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V24-JQ-Independent, CD1d-Restricted Recognition of -Galactosylceramide by Human CD4⁺ and CD8⁺ T Lymphocytes¹

Nuffield Department of Clinical Medicine, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom

	Abstract

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Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid -galactosylceramide (GC), to a highly conserved NKT cell subset expressing an invariant TCR V24-JQ paired with V11 chain (V24⁺V11⁺ invariant NK T cell (NKT^inv)). The developmental pathway of V24⁺V11⁺NKT^inv is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-GC-tetramers, we demonstrate that in humans, TCR variable domains other than V24 and V11 can mediate specific recognition of CD1d-GC. In contrast to V24⁺V11⁺NKT^inv cells, V24^-/CD1d-GC-specific T cells express either CD8 or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8⁺V24^-/CD1d-GC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than V24⁺/CD1d-GC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-GC complex in humans is not uniformly restricted to the V24-JQ/V11 NKT cell subset, but can be mediated by a diverse range of V and V domains. The existence of a diverse repertoire of CD1d-GC-specific T cells in humans strongly supports their Ag-driven selection.

	Introduction

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The non-MHC encoded, ₂-microglobulin-associated CD1 molecules present glycolipids and phospholipids to T lymphocytes (1). According to their amino acid sequence homology, the four CD1 isoforms expressed in humans segregate into two groups. In group 1, CD1 molecules containing CD1a, CD1b, and CD1c are expressed in humans, but they are absent in mice and rats (2). In contrast, the group 2 CD1 molecule, CD1d, is highly conserved in all mammals studied so far (3). Group 1 CD1-molecules present either endogenous or microbial glycolipids to T lymphocytes expressing diverse TCR- and -chains, including various V and J segments (4). In contrast, the major human CD1d-restricted T lymphocyte subset so far identified expresses an invariant TCR V24-JQ chain paired with V11 and recognizes the synthetic, marine sponge-derived glycolipid -galactosylceramide (GC)⁴ (1, 5). The great majority of these V24⁺V11⁺ T cells coexpress the NK locus-encoded C type lectin NKR-P1 (CD161) and therefore are often referred to as invariant NK T cells (NKT^inv) (1). The murine counterpart of human V24⁺V11⁺NKT^inv cells expresses NKR-P1C (NK1.1) and recognizes the CD1d-GC complex through an invariant TCR V14-J281 chain in association with a restricted family of polyclonal V domains (6).

All CD1d-GC-specific NKT^inv cells so far described are either CD4⁺, CD8⁺, or CD4^-CD8^- double negative (DN), while CD8⁺NKT^inv cells have never been described, and are thought to be deleted during ontogeny due to their high binding avidity to CD1d molecules (7). NKT^inv cells are capable of rapidly secreting large amounts of regulatory cytokines, such as IL-4 and IFN-. Consistent with a regulatory role of NKT^inv cells in vivo, development of various autoimmune diseases in mice and humans has been associated with a decrease in the frequency of peripheral NKT^inv cells (8, 9, 10).

Several lines of evidence have recently been generated which suggest that NKT^inv cells derive from common mainstream precursor thymocytes, rather than from separate precursor cells committed to this lineage before variable gene rearrangement. First, the pairing of the invariant V-chain with a particular V-chain is not forced by molecular constraint (11); and second, the unused TCR and loci of NKT^inv cells are indistinguishable from those of mainstream T cells (12, 13). Therefore, the driving force for the selection of these cells may not be genetic programming, but rather Ag specificity. In agreement with a mainstream precursor (or TCR instructive) model, it has recently been suggested that mouse NKT^inv cells go through a CD4⁺CD8⁺ double positive stage during thymocyte development (14).

We reasoned that if Ag-driven selection was responsible for in vivo expansion of NKT^inv cells, then a broad CD1d-GC-specific TCR repertoire should be generated by random rearrangement. Using recombinant human CD1d-tetramers loaded with GC, we tested this hypothesis and investigated whether V24^-/CD1d-GC-specific T cells could be expanded in vitro upon stimulation of human PBMC with -GC. Our results unambiguously demonstrate the existence of human V24^-/CD1d-GC-specific T cells using a wide variety of TCR V- and V-chains. Unlike conventional NKT^inv cells, V24-independent CD1d-GC-specific T cell populations only rarely express CD161, and are either CD4⁺ or CD8⁺.

