Conserved and Heterogeneous Lipid Antigen Specificities of CD1d-Restricted NKT Cell Receptors

Preparation of lipid Ags

The glycolipid Ags α-GalCer, α-GlcCer, and α-mannosylceramide (α-ManCer) were prepared from d-lyxose as described (27). The Sphingomonas wittichii Ag GSL-1′sA was synthesized using a protected galacturonic acid and ceramide lipid moiety as described (16). The end products and synthetic intermediates of all lipids were characterized by ¹H- and ¹³C-nuclear magnetic resonance and electrospray mass spectrometry. The iGb3 glycolipid Ag was a gift from Drs. A. Bendelac and D. Zhou of the University of Chicago (Chicago, IL). Purified and synthetic phospholipids were purchased from Matreya and Avanti Polar Lipids. All lipids were dissolved in DMSO at a concentration of 100 μg/ml and stored frozen at −20°C. Before use, the lipids were sonicated in a heated water bath for 15 min at 37°C.

Preparation of soluble CD1d fusion proteins and tetramers

Soluble human and murine CD1d-Fc fusion proteins and tetramers were produced as described previously (13, 28). Briefly, single-chain β2m-CD1d-Fc fusion proteins were produced in mammalian cells and purified from culture supernatants by protein A affinity chromatography. The resulting dimeric CD1d-Fc fusion proteins were formed into complexes with fluorescently labeled soluble protein A molecules and purified by size exclusion chromatography on a Superose 6 column (Amersham Biosciences).

Derivation of CD1d-restricted T cell clones

T cell clones were established from human tissue samples as described previously (29), and protocols were approved by the Brigham and Women’s Hospital (Boston, MA) Institutional Review Board and the University of Wisconsin Medical School (Madison, WI) Minimal Risk Institutional Review Board. Briefly, T cells sorted using CD1d-tetramers were cultured at 37°C with 5% CO₂ in RPMI 1640 culture medium containing 10% FBS, 2% human AB serum, 1% penicillin and streptomycin, and 1% l-glutamine in the presence of irradiated allogeneic PBMC and PHA. After 5–10 days of culture, 200 U/ml recombinant human IL-2 (Chiron) was added to the medium.

Determination of TCR sequences

TCRβ sequences were obtained by RT-PCR using a Cβ domain primer (5′-GCTGCCTTCAGAAATCCTTTC-3′) paired with a primer specific for Vβ11 (5′-GCCCCAACTGTGCCATG-3′). TCRα sequences were obtained using a Cα domain primer (5′-TCAGCTGGACCACAGCC-3′) paired with a primer specific for Vα24 (5′-CTCTGCAGAATAAAAATGAAAAAGC-3′). To amplify the TCRα sequences of the Vα24-negative TCRs, the constant domain primer was paired with mixes of primers specific for individual TCRα variable genes (30) or with one of the following degenerate primers: 1) 5′-CCGCGGCCGCGTCAYGGTCTCCCYGTGTCTTG-3′; 2) 5′-CCGCGGCCGCTTGGTGATCYTGTGGCTKC-3′; or 3) 5′-CCGCGGCCGCAGRTGATTTTTACSCTGGG-3′. The resulting PCR products were subcloned into the TOPO-TA vector (Invitrogen Life Technologies), and the inserts were sequenced with standard primers located in the vector. A minimum of three subclones were sequenced for each PCR product. To verify the sequences obtained using degenerate primers or primer mixes, nucleotide sequences of the initial PCR products were analyzed and compared with the GenBank database to identify the putative Vα gene segment used. Based on this analysis, the following specific PCR primers were designed and used to amplify complete V/J TCRα products: 1) 5′-CATGTGATAGAAAGACAAGATGGTC-3′ (Vα10 forward); 2) 5′-GGGCAGAAAAGAATGATGAAATC-3′ (Vα2S2 forward); 3) 5′-GGAAGAAGAATGGAAACTCTCC-3′ (Vα3S1 forward); and 4) 5′-TCAGCTGGACCACAGCC-3′ (Cα reverse).⁵

