, represents the first type of T cell to arise in the fetal thymus, is produced for only a few days, and specifically colonizes the epidermis, but is virtually absent elsewhere. The other, the V{gamma}6/V{delta}1 subset, represents the second {gamma}{delta} T cell subset to arise in the thymus, is generated only for a few days in the late fetal and early newborn stages (21), and specifically colonizes the female reproductive tract, the tongue, and the lung (22, 23), although its distribution is less restricted than that of the V{gamma}5/V{delta}1 subset. The existence of specific mechanisms that ensure their generation implies that {gamma}{delta} T cells having these invariant TCRs are of some importance to rodents (24, 25, 26). Although their responses may be beneficial (27, 28), ligands that bind the invariant {gamma}{delta} TCRs and stimulate these cells to respond have not yet been identified.

The tendency of many {gamma}{delta} T cells to limit rather than to maximize the potential diversity of their TCRs implies that the ligands for their TCRs must be equally limited. This may explain why, in contrast to B cells and {alpha}{beta} T cells, it has been difficult to generate Ag-specific {gamma}{delta} T cells by immunization with a defined Ag. Understanding the role of the TCR and defining the ligands it binds are essential to elucidating the immunobiology of these cells. However, although several lines of evidence support the idea that host-derived molecules act as ligands for {gamma}{delta} T cell subsets (2, 4, 27, 29, 30), the identity of only a few of these is known (31, 32). Moreover, a number of studies suggest that {gamma}{delta} TCRs instead or in addition are specialized to recognize common microbial Ags. These include a group of small organic compounds dubbed phosphoantigens that stimulate human V{gamma}9/V{delta}2+ cells (33), as well as certain alkyl amines (34, 35). The question of which stimuli represent ligands for a {gamma}{delta} TCR is complicated by findings showing that {gamma}{delta} T cell responses can also be evoked through receptors other than the TCR (36, 37, 38, 39, 40). In this study, we describe a novel approach toward the discovery of natural ligands for {gamma}{delta} TCRs: the use of recombinant soluble versions of {gamma}{delta} TCRs as direct binding reagents. Using a {gamma}{delta} TCR with a previously defined ligand, we demonstrate the feasibility of the approach and provide additional evidence in support of the notion that the {gamma}{delta} TCR is more Ab-like than is the {alpha}{beta} TCR (32, 41). In addition, we present evidence that the currently unknown ligands for three different {gamma}{delta} TCRs, the V{gamma}6/V{delta}1 and V{gamma}5/V{delta}1 invariant TCRs, and a V{gamma}1/V{delta}6.3 TCR, are expressed on epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of soluble TCR (sTCR)-producing constructs

T cell hybridomas previously generated by our laboratory were used to prepare cDNAs containing the TCR genes expressed by representative members of common {gamma}{delta} T cell subsets (42, 43, 44) (Table I). In addition, spleen cells from a KN6 transgenic/recombination-activating gene 2 knockout mouse (kind gift of D. Kappes, Fox Chase Cancer Center, Philadelphia, PA) were used as a source of cDNA encoding the TCR4 originally expressed by the T22b-reactive {gamma}{delta} T cell hybridoma clone, KN6 (3). For each, primers were designed that would truncate the genes just before the transmembrane regions, by inserting termination codons. The cysteine codon for each chain that forms the interchain disulfide bond of the TCR was preserved in each case, such that the TCR sequence ends directly after this cysteine codon for C{delta}, and two codons below it for C{gamma}. In addition, just before the termination codon, the C{delta} primers contained a 15-codon sequence (a Bir A substrate peptide, or BSP sequence) whose product is recognized with high affinity by the Escherichia coli enzyme BirA, which can be used to add a biotin to the C terminus of the sTCR (see Fig. 1A), although this feature was not used in this study. Amplified {gamma} and {delta} cDNA pairs were then cloned, sequence verified, and transferred into a vector containing dual baculovirus promoters (pAcUW51; BD PharMingen, San Diego, CA) that had been modified to include additional restriction enzyme cloning sites (pBACp10pH; gift of J. Kappler, National Jewish Medical and Research Center). In each construct, the {gamma} gene was cloned into the EcoRI and BamHI sites of the polyhedrin promoter, and the {delta} gene into the XhoI and Bpu 1102 sites adjacent to the p10 promoter. A control {alpha}{beta} sTCR (derived from the OVA/I-Ed-reactive hybridoma DO-11.10) containing a similar BirA site linked to the C terminus of the {beta}-chain (45) was also prepared from baculovirus provided by the Kappler laboratory (National Jewish Medical and Research Center) for comparison.