	Materials and Methods

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Generation of DC

PBMC were purified from healthy donors’ buffy coat by layering over Lymphoprep (Nycomed, Asker, Norway). Monocytes were then positively selected using magnetic beads coated with anti-CD14 mAbs (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). The monocyte-depleted lymphocyte fraction (CD14-negative) was frozen until needed. Monocytes were cultured in cell growth medium (RPMI 1640, Sigma-Aldrich, Dorset, U.K.; 10% FCS, 2 mM L-glutamine; Life Technologies, Paisley, U.K.; 1 mM nonessential amino acids, 1 mM sodium pyruvate, 55 µM 2-ME, penicillin G, and streptomycin; Life Technologies, Paisley, U.K.), containing 50 ng/ml GM-CSF (Novartis, Basel, Switzerland) and 1000 U/ml IL-4 (15). The monocytes were plated in 6-well costar plates at 4 x 10⁵ cells/ml (3 ml/well). After 4 days, maturation was induced in some wells by adding either bacterial LPS (final concentration 1 µg/ml LPS of Salmonella abortus equi; Sigma-Aldrich), 50 ng rTNF- (R&D Systems, Minneapolis, MN), or 4 x 10⁴ irradiated (6 Gy) CD40L-expressing B cells (16). Immature and matured monocyte-derived dendritic cells (Mo-DC) were used for phenotypic analysis and in vitro priming after 6 days in culture, i.e., 40 h after induction of maturation.

T cell in vitro stimulation

Monocyte-depleted or total PBMC were plated in 24-wells at 1 x 10⁶ cells/ml in cell growth medium. The following culturing conditions were chosen: 1) Freshly isolated or thawed PBMC (2 x 10⁶) from a single donor, cultured in the presence of 100 nM GC (KRN7000; Kirin Brewery, Gumna, Japan); 2) Coculture of 2 x 10⁶ monocyte-depleted, thawed lymphoctes and 2 x 10⁵ autologous, GC-pulsed immature or matured Mo-DC, respectively. For pulsing of Mo-DC with GC, cells were cultured for 2 h in 24-wells in a volume of 200 µl RPMI 1640 containing 1 µM GC, followed by addition of 2 x 10⁶ lymphocytes in 1.8 ml of the above medium (i.e., 100 nM final GC concentration). After 5 days, IL-2 was added to cultures (25 IU/ml). Thereafter, cultures were fed every 3–4 days with fresh medium containing IL-2 (1000 U/ml).

Flow cytometry

The following Abs and tetramers were used to stain single-cell suspensions of Mo-DC and in vitro stimulated T cell cultures: purified anti-CD86 (BD Pharmingen, San Diego, CA), -CD83 (Immunotech, Marseille, France), -MHC-class II (mAb L243; American Type Culture Collection, Manassas, VA), -MHC-class I (mAb W6/32; American Type Culture Collection), and PE-conjugated goat-anti-mouse pan IgG (Southern Biotechnology Associates, Birmingham, AL); allophycocyanin- and PE- conjugated human CD1d tetramers loaded with either GC or -mannosylceramide (MC) were generated as previously described (17); FITC- and PE-anti-human TCR V24, FITC anti-human TCR V11 (Serotec, Oxford, U.K.), FITC-anti-CD3, -CD4, -CD8, PE-anti-CD4 (all from DAKO, Kobenhavn, Denmark), FITC-anti-CD161, PerCP-anti-CD8, allophycocyanin-anti-CD8, -CD3 (all from BD Pharmingen), FITC-anti-TCR V1, -V9, -V12, -V16, -V18, -V23, and PE-anti-CD8 (all from Immunotech). Cells were stained on ice for 30 min, washed twice in ice-cold PBS/1% FCS, and directly analyzed. In some experiments (Fig. 4a), cells were first stained with anti-TCR V Abs, followed by CD1d-tetramers. In monomer competition experiments (Fig. 4b), cells were first incubated with CD1d-GC monomers for 20 min at room temperature before addition of CD1d-GC tetramers on ice for another 30 min. Propidium iodide was used to gate out dead cells. For monomer-binding and chase studies (Fig. 9a), cells were first incubated on ice with CD1d-GC monomers for 30 min, washed twice in ice-cold PBS, stained with R-PE-Extraavidin (Sigma-Aldrich) on ice for 30 min, and washed twice with ice-cold PBS. Then, cells were either centrifuged and immediately fixed using 4% formaldehyde in PBS (chase time "0 min") or incubated at 37°C 5% CO₂ in a volume of 200 µl PBS for different chase periods (15, 30, and 60 min) before fixation. Samples were analyzed on a FACSCalibur flow cytometer, and data were processed using CellQuest software (BD Biosciences, San Jose, CA).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Specific binding of CD1d-GC tetramers to V24- T cells. a, Preincubation of the V24-/V1+ T cell line 1B with anti-TCR V1 Ab (bold line) significantly reduced binding by CD1d-GC tetramers, as compared with the intensity of tetramer staining obtained in the presence of anti-TCR V11 Ab (thin line). b, Preincubation of the V24-/V11+ line D5.1 with CD1d-GC monomers partially blocked CD1d-tetramer staining (bold line: 50x excess of monomer over tetramer; thin line: 25x excess; gray area: tetramer only)