Flow cytometric analysis

CD1d tetramers were prepared as described above and loaded at a 40:1 molar ratio with glycolipid Ags dissolved in DMSO or mock treated with DMSO alone and used to stain cells freshly purified from human tissue samples or T cell clones, as described previously (28). Briefly, the cell samples were incubated for 20 min at 4°C with tetramers (10 μg/ml) or directly conjugated Abs in a PBS buffer containing 1 mg/ml BSA and 0.01% NaN₃. The cells were then washed, stained with propidium iodide to identify dead cells, and analyzed by flow cytometry. The mAbs used for costaining were the following: anti-Vα24 (clone C15; Beckman Coulter); anti-Vβ11 (clone C21; Beckman Coulter); anti-CD4 (clone RPA-T4; BD Pharmingen/BD Immunocytometry Systems); and anti-CD3 (clone UCHT1, BD Pharmingen/BD Immunocytometry Systems). Where indicated, CD1d-restricted T cell clones were preincubated with 10 μg/ml unlabeled anti-Vα24 mAb and then washed and stained, or the CD1d tetramer was preincubated before use with 20 μg/ml CD1d42 anti-CD1d-specific mAb (provided by Drs. S. Porcelli, Albert Einstein College of Medicine (Bronx, NY) and M. Exley, Harvard Medical School (Boston, MA).

Human CD1d-Fc dimers were prepared by protein A affinity chromatography as described above and then further purified and desalted into PBS by size exclusion chromatography on a Superose 6 column (Amersham Biosciences). The dimers were loaded at a concentration of 100 μg/ml in PBS containing 100 μg/ml BSA (Sigma-Aldrich) with a 40:1 molar ratio of lipid Ags dissolved in DMSO or mock treated with DMSO alone by incubating for 24 h at 37°C. CD1d-restricted T cells were incubated with lipid-loaded or mock-treated dimers for 30 min at 4°C in a PBS buffer containing 1 mg/ml BSA and 0.05% NaN₃ and then washed and stained with 10 μg/ml phosphatidylethanolamine-labeled goat anti-mouse IgG (BD Biosciences) and detected by flow cytometry.

T cell responses to APCs

APCs were either CD1d-transfected 721.221 cells generated as described (31) or in vitro-derived immature dendritic cells (DCs) generated from human peripheral blood monocytes purified by CD14-positive magnetic bead selection (Miltenyi Biotec) that were cultured for 3 days with 200 U/ml and 300 U/ml rIL-4 and GM-CSF, respectively. DCs were pulsed for 12–16 h with lipid Ags at the indicated concentrations or with vehicle (DMSO) alone and then washed and coincubated with a 1:1 ratio of CD1d-restricted T cells (5 × 10⁴ per well) in sterile 96-well plates in RPMI 1640 culture medium lacking IL-2 (i.e., RPMI 1640 containing 10% iron-supplemented bovine calf serum and penicillin/streptomycin) for 24 h at 37°C and 5% CO₂. Culture supernatants were then withdrawn and tested for the indicated cytokines using a standard, commercially available ELISA and purified recombinant cytokines as standards. Anti-CD1d mAb blocking experiments were performed by coincubating the CD1d59 murine IgM mAb (a gift from Dr. S. Porcelli, Albert Einstein College of Medicine (Bronx, NY)) or the MOPC-104E negative control IgM mAb (Sigma-Aldrich) with the APCs and T cells. The Griffonia simplicifolia I-B₄ lectin (Vector Laboratories; dissolved in 10 mM HEPES, 0.15 M NaCl, 0.08% NaN₃, and 0.1 mM Ca²⁺) was coincubated with the APCs and T cells at the indicated concentrations and compared with mock-treated (diluent buffer alone) APCs.