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  		Table I. {gamma}{delta} sTCRs used in this study

 


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  		FIGURE 1. Generation and testing of sTCRs. A, sTCRs were generated in a baculovirus system, using a vector containing cDNA constructs encoding both {gamma}- and {delta}-chains (example shows the V{gamma}6/V{delta}1 TCR construct). An {alpha}{beta} control sTCR was generated similarly, and grown in our lab from viral stock provided by the Kappler laboratory. B, SDS-PAGE gel, stained with Coomassie brilliant blue, showing the sTCRs used in this study; ~1 µg of protein/lane was loaded. Abbreviations used are based on the {gamma}- and {delta}-chains of the TCRs, respectively, and are as follows: the V{gamma}4/V{delta}5+ KN6-derived TCR, 4:5; a V{gamma}1/V{delta}6.3 TCR derived from the autoreactive hybridoma line BNT, 19.8–1:6.3; the V{gamma}5/V{delta}1 and V{gamma}6/V{delta}1 canonical TCRs, 5:1 and 6:1, respectively; and the TCR derived from the {alpha}{beta} hybridoma DO-11.10, {alpha}{beta}. The sizes of the bands detected matched those predicted from the amino acid sequence, after taking into account N-linked glycosylations. C, {gamma}{delta} sTCRs inhibit the binding of mAbs specific for relevant TCR components and requiring native structure. The mAbs used include: GL3 (anti-C{delta}), 2.11 (anti-V{gamma}1), 17C (anti-V{delta}6.3), F536 (anti-V{gamma}5), H57-597 (anti-C{beta}), and KJ1 (an anti-Id for this DO-11.10-derived TCR). The cell lines used to detect staining inhibition are shown below the graph, and the TCR that each bears is indicated in parentheses. The sTCR used to inhibit the staining is designated on the bottom line. D, mAb-binding inhibition assays showing that different {gamma}{delta} sTCRs are approximately equal in ability to inhibit the binding of anti-C{delta} mAb GL3, and that the various sTCRs are specific in their ability to inhibit mAb binding.

 
Oligonucleotide primers having the following sequences were used to amplify and alter the {gamma}{delta} sTCR cDNAs (all listed 5' to 3'): for the V{gamma}6/V{delta}1 TCR, V{gamma}6L EcoRI+ (GAA TTC TGC AGG ATG GGG GCT TCT) with C{gamma}1 cys- (GGA TCC TTA TTG CCA GCA AGT TGT), and 5'-V{delta}1-XhoI+ (GCC TCG AGG AAA CTA TGC TTT GGA GA) with 3'-C{delta} BSP- (GCG CTC AGC TTA ACG ATG ATT CCA CAC CAT TTT CTG TGC ATC CAG AAT ATG ATG CAG GCC ATA GCA AGG CTC TGA AAT TTG); for the V{gamma}5/V{delta}1 TCR, V{gamma}5L EcoRI+ (GG GAA TTC ACT AAA ATG TCA ACC TCT) with C{gamma}1 cys- (listed above), and 5'-V{delta}1-XhoI+ (listed above) with 3'-C{delta} BSP- (listed above); for the V{gamma}1/V{delta}6.3 TCR, V{gamma}1L-EcoRI+ (GGG AAT TCC TTG GGA TGC TGC TC) with C{gamma}4 Bam(ter)2- (CCG GAT CCT TAT TTC ATG CAA TCT TC), and V{delta}6L-XhoI (CTC GAG ATG CCT CCT CAC AGC CTG TTC TGT G) with 3'-C{delta} BSP- (listed above); for the V{gamma}4/V{delta}5 TCR, V{gamma}4L-EcoRI (GAA TTC CAG ACC ATG AAG AAC CCT GG) with C{gamma}4 Bam (ter) (listed above), and V{delta}5-XhoI (CTC GAG GGA AGG ATG ATT CTT GCC GC) with 3'-C{delta} BSP (listed above).

Purification of sTCRs

The sTCR DNA constructs were cotransfected into the Sf9 moth cell line along with baculovirus helper DNA (BaculoGold, BD PharMingen; or Bacvector 3000, Novagen, Madison, WI) to generate a baculovirus-containing culture supernatant that produces sTCR molecules, as has been previously described (46). An {alpha}{beta} TCR-producing control baculovirus (described in the previous paragraph) was prepared in a similar manner in our laboratory as a negative control.