View larger version (31K): [in this window] [in a new window]   FIGURE 9. Binding of monomeric CD1d-GC complex to DN V24+/V11+ and CD8+V24-/V11+ T cells. Cells were sequentially stained with CD1d-GC monomers and RPE-Extraavidin (a; bold lines), or CD1d-GC tetramers (b; bold lines), or RPE-Extraavidin (a and b; gray area) only, as described in Material and Methods. a, Cells were incubated at 37°C for 0, 15, 30, or 60 min (chase) after RPE-Extraavidin staining. All cells were fixed in formaldehyde before analysis. Percentages of monomer- and tetramer-stained cells are shown for each histogram plot.

Cell sorting and generation of V24^-/CD1d-GC-tetramer⁺ cell lines

For generation of clones and oligoclonal lines, V24^-/CD1d-GC-tetramer⁺ and V24⁺/CD1d-GC-tetramer⁺ cells were sorted by a FACSVantage sorter into 96-well plates coated with 500 ng OKT3 Ab and restimulated with 1 µg/ml PHA (Sigma-Aldrich) and 1 x 10⁵ irradiated feeder cells (allogenic PBMC and B cell line LG2) in medium containing 1000 IU/ml IL-2. Established lines and clones were fed every 3–4 days with fresh medium. To generate a polyclonal V24^-/CD1d-GC-tetramer⁺ line, 3 x 10³ cells were sorted into OKT3-coated 96-wells and cultured in 200 µl of IL-2 containing medium in the presence of feeder cells. Purity of all lines was checked after 3 wk. Clones and oligoclonal lines were restimulated once with PHA and feeder cells, while the polyclonal line was snap frozen in RNAzol (Biogenesis, Poole, U.K.) until further use for spectratype analysis of TCR usage.

Chromium release assays

TCR V24-positive and -negative lines and clones were used as effector cells in a 5-h ⁵¹Cr release assay 14 days after restimulation. Human CD1d-expressing C1R cells (C1R-CD1d) were used as target cells. C1R-CD1d were labeled for 90 min with ⁵¹Cr and at the same time pulsed with either 1 µM GC, 1 µM MC, or vehicle, followed by extensive washing in warm RPMI 1640. In CD8-blocking experiments, cells were cultured in the presence of the CD8-blocking Ab, MF8 (18), or an irrelevant isotype-matched control Ab at 1/100, 1/500, and 1/1000 dilution of ascites. Cells were cultured in triplicate in 96-well round bottom microtiter plates. E:T ratios were 1:1, 3:1, and 9:1. Maximum release was determined from supernatants of cells that were lysed by addition of 5% Triton X-100, and spontaneous release was determined from target cells incubated without effector cells. Percent specific lysis was expressed as (cpm of sample - cpm of spontaneous release)/(cpm of maximum release - cpm of spontaneous release). Anti-CD8-mediated inhibition of specific lysis by D5.1 and D6.1 (Fig. 8b) was expressed as (1 - [% lysis MF8 C1R-CD1d/GC - % lysis MF8 C1R-CD1d/vehicle]:[% lysis CT C1R-CD1d/GC - % lysis CT C1R-CD1d/vehicle].