T cell responses to lipid Ags presented by plate-bound CD1d molecules

CD1-restricted T cell clones were tested for responses to lipid Ags presented by plate-bound CD1d fusion proteins, as described previously (13, 29). Briefly, the CD1d-Fc fusion protein or an isotype-matched negative control mAb was coated in a 10:1 ratio with an anti-LFA-1 mAb onto 96-well microtiter plates. Lipid Ags dissolved in DMSO or vehicle alone were diluted into PBS and incubated with the CD1d fusion protein or negative control mAb at 37°C for 24–72 h. The plates were then thoroughly washed, and CD1d-restricted T cell clones were added in RPMI 1640 culture medium lacking IL-2. The plates were incubated for 24 h at 37°C and 5% CO₂, and the supernatants were then harvested and analyzed for cytokine content using a standard, commercially available ELISA.

Generation of α-GalCer-reactive, Vα24-negative, CD1d-restricted T cell clones

CD1d-restricted T cells were cloned from human PBMCs by single cell flow cytometric sorting using α-GalCer-loaded human CD1d tetramers as described previously (28, 29). The tetramer-derived clones were tested for staining by mAbs specific for TCR Vα24 or Vβ11. Of eight CD1d-restricted T cell clones derived in an initial experiment, seven (J3N.4, J3N.5, J3N.11, C1N.4, C1N.5, C1H.3, and CAD1.1) stained positively using both the anti-Vα24 and anti-Vβ11 mAbs (Fig. 1⇓A and data not shown), whereas one clone (J3N.1) stained positively with the anti-Vβ11 mAb and showed no specific staining with the anti-Vα24 mAb (Fig. 1⇓B). Hence, this clone appeared to possess a Vα24-negative, Vβ11-positive TCR. To confirm that the staining was specific, we investigated whether the CD1d tetramer staining could be blocked by Ab binding. The anti-Vα24 mAb completely prevented binding of the α-GalCer-loaded CD1d tetramer to a clone that appeared Vα24-positive (Fig. 1⇓C) but had no effect on tetramer binding to clone J3N.1 (Fig. 1⇓D). In contrast, prebinding an anti-CD1d mAb to the CD1d tetramer inhibited its binding to clone J3N.1 as well as to a Vα24-positive clone, indicating that the staining was CD1d-dependent (Fig. 1⇓, C and D).

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FIGURE 1.

A–D, Flow cytometric staining of CD1d-restricted T cells. Staining of clone J3N.5 (A) and clone J3N.1 (B) with the reagent shown on the x-axes (solid lines) or with negative control tetramers or Abs (dashed lines). Clone J3N.5 (C) and clone J3N.1 (D) were stained with an α-GalCer-loaded CD1d tetramer (solid lines) or vehicle-treated CD1d tetramer (dashed lines). Top panels show staining of unblocked samples, middle panels show staining after the T cells were preincubated with an anti-Vα24 Ab, and bottom panels show staining with an α-GalCer-loaded tetramer that was preincubated with an anti-CD1d mAb. E, Staining of a B cell-depleted PBMC sample gated on the lymphocyte subset by forward and side scatter. The left panel shows the staining using vehicle-treated CD1d tetramer, the middle panel shows staining using α-GalCer loaded tetramer, and the right panel shows staining using α-GalCer-loaded tetramer after blocking with anti-Vα24 mAb.

A previous analysis reported polyclonal Vα24-negative T cell lines that stained with α-GalCer-loaded CD1d tetramers, but these cells were detected after expansion in vitro by stimulation with α-GalCer (10). To investigate the presence of such cells in unstimulated PBMC samples directly ex vivo, 20 healthy donors were tested to evaluate staining by α-GalCer-loaded CD1d tetramers after blocking with the anti-Vα24 mAb. For almost all donors the percentage of CD1d tetramer-stained events was significantly reduced in samples blocked with the anti-Vα24 mAb; however, some tetramer-positive events remained and usually included both CD4-positive and CD4-negative T cells (Fig. 1⇑E). There was substantial donor-to-donor variation in the percentage of CD1d tetramer-stained events detected after anti-Vα24 mAb blocking, ranging from 3 to 100% of the unblocked percentage with a mean of 22% (SD of 23%) and a median of 14%. In most cases, after blocking with the anti-Vα24 mAb some tetramer-positive cells had reduced fluorescence intensity compared with unblocked samples, whereas the fluorescence intensity of some cells was similar to that of unblocked samples (Fig. 1⇑E). This result suggested that in some cases the anti-Vα24 mAb did not compete at all with the tetramer for TCR binding, and in other cases it either competed inefficiently or facilitated the detection of increased numbers of T cells that have low affinity for α-GalCer-loaded CD1d. Thus, this analysis demonstrated that most donors possessed a significant fraction of CD1d tetramer-positive T cells for which staining was not efficiently blocked by the anti-Vα24 mAb, but it was not clear what fraction were Vα24-negative T cells as opposed to Vα24-positive T cells, that have low affinity for the anti-Vα24 mAb.