For protein production, sTCR/baculovirus-containing culture supernatants were produced in High Five insect cells, using a multiplicity of infection of ~5–10 infectious units per insect cell. The cells were then cultured for 6 days, at 27°C for the first day, then at 19°C for the remainder. Culture supernatants containing {gamma}{delta} sTCRs were then purified by passing them over anti-C{delta} (GL3) (47) Sepharose affinity columns. For the {alpha}{beta} control sTCR, the culture supernatant was similarly purified by passage over an anti-C{beta} (H57-597) (48) column. These columns were prepared using CNBr-activated Sepharose CL-4B beads (Sigma-Aldrich, St. Louis, MO) in accordance with the manufacturer’s directions. However, to preclear the supernatant and obtain cleaner preparations, we first passed those supernatants containing {gamma}{delta} sTCRs over an anti-C{beta} column, and those containing the {alpha}{beta} sTCR over an anti-C{delta} column, running the flow-through immediately afterward over the correct affinity column for the TCR type. Next, the affinity columns were washed with ~25 column volumes of 50 mM NaCl/100 mM Tris, pH 7.4, and the bound molecules were eluted with 50 mM diethylamine in distilled water, pH 11.5. Fractions of 0.9 ml, up to a total of ~10 ml, were serially collected into tubes containing 0.1 ml of 1 M Tris, pH 6.5, to neutralize them. Fractions containing the eluted protein were identified by OD at 280 nM, combined, and dialyzed overnight to PBS. The products were then concentrated with Centricon-30 or Amicon Ultra filter devices (Amicon Bioseparations; Millipore, Beverley, MA), and stored at 4°C. Average yields differed for the various TCRs; the V{gamma}6/V{delta}1 TCR averaged ~0.4 µg/ml of supernatant, whereas the V{gamma}5/V{delta}1 TCR gave a yield of ~0.7 µg/ml, the V{gamma}1/V{delta}6.3 and V{gamma}4/V{delta}5 sTCRs ~0.3 µg/ml, and the {alpha}{beta} sTCR ~1 µg/ml.

mAb-binding inhibition assay (quality test for sTCRs)

To determine whether each sTCR retained native conformation sufficient to bind to anti-TCR mAbs, a competition assay was conducted. For each test, 40 ng per sample of an anti-TCR mAb was incubated for 30–60 min either alone or together with a sTCR, in 96-well plates, using 1 µg of sTCR in a total volume of 50 µl (e.g., see Fig. 1C). A total of 45 µl of the diluted mAb or mixture was then transferred into wells of a 96-well plate containing 105 cells of a T cell hybridoma bearing a TCR recognized by the mAb. The degree of binding of the sTCR to the mAb was determined by a reduction in the amount of mAb available in the mixture to stain the T cell hybridoma cells, as compared with the mAb alone at the same concentration. Bound mAb was detected using a fluorescently labeled secondary Ab (goat anti-rat IgG or rabbit anti-hamster IgG; Jackson ImmunoResearch Laboratories, West Grove, PA).

To verify that the mAbs used indeed recognize the TCR components in question only when undenatured, in some experiments a second sample of each TCR was also tested at the same time, which had been deliberately denatured. In this study, the sTCR was first boiled for 10 min, snap cooled in ice water, and then centrifuged for 5 min at top speed in a microfuge before adding it to the mAb. The top half of the supernatant in the tube was removed to use for staining, while the bottom half was vortexed and retained for gel analysis, to ensure that any loss in the ability of the boiled sTCR to absorb out the mAb was not simply due to its precipitation during boiling. In all cases, this explanation was ruled out because the amount of protein detected by SDS-PAGE in the boiled preparations looked comparable to that from nonboiled controls prepared in otherwise the same way.

Preparation of multimeric sTCRs

Octameric versions of the {gamma}{delta} sTCRs were generated using a new method recently developed by J. Kappler (58). Briefly, using a low-level biotinylation protocol, a biotin group was first added to the anti-C{delta} mAb GL3. This was accomplished using a long-chain biotinylation reagent (EZ-Link Sulfo-LC-Biotin; Pierce, Rockford, IL), using the manufacturer’s standard protocol, with the stoichiometry of the reagents adjusted such that the ratio of biotin to Ab was less than 1. The mAb was then tetramerized by adding a limiting amount of labeled streptavidin (streptavidin-Alexa Fluor 647; Molecular Probes, Eugene, OR), and the tetrameric form of the mAb was separated from the monomeric by FPLC. The tetrameric mAb was then mixed with the desired {gamma}{delta} sTCR such that the final concentrations were 1 mg/ml for the sTCR and 0.2 mg/ml for the mAb, and the molecules were allowed to complex overnight at 4°C before use. Because each tetramerized mAb subunit is divalent, a multimeric sTCR will then be formed, containing up to eight TCR heterodimers. We chose this method because the conventional tetramerization protocol (which involves biotinylating the BSP peptide via the E. coli enzyme BirA (49)) resulted in poor yields, presumably because the BSP peptide tag on our sTCRs is unstable. Although octamerization yields a product that is not as well defined as is possible with the conventional tetramerization method, because the biotin group may not always be added to the same site on every molecule and unoccupied portions of the anti-C{delta} mAb may persist, this method vastly improved our yields and worked well for cell staining.