View larger version (14K): [in this window] [in a new window]   FIGURE 8. Role of CD8 coreceptor in V24-independent specific cytotoxicity against GC-pulsed C1R-CD1d. C1R-CD1d cells pulsed with GC or vehicle were used in a 51Cr-release assay as targets for CD8+V24-/CD1d-tetramer+ T cell lines D5.1 (a and b), and D6.1 (b) in the presence of either the anti-CD8 blocking Ab MF8 (GC-pulsed: ; vehicle-pulsed: ) or irrelevant isotype-matched control Ab CT (GC-pulsed: ; vehicle-pulsed: ) at 1:100 (a and b) or 1:1000 (b) dilutions. b, Results were obtained at a 9:1 E:T ratio.

A total of 2.5 x 10⁵ T lymphocytes were cultured in 48-well plates in the presence of either glycolipid-pulsed C1R-CD1d (see Materials and Methods) or 10^-7 M PMA (Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich). After 90 min, 10 µg/ml brefeldin A (Sigma-Aldrich) was added to the cultures to block cytokine secretion. After 6 h in culture, cells were washed twice in PBS and fixed in 2% paraformaldehyde. Intracellular cytokine staining was performed after permeabilization of cells with FACS permeabilization buffer (BD Biosciences), using the following Abs from BD Pharmingen: FITC-anti-IFN-, PE-anti-IL-4, PE-anti-IL-13, and allophycocyanin-anti-IL-2. Four-color analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).

Spectratyping of TCR repertoire

Total RNA was extracted from a pure TCR V24^-/CD1d-GC-tetramer⁺ polyclonal cell line (see Materials and Methods) using RNAzol reagent according to the manufacturer’s instruction, and sscDNA was synthesized by reverse transcription using Moloney murine leukemia virus reverse transcriptase and an oligo(dT) adaptor in a reaction volume of 50 µl. Oligonucleotides used to analyze the 32 different TCR V and 24 different TCR V families, as well as the C- and C-specific primers, have been described (19, 20). Each TCR V- and TCR V-PCR product was then used as a template for extension, or run-off, reactions using C- and C-specific nested fluorescent primers, respectively. The fluorescent run-off products were subjected to gel electrophoresis in an automated DNA sequencer (PerkinElmer, Bucks, U.K.), and CDR3 size distribution and signal intensities were analyzed with GeneScan software (PerkinElmer). Analysis of V-joining (J) segments was conducted in the same way using previously described fluorescent J-specific oligonucleotide probes (20).

	Results

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Expansion of CD1d-GC-specific V24-negative T cells in vitro from healthy donors’ PBMC

We and others have recently demonstrated that CD1d-tetramers loaded with GC can be used for sensitive detection of human and mouse CD1d-GC-specific NKT^inv cells (17, 21, 22). Among freshly isolated PBMC from healthy donors, the frequency of V24⁺V11⁺NKT^inv cells ranges from 0.01 to 1.0%. The frequency of V24-negative/CD3⁺ cells binding to CD1d-GC-tetramer in fresh PBMC samples from healthy donors was very low, and could not be confidently distinguished from background staining (data not shown). In contrast, distinct TCR V24-negative T cells stained by CD1d-GC-tetramers (V24^-/CD1d-GC-tetramer⁺ T cells) could be expanded in vitro from all seven donors tested, to frequencies ranging from 1.0 to 5.5% (Fig. 1, and data not shown). The percentage of V24^-/CD1d-GC-tetramer⁺ T cells varied significantly with different stimulation protocols. Interestingly, stimulation with GC-pulsed mature autologous Mo-DC was required for efficient expansion of V24^-/CD1d-GC-tetramer⁺ T cells in some donors (Fig. 1, a–c), while addition of GC alone (without Mo-DC) was sufficient to visibly expand V24^-/CD1d-GC-tetramer⁺ T cells in other subjects (Fig. 1d). In all donors, we observed a greater expansion of V24^-/CD1d-GC-tetramer⁺ cells when mature, rather than immature, GC-pulsed Mo-DC were used for stimulation (Fig. 1 and data not shown). CD1d-tetramers loaded with -mannosylceramide (MC) failed to stain both V24⁺V11⁺NKT^inv cells and V24^- T cells, confirming the specificity of binding of CD1d--GC-tetramers (data not shown). These results demonstrate that recognition of CD1d-GC in humans is not limited to the invariant V24⁺V11⁺ TCR, and they are consistent with the possibility that a broader T cell population may be capable of specifically recognizing GC presented by CD1d molecules. To address this hypothesis, we analyzed the TCR usage and Ag specificity of V24^-/CD1d-GC tetramer⁺ cells.