To further investigate the characteristics of the inefficiently blocked population, CD1d tetramer-stained T cells were cloned from an anti-Vα24 mAb-pretreated PBMC sample. Four CD1d-restricted T cell clones were obtained that, upon subsequent analysis, showed no positive staining with the anti-Vα24 mAb (J24N.16, J24N.22, J24N.43, and J24N.70), and three clones (J24L.10, J24L.17, and J24L.28) were obtained that stained weakly positive with the anti-Vα24 mAb (data not shown). All of the clones showed clearly positive staining with the anti-Vβ11 mAb (data not shown). Thus, all together the panel of T cell clones generated using α-GalCer-loaded human CD1d tetramers consisted of five clones that showed no staining with an anti-Vα24 mAb, but were stained with anti-Vβ11, and thirteen that were stained positively by both mAbs. Two of the Vα24-negative clones (J3N.1 and J24N.70) were negative for both CD4 and CD8β (double negative) and the other three were CD4-positive, whereas 12 of the 13 Vα24-positive clones were CD4-positive and one was double negative (data not shown).

TCR sequences of CD1d-restricted T cell clones

TCR α-chain and β-chain sequences of the Vα24-positive and Vα24-negative clones were determined by RT-PCR. Analysis of the TCR α-chain sequences of the CD1d-restricted T cell clones revealed three types (see Table I⇓): 1) nine clones with “invariant” TCR α-chains that use the previously described canonical rearrangement of Vα24 with Jα18 (2, 3, 4, 5); 2) four clones having “variant” Vα24-positive TCR α-chains that consist of Vα24 rearranged with Jα18 but containing single amino acid substitutions at position 92 or 93 at the end of the V gene segment; and 3) five clones that do not stain with the anti-Vα24 mAb and use V genes other than Vα24. These clones were found to use one of three Vα genes: AV10S1, AV2S2, or AV3S1. Three of the five Vα24-negative clones used AV10S1, but, notably, two different alleles were used, and each clone also had unique substitutions at the end of the V gene segment, presumably resulting from N region changes (Table I⇓). Remarkably, all of the Vα24-negative T cell clones use the same Jα segment (Jα18) that is used by the Vα24-positive clones and maintain identical CDR3 length. Hence, the primary sequence of the CDR3 region of the TCR α-chains is highly conserved among all of the clones (Table I⇓).

Analysis of the TCR β-chain sequences confirmed the use of the Vβ11 gene segment paired with a variety of Jβ segments (see Table I⇑). Most clones contained unique TCRβ junctional rearrangements, but two independently derived clones were identical, and one clone appeared to have two different Vβ11-positive rearrangements (Table I⇑). Because of the usage of different D and Jβ segments and substantial N region alterations, the TCR β-chains of the clones are predicted to have CDR3 loops that are highly heterogeneous in length, charge, polarity, and inclusion of aliphatic residues. Notably, the CDR3 regions of the TCR β-chains from the Vα24-negative clones did not appear distinct from those of the Vα24-positive clones in any of these parameters.