Cell culture

T cell hybridomas, B cell hybridomas, and all other mammalian cell lines except XB-2 were cultured in IMDM (Sigma-Aldrich), supplemented with sodium pyruvate, sodium bicarbonate, 2-ME, L-glutamine, essential and nonessential amino acids, glucose, penicillin, streptomycin, gentamicin, and 10% FBS. XB-2 was grown in defined keratinocyte serum-free medium (Life Technologies/Invitrogen, Grand Island, NY) supplemented with 15% FBS, and before staining was removed from culture flasks by incubating the cells for 20–30 min at 37°C in PBS containing 2 mM EDTA. Insect cells (Sf9 and High Five) were cultured in Grace’s insect medium (Life Technologies/Invitrogen, Grand Island, NY) supplemented with 10% FBS and an antibiotic/antimycotic (Life Technologies/Invitrogen), at either 27°C or 19°C.

sTCRs as staining reagents

For staining with nonmultimerized sTCRs, cells (~2 x 105 cells/well in 96-well culture plates) were incubated for 15–20 min at 4°C with 5 µg/ml mAb 2.4G2 to block Fc{gamma} receptors, then washed (with balanced salt solution (BSS) containing 2% FBS plus 0.1% sodium azide) one time and incubated with 40–100 µg/ml sTCR, as indicated, for 50–60 min at 4°C. To detect bound {gamma}{delta} sTCRs, the cells were then washed three times, incubated with a FITC-labeled anti-C{delta} mAb (GL3) for 15–20 min at 4°C, and washed three more times. For staining with octameric sTCRs, any potential Fc receptors were preblocked, as described above, with unlabeled 2.4G2 mAb. The cells were then incubated with sTCR octamers at the concentrations indicated in each figure at 4°C for 50–60 min, then washed three times. Binding of the sTCR octamers was directly visualized via the labeled streptavidin component. Stained cells were analyzed on a FACScan or FACSCalibur flow cytometer using CellQuest software; the histograms and scatterplots shown in this study were generated using FlowJo software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Integrity of sTCRs

The sTCRs used in this study include a canonical V{gamma}6/V{delta}1 TCR, a canonical V{gamma}5/V{delta}1 TCR (related to the V{gamma}6/V{delta}1 in having an identical {delta}-chain and J{gamma}-C{gamma}, but a different V{gamma}), a V{gamma}1/V{delta}6.3 TCR representative of a common subset found in the spleen and lymphoid organs, and the V{gamma}4/V{delta}5 TCR expressed by the KN6 hybridoma (50), whose ligand has been already identified as the MHC class I-like molecule T22b (3, 32) (Fig. 1B). An {alpha}{beta} sTCR was also used in some experiments as a control. We examined the integrity of the purified {gamma}{delta} sTCRs by testing whether they retain the ability to bind to anti-TCR mAbs recognizing native structures. In this study, we used a competition assay to determine whether adding sTCR to an anti-TCR mAb could affect the ability of mAb to stain a cell line bearing an appropriate TCR (Fig. 1C). Retention of the ability of a sTCR to bind a mAb is therefore indicated by a reduction in the subsequent degree of staining with the mAb. As shown in Fig. 1C ({blacksquare}), we found that each sTCR was able to block staining with appropriate anti-TCR mAbs. However, the various sTCRs had little or no effect on the staining of irrelevant anti-TCR mAbs under the conditions tested, and {gamma}{delta} sTCRs were approximately equal to one another in their ability to inhibit the binding of the anti-C{delta} mAb GL3 (Fig. 1D).

Because in most instances we were not certain whether these mAbs require native structure in the TCRs they recognize, we also determined whether deliberately denatured (boiled) sTCRs could still bind to these mAbs (Fig. 1C,

). This treatment caused the sTCRs to lose all or most of their ability to bind to the relevant mAbs, with the exception of the control {alpha}{beta} sTCR, which still retained nearly 50% blocking ability for its anti-idiotypic mAb. Thus, for all of the {gamma}{delta} TCR-specific mAbs we tested, denatured forms of the {gamma}{delta} sTCRs were recognized very poorly or not at all.

A {gamma}{delta} sTCR specifically detects cells expressing its known ligand

To examine the feasibility of using {gamma}{delta} sTCRs as reagents to detect their own ligands, we generated a soluble version of a {gamma}{delta} TCR whose ligand has already been identified. This TCR, originally derived from hybridoma line KN6 (3), recognizes a nonclassical class I molecule, T22b, expressed during embryonic development and in some adult tissues following stress or activation (32). Although the affinity of a related {gamma}{delta} TCR for T22b was found to be higher than the very low affinities typical of {alpha}{beta} TCRs (32), we feared that the affinity of {gamma}{delta} sTCRs for their ligands might generally be low, and therefore also generated an octameric version of this sTCR to test for ligand staining. However, we found that even without multimerization, the V{gamma}4/V{delta}5 sTCR stained the T22b transfectant, but did not stain the untransfected parent line, T2 (Fig. 2A). When multimerized, V{gamma}4/V{delta}5 sTCR showed an enhanced ability to stain the T22b transfectant (Fig. 2B). In contrast, irrelevant {gamma}{delta} sTCRs failed to stain this cell line, either before (data not shown) or after multimerization (Fig. 2C). When the T2-T22b transfectant was double stained with both the anti-T22b mAb and the soluble KN6-derived sTCR octamer (Fig. 2D), staining with the V{gamma}4/V{delta}5 sTCR was weak on individual cells expressing lower levels of T22b, but bright for those expressing high levels, a predictable correlation if the mAb and the sTCR detect the same molecule.