View larger version (45K): [in this window] [in a new window]   FIGURE 1. Expansion of V24-/CD1d-GC-tetramer+ T cells. PBMC from Donor 6 were cocultured with 100 nM GC alone (a), GC-pulsed immature Mo-DC (b), or CD40L-matured, GC-pulsed Mo-DC (c). d, PBMC from donor 7 were cultured in the presence of 100 nM GC without added Mo-DC. Percentages at day 16 of the T cell culture of V24+/CD1d-GC-tetramer+ and V24-/CD1d-GC-tetramer+ T cells are shown.

Diverse TCR V and TCR V repertoire of V24^-/CD1d-GC-specific T lymphocytes, with frequent usage of TCR V11

Human V24⁺V11⁺NKT^inv lymphocytes use TCR V11 domains with various CDR3 regions (23). In contrast, the murine equivalent to human V24⁺V11⁺NKT^inv lymphocytes, V14-J281⁺NKT^inv lymphocytes, use at least five different TCR V families with polyclonal CDR3 regions (6). For these reasons, it has been speculated that the V-chain does not contribute to the specific recognition of CD1d-GC (21). When we compared staining of GC-stimulated in vitro cultures with CD1d-GC-tetramers and Abs against TCR V24 or V11, we found that in all seven donors, a substantial proportion of V24^-/CD1d-GC-specific T cells expressed V11 (Fig. 2). In some donors, V24^-V11⁺/CD1d-GC-tetramer⁺ cells comprised >90% of all V24^-/CD1d-GC-specific T cells (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. A high proportion of V24-/CD1d-GC-specific T cells express TCR V11. PBMC from donor 1 were cocultured with LPS-matured, GC-pulsed Mo-DC. Stainings were conducted with a, CD1d-GC-tetramers and either V24-specific Abs; or b, A mixture of V24- and V11-specific Abs. Percentages (on day 16 of the T cell culture) are shown for V24+/CD1d-GC-tetramer+ (a), V24-/CD1d-GC-tetramer+ (a), V24-V11-/CD1d-GC-tetramer+ (b), and CD1d-GC-tetramer+/(V24 V11)+ T cells (b). All density plots were gated on propidium iodide negative, CD3+ lymphocytes.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. TCR V, V, and J usage of V24-independent CD1d-GC-specific T lymphocytes. A polyclonal V24-/CD1d-GC tetramer+ line from Donor 4 was generated as described in Material and Methods. a, The V24-/CD1d-GC tetramer+ phenotype and purity of this line was confirmed before isolation of mRNA for spectratype analysis. b, Spectratype analysis on cDNA from this line ruled out contamination by TCR V24-expressing cells. c, Positive results of the spectratype analysis are shown for each V or V family, together with the amino acid length of the corresponding CDR3 region. c, Positive results for J segments are also shown. a, The density plot was gated on propidium iodide negative, CD3+ cells.

Taken together, these results demonstrate that GC not only stimulates V24⁺V11⁺NKT^inv lymphocytes, but can also efficiently induce a CD1d-GC-specific polyclonal, V24-independent T cell response. TCR V11 is overrepresented among V24^-/CD1d-GC-specific T cells, suggesting either an inherent affinity of V11 to CD1d or its direct involvement in specific recognition of CD1d-GC.