Conserved recognition of bacterial α-GSL

As expected from their specific binding of α-GalCer-loaded CD1d tetramers, the Vα24-negative T cell clones resembled Vα24-positive T cells by responding functionally to CD1d-mediated presentation of α-GalCer (see Figs. 2⇓ and 3⇓). The Vα24-positive and Vα24-negative clones showed similar dose-response curves to α-GalCer presented by CD1d-positive APCs or by plate-bound recombinant CD1d molecules, suggesting that they have a similar sensitivity to this Ag (Figs. 2⇓ and 3⇓). For both types of clone, the responses to α-GalCer could be inhibited by addition of an anti-CD1d mAb, and neither type showed significant responses to CD1d-negative APCs that were pulsed with α-GalCer (data not shown), demonstrating the CD1d-dependence of the α-GalCer recognition.

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FIGURE 2.

Cytokine secretion by Vα24-positive and Vα24-negative CD1d-restricted T cell clones in response to APCs pulsed with the indicated concentrations of α-GalCer (filled circles), α-GSL (open circles), or vehicle alone (open squares). Filled squares show the responses of each T cell to the nonspecific stimulator PHA-m. Values that were below the detection limit of the IL-4 ELISA were assigned a value of 1 pg/ml for plotting on the logarithmic scale. Assays were performed in triplicate, and error bars represent the SD values of the means (in some cases these values are too small for the bars to be visible on the plots). Similar results were obtained in three independent experiments.

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FIGURE 3.

Cytokine secretion by Vα24-positive and Vα24–negative clones in response to plate-bound recombinant CD1d molecules pretreated with α-GalCer (filled circles), α-GlcCer (open circles), or vehicle alone (open squares). Assays were performed in triplicate, and error bars show the SD values of the mean. Similar results were obtained in three independent experiments.

Bacterially produced α-GSLs have recently been identified that are structurally similar to α-GalCer but contain modifications of the sugar and lipid moieties (16). These compounds were significantly less potent activators of canonical CD1d-restricted NKT cells than α-GalCer, suggesting that the modifications reduce the efficiency of CD1d loading or TCR binding (16, 17, 18). We investigated the responses of Vα24-negative and Vα24-positive CD1d-restricted T cell clones to GSL-1′sA, a bacterial α-GSL related to α-GalCer, that has a keto group attached to carbon 6 of the galactose sugar head group and lacks one hydroxyl group on the lipid’s sphingosine chain (16, 18). DCs (GM-CSF and IL-4 cultured human monocytes) were pulsed with α-GSL or α-GalCer or vehicle alone and then washed and cocultured with the T cell clones. The Vα24-negative and Vα24-positive clones had dose-response curves essentially identical to that of the GSL-1′sA lipid (Fig. 2⇑). Thus, in addition to their shared reactivity for α-GalCer, Vα24-negative and Vα24-positive clones appear equivalently able to respond to a bacterial α-GSL.

Discrimination of α-linked sugar moieties

CD1d-restricted T cells with Vα14- or Vα24-invariant TCR α-chains have been previously characterized as responding similarly to α-GalCer and α-GlcCer but failing to respond to α-ManCer (14, 15). The Vα24-negative and Vα24-positive T cell clones were tested in parallel for their ability to respond to the α-GalCer and α-GlcCer lipids presented by plate-bound recombinant CD1d molecules (29). All of the Vα24-positive CD1d-restricted T cell clones responded robustly to both α-GalCer and α-GlcCer, whereas all of the Vα24-negative clones responded strongly to α-GalCer but showed markedly lower responses to α-GlcCer (Fig. 3⇑). There was little or no cytokine secretion in response to α-ManCer or to vehicle-treated CD1d molecules by any of the clones, and no detectable response was observed to a negative control protein treated with α-GalCer, demonstrating the specificity and CD1d-dependence of the α-GalCer and α-GlcCer responses (data not shown). These results indicated that the Vα24-negative and Vα24-positive T cell clones differ in their specificity for sugar residues of α-GSLs.