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  		FIGURE 2. {gamma}{delta} sTCRs can be used as staining reagents to detect ligands on cell lines. A, Staining of the human T cell line (T2) transfected with T22b (T2-T22), and of untransfected T2, using the KN6-derived V{gamma}4/V{delta}5 sTCR monomer, at a concentration of 40 µg/ml. Binding of the V{gamma}4/V{delta}5 sTCR monomer was detected with an anti-C{delta} FITC labeled secondary. B, An octameric version of the V{gamma}4/V{delta}5 sTCR shows an enhanced ability to stain T2-T22. Staining is shown using serial dilutions of octameric KN6-derived sTCR. C, The V{gamma}4/V{delta}5 KN6-derived sTCR octamer stains T2-T22, but other {gamma}{delta} sTCR octamers do not. Staining was conducted using 10 µg/ml of each sTCR octamer. D, Double staining (right panel) of T2-T22 with the anti-T22b-specific mAb 7H9 (FITC labeled) and V{gamma}4/V{delta}5 KN6-derived sTCR octamer (AF-647 labeled). Unstained cells and single-color profiles are shown for comparison (left three panels).

 
{gamma}{delta} sTCRs having unknown ligands detect their putative ligands on cultured cell lines

We next tested mouse cell lines from different sources for their ability to stain with our sTCRs. Staining was evident on several different cell lines (see Table II). The keratinocyte cell line XB-2 typically stained more brightly than the others, and simultaneously for both the V{gamma}6/V{delta}1 and the V{gamma}1/V{delta}6.3 sTCRs (Fig. 3A). These two sTCRs also stained several other epithelial cell lines, including another keratinocyte cell line, PAM-212, but typically did not stain lymphocyte cell lines (Table II and Fig. 3B). The XB-2 and PAM-212 keratinocyte cell lines were likewise detected with the V{gamma}5/V{delta}1 sTCR, but the staining was weak (e.g., Fig. 4A; discussed further below). When octamerized, the V{gamma}6/V{delta}1 and the V{gamma}1/V{delta}6.3 sTCRs showed an enhanced ability to stain XB-2, even at a dilution as low as 2.5 µg/ml (Fig. 3C). In contrast, the V{gamma}4/V{delta}5 KN6-derived sTCR octamer, as expected, showed virtually no staining of these cell lines (e.g., Fig. 3D).


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  		Table II. Staining of various cell lines with {gamma}{delta} sTCR monomers

 


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  		FIGURE 3. {gamma}{delta} TCRs having unknown ligands can be used in soluble form to directly detect their own ligands. A, Staining of the XB-2 keratinocyte cell line with monomeric V{gamma}1/V{delta}6.3 and V{gamma}6/V{delta}1 sTCRs. B, Staining of various cell lines with the V{gamma}6/V{delta}1 sTCR monomer. XB-2 and PAM-212 are mouse keratinocyte cell lines; D0–11.10 and BE{alpha}-16.3 are mouse T cell hybridomas. Two other mouse lymphocyte-derived cell lines also failed to stain with this sTCR: HT-2 (a T cell line (56 )) and WEHI-5 (a B cell lymphoma (57 ); data not shown). Staining with secondary only is also shown for XB-2; the other cell lines looked similar for this control (data not shown). C, Staining of the XB-2 cell line with titered amounts of octamerized versions of two different sTCRs. D, Comparison of profiles of XB-2 cells stained with 20 µg/ml of the V{gamma}1/V{delta}6.3 octamer vs the KN6-derived V{gamma}4/V{delta}5 sTCR octamer.

 


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  		FIGURE 4. Anti-V region mAbs specifically inhibit sTCR staining. A, PAM-212 keratinocyte cells were stained with V{gamma}5/V{delta}1 sTCR monomer that had been pretreated for 30 min at room temperature with the mAb shown (indicated by square brackets), using 100 µg/ml of each reagent. Bound sTCR/mAb complex was then detected with rabbit anti-hamster Ig FITC (anti-H-FITC) or goat anti-rat IgM FITC (anti-R-FITC), as appropriate. As a control, staining with the same concentration of sham (PBS)-treated V{gamma}5/V{delta}1 sTCR is also shown, as detected by subsequent staining with anti-C{delta} and then anti-hamster FITC, in three separate staining steps (5:1 + anti-C{delta} + anti-H-FITC). B, T2-T22 cells stained with the KN6-derived V{gamma}4/V{delta}5 sTCR, either alone or after preincubation for 1 h with the indicated mAb. Staining was conducted using 40 µg/ml sTCR octamer that had first been incubated with or without 50 µg/ml of the mAb indicated for 1 h at room temperature. C and D, Staining of the XB-2 keratinocyte cell line with two different sTCR octamers having unknown ligands, using either sTCR octamer alone or following preincubation for 1 h with the indicated mAb. Staining was conducted as in B, except that 25 µg/ml sTCR octamer was used with 50 µg/ml mAb, and the preincubation was conducted for 45 min at room temperature.