Functional capacities of V24^-/CD1d-GC-specific T lymphocytes

To further assess Ag specificity of V24^-/CD1d-GC-tetramer⁺ T lymphocytes, we studied their capacity to specifically lyse CD1d-transfected C1R cells (C1R-CD1d) pulsed with GC, MC, or vehicle (Fig. 5). These results demonstrated that both CD4⁺ and CD8⁺V24^-/CD1d-GC-tetramer⁺ T cells are highly efficient in specifically lysing GC-pulsed, but not unpulsed or MC-pulsed, C1R-CD1d (Fig. 5). In the same experiment, levels of specific lysis were similar for V24^-/CD1d-GC-tetramer⁺ T cells and V24⁺V11⁺NKT^inv (data not shown).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Specific cytotoxicity of V24-/CD1d-GC-tetramer+ T lymphocytes. C1R-CD1d cells, pulsed with -GC (), -MC (), or vehicle (•) were used as targets in a standard 5-h 51Cr-release assay. The CD4+V24-/V11+ clone 2A (left panel), the CD4+ V24-/V1+ line 1D (>99% V24-/CD1d-GC tetramer+; middle panel), and the CD8+V24-/V11+ line D5.1 (>99% V24-/CD1d-GC tetramer+; right panel) were used as effector T cells at the indicated E:T ratios.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Intracellular cytokine staining of V24-/CD1d-GC-tetramer+ T lymphocytes. C1R-CD1d cells pulsed with GC (top panel) or MC (middle panel) were used to stimulate V24-/CD1d-GC-tetramer+ T cells as described in Materials and Methods. For maximum stimulation, T cells were incubated with PMA and ionomycin (P+I; bottom panel). Results shown were obtained using line 2C (>99.8% pure for V24-/V1+CD1d-GC-tetramer+ T cells), and are representative of two separate experiments.

CD4/CD8-coreceptor use and CD161-expression in V24^-/CD1d-GC-specific T cells

Mouse and human NKT^inv cells are either CD4⁺CD8^- or CD4^-CD8^- (DN), whereas to date no CD8-expressing GC-specific NKT^inv cells have been described (1, 24). We observed that a significant proportion of V24^-/CD1d-GC-specific T cells in several donors were CD8⁺CD4^- (Fig. 7), while all other V24^-/CD1d-GC-specific T cells were CD4⁺CD8^- (data not shown). In contrast, DN V24^-/CD1d-GC-specific T cells were not found in any of the seven donors analyzed (data not shown). To investigate the role of CD8 in V24^-/CD1d-GC-specific T cell lines, we assessed whether lysis of GC-pulsed C1R-CD1d cells by CD8⁺V24^- cells could be inhibited by the presence of an anti-CD8 blocking Ab (18). The results of these experiments showed that incubation with anti-CD8 blocking Ab, but not with an irrelevant isotype-matched control Ab, significantly reduced specific lysis of GC-pulsed C1R-CD1d cells (Fig. 8).

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Phenotype of CD1d-GC-specific T lymphocytes. Cells from donor 6 were stained with CD1d-GC-tetramer, anti-V24, and either anti-CD8 (top panel), anti-CD4 (middle panel), or anti-CD161 (bottom panel) Abs, 2 wk after in vitro stimulation with 100 nm GC. Histogram plots were gated on V24-/CD1d-GC-tetramer+, V24+/CD1d-GC-tetramer+, or CD1d-GC-tetramer-negative cells as indicated. Percentages of CD8+, CD4+, and CD161+ cells in the gated population are shown. Propidium iodide was used to gate out dead cells.

Different binding of CD1d-GC monomeric complexes to CD8⁺V24^- and DN V24⁺CD1d-GC-specific T cells

Mouse V14⁺NKT^inv cells have been previously shown to bind CD1d-GC monomers (21). Our results demonstrate that human CD1d-GC monomers were capable of binding to a DN V24⁺V11⁺ line, while they failed to stain a panel of CD8⁺V24^-CD1d-GC-specific T cell clones and lines (Fig. 9a and data not shown). As a control, we showed that CD3 expression levels and intensity of tetramer-staining for CD8⁺V24^- and DN Va24⁺CD1d-GC-specific cells were identical (Fig. 9b and data not shown). Monomeric CD1d-GC complexes were capable of staining a large fraction of the DN V24⁺V11⁺ line and dissociated with a half-life of 50 min (Fig. 9a and data not shown). Together these results suggested that compared with DN V24⁺V11⁺ cells, TCRs of CD8⁺V24^-CD1d-GC-specific cells have a lower affinity for CD1d-GC.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The glycolipid-presenting CD1d molecule is highly conserved among mammalian species and exhibits distinct cross-specific recognition by conserved DN or CD4⁺ lymphocyte subsets coexpressing an invariant TCR V24-JQ segment paired to V11 and CD161 (NKR-P1) (3). These NKT^inv specifically recognize the marine-sponge-derived, synthetic glycolipid GC, while the natural ligand seen by these cells in vivo remains unknown (5).