To investigate this sugar specificity difference further, three Vα24-positive and three Vα24-negative T cell clones were tested for staining by human CD1d-Fc dimers loaded with either α-GalCer or α-GlcCer or treated with vehicle alone. The Vα24-positive T cells stained similarly with α-GalCer- and α-GlcCer-loaded CD1d dimers, whereas the Vα24-negative T cells showed significant positive staining only with the α-GalCer-loaded CD1d dimer (Fig. 4⇓). The titration curves for clones J3N.1 and J24N.70 appeared to approach saturation at a similar concentration of the α-GalCer-loaded CD1d dimer as that observed for the Vα24-positive clones tested, suggesting that the affinity of these Vα24-negative TCRs for α-GalCer may be close to that of canonical Vα24-positive TCRs (Fig. 4⇓). In contrast, the titration curve for clone J24N.22 reproducibly appeared to require higher dimer concentrations to reach saturation, suggesting that the TCR of this clone may have a lower affinity for α-GalCer (Fig. 4⇓). Thus, variation in Vα-encoded TCR α-chain regions may affect the strength of the interaction with α-GalCer, but the most significant effect appears to be on the ability to bind α-GlcCer. These results suggest that the weaker functional response of the Vα24-negative T cell clones to α-GlcCer is due to lower TCR affinity for this glycolipid compared with α-GalCer, whereas Vα24-positive TCRs appear to have similar affinity for both glycolipids.

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FIGURE 4.

Flow cytometric staining of Vα24-positive and Vα24-negative T cell clones by CD1d-Fc dimers loaded with α-GalCer (filled circles) or α-GlcCer (open circles). Similar results were obtained in three independent experiments.

Autoreactive responses

Many CD1d-restricted T cells are autoreactive to CD1d-positive APCs in that they respond functionally to contact with such APCs in the absence of exogenously added Ags. A number of studies have shown that this effect is apparently due to recognition of specific cellular glycolipids presented at the cell surface by CD1d (9, 11, 12, 13). The autoreactive responses of selected Vα24-positive and Vα24-negative clones were investigated by testing cytokine secretion in response to CD1d-transfected 721.221 cells compared with the untransfected parent cells in the absence of added Ags, and the CD1d dependence was confirmed by blocking with an anti-CD1d mAb. A CD1d-dependent response could be detected for all of the clones tested, but the magnitude of the response varied markedly (Fig. 5⇓A). The Vα24-negative clones J3N.1 and J24N.70 reproducibly showed only modest cytokine secretion in response to the CD1d-transfected APCs, whereas clone J24N.22 showed a somewhat higher cytokine secretion in response to the transfectants that was similar to that of the Vα24-positive clone J3N.4 (Fig. 5⇓A). Two other Vα24-positive clones, J3N.5 and J24L.17, had progressively greater cytokine secretion responses, respectively (Fig. 5⇓A). The level of the autoreactive responses did not correlate with cytokine secretion stimulated by the nonspecific stimulator PHA (Fig. 5⇓A), suggesting that the differences among the clones were not simply due to differing activation states or cytokine production capacity.

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FIGURE 5.

A, Cytokine secretion by the indicated T cell clones in response to untransfected 721.221 cells (hatched bars), CD1d-transfected 721.221 cells (black bars), CD1d-transfected 721.221 cells in the presence of a negative control mAb (light gray bars), the CD1d59 blocking mAb (dark gray bars), or the nonspecific stimulator PHA-m (open bars). B, Cytokine secretion by the indicated clones in response to CD1d-transfected 721.221 cells alone (black bars) or with mock buffer (light gray bars), 200 ng/ml I-B₄ (dark gray bars), or an anti-CD1d mAb (stippled bars). Assays were performed in triplicate, and error bars represent the SD values of the means. Similar results were obtained in three independent experiments.