 
Specificity of the sTCR staining

Our ability to enhance sTCR staining by aggregating the C region with an anti-C{delta} mAb suggested that this part of the sTCR is not involved in ligand binding, as would be expected. We therefore next tested whether complexing a sTCR with an anti-V region mAb would in contrast have an inhibitory effect on its ability to stain, as would be expected if the V region of a {gamma}{delta} TCR confers its specificity. Fig. 4A shows the results of this analysis using the V{gamma}5/V{delta}1 sTCR to stain the PAM-212 keratinocyte cell line. As can be seen, although dimerizing the sTCR by preincubating it with an anti-C{delta} mAb enhanced its ability to stain, pretreatment with two different anti-V region mAbs (17D1, discussed further below, and F536, an anti-V{gamma}5 reagent) in contrast inhibited the staining. This result suggests that the binding of the V{gamma}5/V{delta}1 sTCR to PAM-212, although weak, specifically involves the V region of the TCR.

Our ability to stain certain cell lines using quite low concentrations of sTCR octamers (near the range generally used for mAbs) suggested that the binding with octamers was also specific. However, the possibility remained that {gamma}{delta} sTCRs, particularly when octamerized, are simply sticky. To test the octamer binding for specificity, we similarly preincubated the sTCR octamers with mAbs specific for various V{gamma} and V{delta} chains before staining. To test this approach, we first used the V{gamma}4/V{delta}5+ KN6-derived octamer as a control on T2-T22 cells (Fig. 4B), and found that its binding was indeed inhibited by both a V{gamma}4- and a V{delta}5-specific mAb, whereas an irrelevant mAb specific for V{gamma}7 (as well as irrelevant mAbs specific for V{gamma}1, V{gamma}5, V{delta}6.3, or one recognizing both V{gamma}6/V{delta}5 and V{gamma}5/V{delta}1; data not shown) had virtually no effect.

We then similarly tested the specificity of the binding of {gamma}{delta} sTCR octamers on the XB-2 cell line, as shown in Fig. 4C. The V{gamma}1/V{delta}6.3 sTCR was largely inhibited by an anti-V{delta}6.3 mAb, but not by irrelevant mAbs. The V{gamma}6/V{delta}1 sTCR was next tested using the mAb 17D1, which detects both the V{gamma}5/V{delta}1 TCR (51) and the V{gamma}6/V{delta}1 TCR (52), although the latter only when it is first bound to an anti-C{delta} mAb. This IgM mAb has also been found to have a relatively lower affinity for V{gamma}6/V{delta}1 under such conditions, but it represents the only mAb currently available that recognizes the variable portion of this TCR. The requirement of 17D1 for anti-C{delta} cobinding in recognition of this TCR is met because an anti-C{delta} mAb is already complexed to this sTCR in its octameric form. We found a measurable (~50%) reduction in staining with the V{gamma}6/V{delta}1 sTCR octamer using 17D1 (Fig. 4C). In contrast, the 17D1 Ab had no effect on the staining of the irrelevant V{gamma}1/V{delta}6.3 sTCR octamer, but predictably also inhibited the binding of another relevant sTCR, the V{gamma}5/V{delta}1 monomer, on PAM-212 cells (Fig. 4A). Therefore, the staining observed with the V{gamma}6/V{delta}1, V{gamma}5/V{delta}1, and V{gamma}1/V{delta}6.3 sTCRs most likely is the result of specific interactions requiring the V region of the TCR.

Interestingly, a mAb specific for the V{gamma}1-J{gamma}4-C{gamma}4 chain, mAb 2.11 (53), strongly enhanced binding of the V{gamma}1/V{delta}6.3 sTCR to XB-2 (Fig. 4D), rather than blocking it, as we originally expected. The same mAb had, however, little or no effect on the binding of irrelevant {gamma}{delta} sTCR octamers such as V{gamma}6/V{delta}1 (Fig. 4D). Because both J{gamma}4 and C{gamma}4 are expressed almost exclusively in conjunction with V{gamma}1, it has not been clear whether mAb 2.11 is specific for the variable or constant portions of this TCR chain (54). Although we have yet to fully define the 2.11 epitope, our prelimary investigations show clearly that it must at least partially include V{gamma}1 components (because 2.11 fails to stain cells bearing the closely related V{gamma}2 chain, including one {gamma}{delta} T cell hybridoma expressing V{gamma}2 aberrantly rearranged to J{gamma}4/C{gamma}4 (42); data not shown). If mAb 2.11 nonetheless recognizes an epitope that is far removed from the Ag binding site of the V{gamma}1/V{delta}6.3 TCR, it could instead of blocking further aggregate the V{gamma}1/V{delta}6.3 TCR octamer and cause a still greater enhancement of its avidity and ability to stain.