Recent evidence from mouse studies indicates that NKT^inv cells do not develop from separate precursor cells committed to this sublineage before variable-gene rearrangement, but branch off the mainstream developmental pathway because of their TCR specificity (11, 12, 13, 14). Therefore, we reasoned that recognition of CD1d-GC may not be uniformly restricted to NKT^inv cells bearing the canonical TCR V24-JQ chain, but may also be mediated by other randomly generated TCRs.

We and others have recently described the use of rCD1d-tetramers loaded with GC for sensitive and specific identification of both murine invariant V14-J281⁺ or human V24⁺V11⁺NKT^inv cells (17, 21, 22). Using different in vitro culture conditions in combination with the use of human CD1d-GC-tetramers, we tested the hypothesis that human T lymphocytes, other than the "conventional" CD161⁺V24⁺V11⁺NKT^inv subset, can recognize CD1d-bound GC.

Distinct V24-negative T cell populations binding to CD1d-GC-tetramers were detected in all seven healthy donors’ PBMC after in vitro stimulation with GC-pulsed matured Mo-DC (Fig. 1). In two donors, addition of GC alone (without Mo-DC) was sufficient to visibly expand V24^-/CD1d-GC-specific T cells (Fig. 1d and data not shown), suggesting that these cells were expanded in vivo, albeit at a frequency below the threshold required for tetramer staining. Specificity of these novel T cell subsets for CD1d-GC complex was demonstrated in several ways. First, V24^-/CD1d-GC tetramer⁺ lines and clones specifically lysed GC-pulsed, but not unpulsed or MC-pulsed C1R-CD1d cells, ruling out ligand-independent recognition of CD1d (Fig. 5). Second, addition of monomeric CD1d-GC complexes efficiently prevented staining by CD1d-GC tetramers (Fig. 4b). Finally, preincubation with TCR-specific Abs significantly reduced specific staining by CD1d-GC tetramers (Fig. 4a).

Consistent with a mainstream model of NKT^inv cell selection, spectratype analysis demonstrated that V24^-/CD1d-GC-specific T cells can use a wide variety of V and V segments (Fig. 3). However, a marked bias was observed for the use of V11 (Fig. 2 and data not shown), with >90% of V24^-/CD1d-GC-specifc T cells using V11 in some donors (data not shown). Previous tetramer-based analysis of NKT^inv cells in mice have suggested that the CDR3 regions are permissive, but do not specifically contribute to the recognition of CD1d-GC complexes (21). Similar to the observation that some V regions have inherent affinity for MHC class II molecules (27), human V11 regions may have inherent affinity for CD1d molecules. Alternatively, the V11 regions in V24^-/CD1d-GC-specifc T cells may contribute more specifically to the recognition of CD1d-GC complexes.

"Conventional" NKT^inv in humans and mice exhibit a CD4⁺ or a DN phenotype (1). In contrast, all Va24^-/CD1d-GC-specific T cells in our seven donors were either CD4⁺ or CD8⁺, but they were never DN (Fig. 7 and data not shown). Importantly, specific lysis of GC-pulsed CD1d-expressing targets by CD8⁺V24^-/CD1d-GC-specific lines was significantly reduced in the presence of an anti-CD8 blocking Ab (Fig. 8), demonstrating that CD8 can act as a coreceptor for human glycolipid-specific CD1d-restricted T cells.

Previous studies in mice have provided evidence that CD1d binds CD8 and not CD4 (7, 11, 12). Lantz and Bendelac (11) have shown that in V14-J281-transgenic mice, the CD8-compartment is selectively depleted of V7 and V8, i.e., the main V-chains used by mouse V14-J281 NKT^inv cells. The same group found that V14-J281 NKT^inv were lost in CD8 transgenic mice, suggesting that CD8-expressing NKT^inv cells are negatively selected during thymic development due to excessive avidity (12). This model predicts that NKT^inv cells bear TCRs with a high inherent affinity for CD1d loaded with either GC or its natural "GC-like" ligand. Our observation that monomeric CD1d-GC complex can efficiently bind to DN NKT^inv cells, but not to CD8⁺V24^-CD1d-specific T cells (Fig. 9a) is highly consistent with such a model. In addition, it has been previously demonstrated that higher affinities of the TCR-peptide/MHC interaction favor the development of a CD4⁺ phenotype, whereas lower affinities result in a CD8⁺ phenotype (28, 29), and that TCR binding energetics can determine the expression of NKR-P1 by NKT^inv cells (30, 31, 32). Among V24^-/CD1d-GC-specific T cell subsets in the subjects we studied, CD161 was rarely expressed on CD4⁺ cells (data not shown), and was absent from CD8⁺ cells (Fig. 7).