The G. simplicifolia I-B₄ isolectin has previously been shown to inhibit autoreactive responses by a murine NKT cell hybridoma and by a human Vα24-positive NKT cell line, but not by murine CD1d-restricted T cells with diversely rearranged TCRs (22). This effect was thought to be due to the binding of the lectin to terminal α-linked galactose moieties of cellular glycolipids presented by CD1d molecules, which blocks Ag recognition by the T cells (22). We therefore investigated whether I-B₄ could block the responses of the Vα24-positive and Vα24-negative CD1d-restricted T cell clones to the CD1d/721.221 transfectants. One clone, J24L.17, reproducibly showed a slight but statistically significant decrease in cytokine secretion in the presence of the I-B₄ lectin, but none of the other clones tested showed any specific inhibition by the lectin (Fig. 5⇑B). Cytokine secretion by clone J24L.17 was diminished by ∼15% in the presence of 20 ng/ml I-B₄ lectin, and titrating the concentration of I-B₄ up to 1 mg/ml resulted in no further inhibition of cytokine secretion (Fig. 5⇑B and data not shown). Hence, a fraction of the CD1d-presented self Ags recognized by clone J24L.17 may contain terminal α-linked galactose residues that are available for binding by the I-B₄ lectin, but the self Ags recognized by the five other clones tested in these experiments are not sensitive to this method of blocking.

Recognition of iGb3

The iGb3 glycolipid has recently been identified as a candidate self Ag that may select canonical murine CD1d-restricted T cells in the thymus and may also be responsible for their autoreactive responses (22). Three Vα24-negative and three canonical Vα24-positive clones were tested in parallel for responses to iGb3 presented by CD1d-transfected 721.221 cells. The Vα24-negative clones showed no responses to the iGb3 lipid despite responding strongly to the α-GalCer used as a positive control (Fig. 6⇓, right panels). One Vα24-positive clone, J3N.4, showed marked responses to iGb3, indicating that this Ag was presented by the APCs (Fig. 6⇓, top left panel). Surprisingly, two other Vα24-positive clones showed no significant responses to iGb3 (Fig. 6⇓, middle and bottom left panels). These two Vα24-positive clones had strong autoreactive responses to the CD1d-transfected APCs (compare cytokine production in response to vehicle alone with that in response to PHA or α-GalCer; Fig. 6⇓), and, therefore, it is possible that their responses to iGb3 were not detectable above the self Ag signal. Hence, it is not clear from these experiments whether only one or all three of the Vα24-positive clones tested were capable of recognizing iGb3. However, the Vα24-negative clones tested were not highly autoreactive and were functionally active, as demonstrated by their robust responses to α-GalCer. Therefore, these results suggest that the Vα24-negative TCRs are not able to recognize iGb3, and, hence, their positive selection in vivo was likely to have been mediated by a different self Ag.

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FIGURE 6.

Cytokine secretion by Vα24-positive and Vα24-negative clones in response to APCs pulsed with the indicated concentration of α-GalCer (filled circles), iGb3 (open circles), or vehicle alone (open squares). Filled squares show the response of the T cell clones to the nonspecific stimulator PHA-m. Values that were below the detection limit of the ELISA were assigned a value of 1 pg/ml for plotting on the logarithmic scale. Assays were performed in triplicate, and error bars represent the SD values of the means. Similar results were obtained in three independent experiments.

Phospholipid recognition

We have shown previously that an autoreactive Vα14-positive, murine CD1d-restricted T cell specifically responded to certain phospholipids and that cellular Ags recognized by this T cell include a form of phosphatidylethanolamine (13, 32). Therefore, we investigated the reactivity of twelve Vα24-positive and Vα24-negative T cell clones to a series of purified phospholipids presented by plate-bound recombinant CD1d molecules. One Vα24-positive clone (BM2a.5) responded specifically to purified phosphatidylethanolamine and phosphatidylinositol but not to other phospholipids (Fig. 7⇓A). The other Vα24-positive or Vα24-negative clones showed no detectable responses to any of the purified phospholipids (data not shown). In addition to showing modest but clearly detectable responses to phosphatidylinositol and phosphatidylethanolamine, clone BM2a.5 responded strongly to CD1d molecules treated with α-GalCer, suggesting that the phospholipid reactivity is an additional lipid specificity that does not replace the ability to recognize CD1d-presented α-GalCer (Fig. 7⇓A).

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FIGURE 7.