The V{gamma}1/V{delta}6.3 and V{gamma}6/V{delta}1 cell surface ligands on XB2 cells are protease sensitive

To investigate the chemical nature of {gamma}{delta} TCR ligands, we have attempted to treat cells in a number of ways that might denature or destroy the ligand. Fig. 5 shows the effect of treating T2-T22 and XB-2 cells with enzymes under conditions that preserve cell viability. The binding of the V{gamma}4/V{delta}5 KN6-derived sTCR octamer to the T2-T22 transfectant was diminished by treatment with the proteolytic enzymes trypsin and chymotrypsin (Fig. 5A), as expected, because the T22b molecule is a cell surface glycoprotein.



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  		FIGURE 5. The ligands detected by two {gamma}{delta} sTCRs are protease sensitive. A, XB-2 or T2-T22 cells were either treated with proteases, or left untreated, before staining with sTCR octamers, as indicated. Trypsin was used at 1.0 mg/ml, and chymotrypsin at 5 U/ml. Each enzyme was allowed to act for 30 min at 37°C. The cells were then washed three times in BSS containing 5% FBS before staining with sTCR octamers. B, Example control experiment to rule out the possibility that residual protease in the treated cells (example shown is for pronase) digests the sTCRs and destroys their ability to stain. Staining was conducted with V{gamma}6/V{delta}1 sTCR monomer on XB-2 cells. Pronase was used at a concentration of 0.5 mg/ml, and allowed to act on the XB-2 cells for 30 min at 37°C, which were then washed as described in A. Similar results were obtained with supernatants containing V{gamma}6/V{delta}1 sTCR monomer first used to stain chymotrypsin- and trypsin-treated XB-2 cells (data not shown). C, Staining of XB-2 cells with the V{gamma}6/V{delta}1 sTCR monomer is diminished by protease treatment, but not by neuraminidase treatment. Trypsin was used at a concentration of 2.5 mg/ml, chymotrypsin at 5 U/ml, and neuraminidase at 2.5 U/ml; all enzymes were allowed to act for 30 min at 37°C, then washed as described in A. D, Staining of XB-2 cells with neither the V{gamma}6/V{delta}1 nor the V{gamma}5/V{delta}1 sTCR monomer is diminished by pretreatment with N-glycosidase. Cells were treated with 1 U/ml N-glycosidase F in medium without FBS, while bound to petri plates in culture at 37°C overnight. Treatment of XB-2 with the same enzyme, but with the cells in suspension, was similarly ineffective (data not shown).

 
Staining of the XB-2 keratinocyte cell line with the V{gamma}1/V{delta}6.3 sTCR octamer was likewise protease sensitive (Fig. 5A), and in fact this ligand seems to be more sensitive to chymotrypsin than is the T22b molecule (however, increasing the chymotrypsin level by 10-fold completely abolished staining of the T2-T22 cells with the KN6-derived sTCR octamer; data not shown). Staining of the XB-2 cell line using either V{gamma}5/V{delta}1 or V{gamma}6/V{delta}1 sTCR octamers and monomers was also diminished by pretreatment of the cells with trypsin, chymotrypsin, or pronase (e.g., see Fig. 5, B and C), with sensitivities similar to those seen with the V{gamma}1/V{delta}6.3 sTCR. We verified that the sTCRs used in these experiments were not simply being degraded by residual protease by saving the sTCR-containing supernatants used to stain the protease-treated cells, and reusing them to stain untreated cells. For all enzymes, the reused supernatants stained the untreated cells with undiminished brightness (e.g., Fig. 5B). These results indicate that the ligands recognized by our {gamma}{delta} sTCRs are proteins or protein-associated molecules. For the V{gamma}6/V{delta}1 and V{gamma}5/V{delta}1 sTCRs, we have also attempted to inhibit staining by pretreating cells with other enzymes, including neuraminidase and N-glycosidase (Fig. 5, C and D). However, these two enzymes had no effect, indicating that the ligand(s) recognized is either not glycosylated, or that the removal of sialic acid or N-linked sugar groups does not significantly affect the binding.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the possibility that multimerized {gamma}{delta} sTCRs could be used as reagents to detect their own ligands. The ability of our {gamma}{delta} sTCR preparations to bind mAbs recognizing certain TCR components when not deliberately denatured suggested that they retain at least to some degree their native configuration in both C and V region domains, an expected prerequisite of preserving specificity for their natural ligands. Using the {gamma}{delta} sTCRs as staining reagents, we found that they were able to detect molecules on the surface of some, but not all cell lines, with or without prior multimerization. Moreover, we found by titration that the degree of sensitivity of the staining for two multimerized {gamma}{delta} sTCRs whose ligands are unknown was comparable to that of a multimerized {gamma}{delta} sTCR whose ligand is known, the KN6-derived sTCR, in detecting its ligand T22b on the surface of a transfected cell line. This implies that these sTCRs have a ligand affinity similar to that of the KN6-derived sTCR (32). Three observations indicate that the staining observed with these sTCRs is the result of an interaction with a specific ligand, rather than nonspecific stickiness. First, the binding seen for all four of our {gamma}{delta} sTCRs was inhibited by pretreating them with relevant, but not irrelevant, anti-V region mAbs. In contrast, deliberate multimerization of the {gamma}{delta} sTCRs with an anti-C region mAb enhanced their ability to stain. This implies that the staining is due to specific interactions requiring the V, but not the C region of each TCR. Second, irrelevant {gamma}{delta} sTCRs were unable to stain the T22b-transfected cell line, and conversely the KN6-derived {gamma}{delta} sTCR failed to stain the keratinocyte cell lines detected by three other {gamma}{delta} sTCRs. Third, although a single cell line, XB-2, stained strongly with two different {gamma}{delta} sTCRs, V{gamma}6/V{delta}1 and V{gamma}1/V{delta}6.3, the shape of the staining profile obtained with each was unique (e.g., compare profiles of each in Fig. 3, A and C), suggesting that each detects a different molecule.