Based on these results, we hypothesize that in humans, a wide variety of CD1d-GC-specific TCRs are generated by random TCR rearrangement, and that the binding affinity of a given TCR-CD1d/GC interaction is a key determinant in CD4/CD8/DN lineage commitment and CD161 expression. The fact that V24^-/CD1d-GC-specific T cells can express CD8 and exhibit CD8-dependent cytotoxicity (Figs. 7 and 8) is consistent with a lower affinity TCR for CD1d-GC in these CD8⁺ cells compared with conventional CD8^-V24⁺/V11 NKT^inv cells, and the clear difference in CD1d-GC monomer staining between CD8⁺ and DN V24⁺CD1d-specific T cells (Fig. 9a) supports this hypothesis.

Hence, it is possible that higher surface density of CD1d loaded with -GC (or its natural ligand) may be required for expansion of V24^-/CD1d-GC-specific T cells compared with NKT^inv. Although the identity of the -GC-like natural ligand is not known, it is tempting to speculate that conditions associated with an increased synthesis of the "-GC-like" natural ligand in vivo may lead to the expansion of V24^-/CD1d-GC-specific T cells. Likewise, injection of GC in vivo for therapeutic reasons may induce expansion of V24^-/CD1d-GC-specific T cells. Phase I clinical trials, investigating the safety profile of weekly i.v. GC injections in patients with solid malignant tumors are currently underway (33). Based on our findings, it is possible that CD4⁺ and CD8⁺Va24^-/CD1d-GC-specific T cells expand in a proportion of patients receiving GC. Because the in vivo function of these cells is still unknown, we suggest that tetramer-based monitoring of V24^-/CD1d-GC-specific T cells as well as NKT^inv cells should be considered in patients receiving GC.

In conclusion, we have demonstrated that specific recognition of CD1d-GC complex can be mediated by human V24-JQ-independent T cell subsets, which use a variety of TCR V and TCR V families. In contrast to conventional NKT^inv cells, V24-independent T cells are never found in the DN compartment, while they can express CD8 and exhibit CD8 coreceptor-dependent specific cytotoxicity. Furthermore, CD161 is only very rarely expressed by V24^-/CD1d-GC-specific T cells expanded in vitro. Finally, the existence of a diverse repertoire of CD1d-GC-specific T cells in humans strongly supports their Ag-driven selection. It remains to be assessed whether V24-independent CD1d-GC-specific T cells are elicited as a result of an adaptive immune response, more similar to MHC/peptide T cell responses, and whether they may have a different physiological role than conventional NKT^inv cells.

	Acknowledgments

 
We thank Xiao-Ning Xu for providing the MF8 and CT Abs; Dawn Shepard for technical help; and Michael J. Palmowski, Uzi Gileadi, and Rod Dunbar for helpful discussions and critical reading of the manuscript. -GC (KRN7000) was generously provided by Kirin Brewery (Gunma, Japan).

	Footnotes

 
¹ This study was funded by research grants from the Cancer Research U.K. and the Cancer Research Institute of United States of America. S.D.G. was funded by grants from the Swiss National Science Foundation, the Roche Research Foundation, and Novartis Stiftung. N.D. was funded by the French Institute National de la Santé et de la Recherche Médicale.

² Current address: Department of Rheumatology and Clinical Immunology, University Hospital Bern, Inselspital, CH-3010 Bern, Switzerland. E-mail address: stephan.gadola{at}insel.ch

³ Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Weatherall Institute of Molecular Medicine, OX3 9DS Oxford, U.K. E-mail address: vincenzo.cerundolo{at}imm.ox.ac.uk

⁴ Abbreviations used in this paper: GC, -galactosylceramide; NKT^inv, invariant NK T cell; DN, double negative; Mo-DC, monocyte-derived dendritic cell; MC, -mannosylceramide; J, V-joining.

Received for publication January 3, 2002. Accepted for publication March 25, 2002.

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