A, Cytokine secretion by clone BM2a.5 (Vα24-positive) in response to the CD1d-Fc fusion protein treated with DMSO (vehicle), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), or α-GalCer. B, Cytokine secretion by clone BM2a.5 in response to plate-bound CD1d-Fc fusion protein (filled circles) or a negative control mAb (open circles) that was pretreated with purified phosphatidylethanolamine (PE), or vehicle alone (open square). C, Cytokine secretion by clone BM2a.5 (left panel) and clone J3N.5 (right panel) in response to CD1d-Fc molecules treated with the indicated concentrations of synthetic phosphatidylethanolamine (PE) preparations containing acyl chains that have no unsaturations (di 18:0), two unsaturations (di 18:2), or three unsaturations (di 18:3). Assays were performed in triplicate, and error bars represent the SD values of the means.

Further analysis showed that cytokine secretion in response to phosphatidylethanolamine-treated CD1d was dependent on the presence of the recombinant CD1d molecules, because a plate-bound negative control IgG2a molecule similarly pretreated with phosphatidylethanolamine elicited no significant response (Fig. 7⇑B). Additionally, the response was highly specific, because synthetic phosphatidylethanolamine molecules containing two or three unsaturations in their acyl chains were recognized, but phosphatidylethanolamine containing no unsaturations was not recognized (Fig. 7⇑C). A similar specific reactivity to phosphatidylethanolamine containing two or three acyl chain unsaturations was observed previously for an autoreactive murine NKT cell (32). Moreover, none of the synthetic phosphatidylethanolamine preparations stimulated a different Vα24-positive clone, J3N.5, demonstrating that these compounds do not nonspecifically activate NKT cell clones (Fig. 7⇑C). Thus, clone BM2a.5 was able to specifically respond to two phospholipids presented by CD1d, whereas 11 other CD1d-restricted clones appeared unable to recognize these lipids. These results demonstrate directly that lipid reactivity can vary among canonical Vα24-positive NKT cells that share an ability to respond to α-GalCer and indicate that the ability to recognize CD1d-presented phospholipids may be a feature of a subset of human as well as murine CD1d-restricted T cells.

Correlations with TCR sequences

Both the Vα24-positive and Vα24-negative clones use Jα18 and, thus, the sequences of their CDR3α loops are nearly identical, but the CDR1, CDR2, and fourth hypervariable loops of the TCR α-chain differ substantially (Table II⇓). Additionally, the Vα24-positive and Vα24-negative clones all use Vβ11, so the sequences of their TCR β-chain CDR1 and CDR2 loops are the same, but each clone has a unique TCRβ CDR3 (Table I⇑). Hence, the specificity differences among the clones could be explained by Vα-encoded sequence differences, sequence differences in the TCR β-chain CDR3, or conformational differences in other critical TCR regions (e.g., the TCRα CDR3 loop) resulting from differences in these regions.

Clone BM2a.5 was the only one that used Jβ2.6 (Table I⇑). Hence, the phospholipid reactivity of this clone could be due to its use of this Jβ segment or could result from residues within its unique N region-encoded CDR3β sequence. None of the other specificity differences among the clones showed any readily discernible correlation with the CDR3β sequence, suggesting that these are more likely to be due to differences in the TCR α-chain.

The presence of a conserved amino acid motif in the CDR1α loop appeared to correlate with the ability to respond robustly to both α-GalCer and α-GlcCer. Murine Vα14 and human Vα24 share expression of valine at position 26 and proline at position 28 of the CDR1α loop, and this motif is not present in the Vα24-negative TCRs (Table II⇑). Because murine Vα14-positive and human Vα24-positive NKT cells share the ability to respond to both α-GalCer and α-GlcCer, whereas the Vα24-negative cells showed reduced responses to α-GlcCer, this observation suggests that the presence of this CDR1 motif could be important for the ability of canonical NKT TCRs to accommodate α-GSLs containing either glucose or galactose. Notably, one of the Vα24-negative TCRs (J24N.70) has a CDR1α loop that is predicted to have a similar structure to that of Vα24 (33), and, therefore, the correlation appears to be specific to the amino acids in the CDR1α loop rather than to its overall structure.