Because XB-2 and PAM-212 are keratinocyte cell lines, and stressed keratinocytes (including PAM-212) have been shown to express a ligand specific for V{gamma}5/V{delta}1+ {gamma}{delta} T cells (4), their detection by the V{gamma}5/V{delta}1 sTCR was predicted, but the finding that ligands for different sTCRs may be simultaneously present on a single cell line was unexpected. However, perhaps the simultaneous expression of ligands for different {gamma}{delta} TCRs is unusual and associated with the transformed nature of certain cell lines.

Finally, we found that the ligands detected by three {gamma}{delta} sTCRs were protease sensitive, indicating that the ligands are themselves proteins, or are bound to a cell surface protein. Moreover, previous experiments have already implied that the unidentified ligands of these three {gamma}{delta} TCRs are inducible host molecules: for the V{gamma}6/V{delta}1 TCR, a molecule induced in several different tissues during inflammation evoked by bacterial infection (38, 42), by autoimmune attack (27), and by toxic drugs (20); for the V{gamma}5/V{delta}1 TCR, a molecule inducible on stressed keratinocytes (4); and for the V{gamma}1/V{delta}6.3 TCR, an autologous molecule most likely expressed by certain tumor cells (2, 55). Our finding that {gamma}{delta} TCRs can be used to detect their own ligands will likely prove useful in the full characterization of these ligands, and may as well identify the cells with which {gamma}{delta} T cells and their subsets interact.


   Acknowledgments
 
We thank John Kappler and Fran Crawford (National Jewish Medical and Research Center) for help with growing and purifying sTCRs and for the DO-11.10-derived {alpha}{beta} TCR-producing baculovirus; Yueh-Hsiu Chien (Stanford University, Stanford, CA) for providing the T22b transfectant and the anti-T22b mAb 7H9; Dietmar Kappes (Fox Chase Cancer Center) for providing KN6/recombination-activating gene-/- mice; Pablo Pereira (Pasteur Institute, Paris, France) for his gift of the anti-V{delta}5 mAb F45-152; Chris Reardon (University of Colorado Health Sciences Center) for providing and assistance in growing the keratinocyte cell lines; Philippa Marrack (National Jewish Medical and Research Center) for the BE{alpha}-16.3 and DO-11.10 T cell hybridomas; and Bill Townend, Shirley Sobus, and Josh Loomis (National Jewish Medical and Research Center) for flow cytometry assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01AI44920 to R.L.O., and by National Institutes of Health Grants RO1AI40611 and RO1HL65410 to W.K.B. In addition, salary support was partially provided to M.K.A. by National Institutes of Health Training Grant T32AI07405 postdoctoral position, to C.L.R. by an Investigator Award from the Arthritis Foundation, and to M.K.A. and C.L.R. by the Robert William Gitzen Jr./Christopher Peter Gitzen and Janet S. Lewald fellowships, respectively, from the National Jewish Medical and Research Center. Back

2 M.K.A. and C.L.R. contributed equally to this study and should be considered dual first authors. Back

3 Address correspondence and reprint requests to Dr. Rebecca L. O’Brien, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: obrienr{at}njc.org Back

4 Abbreviations used in this paper: sTCR, soluble TCR; BSP, Bir A substrate peptide; BSS, balanced salt solution. Back

Received for publication September 8, 2003. Accepted for publication January 15, 2004.


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