Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor β-chain repertoire and how α-galactosylceramide (α-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-αGalCer- and αGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional αβ TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.

Fundamental to the initiation of a cellular immune response is cell-to-cell communication through receptor triggering and the dynamic formation of an immunological synapse. Central to this process is the interaction between the Ag and its cognate receptor, which relays the specificity of recognition. Although much has been learned regarding the interactions between peptide Ags and their cognate TCR, comparatively little is known about the recognition of CD1-restricted glycolipid Ags by specific T cells.

Va14Ja18 natural T (iNKT) 3 cells are a unique subset of CD1d1-restricted T lymphocytes whose invariant α-chain preferentially pairs with Vb8.2 β-chain and less commonly with Vb7. Remarkably, in vivo iNKT cell activation through the TCR results in rapid (i.e., within 60–90 min) and robust IL-4 response and a spectrum of Th1 and Th2 cytokines (reviewed in Ref.1). In striking contrast, conventional T lymphocytes require up to a day to produce significant amounts of cytokines in response to Ag.

The natural Ag recognized by iNKT cells remains unknown. A variety of CD1d-positive cells activate freshly isolated thymic iNKT cells and derived hybridomas without the addition of any exogenous Ag (2, 3, 4, 5, 6), which suggests the recognition of self-Ags. Moreover, presentation of self-Ags requires CD1d trafficking through the late endosomes/lysosomes (3, 4, 7, 8, 9). The recognition of α-galactosylceramide (αGalCer) and α-glucosylceramide by iNKT cells (10, 11, 12, 13, 14) suggests a glycosphingolipid nature of the elusive Ags. Although αGalCer is a nonphysiological Ag, our recent studies indicate that it may be a very close mimic of at least one natural iNKT cell ligand (15). Consistent with this conclusion is the fact that αGalCer and/or its close analogue OCH, with a shortened long-chain sphingosine base and acyl chain, exhibit immunopharmacological effects in vivo. Thus, αGalCer acts like an adjuvant enhancing immunity to malaria and other infectious pathogens (16). Furthermore, αGalCer and/or OCH can prevent autoimmune diseases in mouse models of type I diabetes and multiple sclerosis (17, 18, 19, 20, 21). Interestingly, the ability of OCH to induce IL-4 alone and no IFN-γ appears to underlie its pharmacological action (19). Thus, delineating the biochemical parameters of Va14Ja18 TCR/Ag interactions is of paramount pharmacological significance.

Interactions of soluble Ag receptors of conventional T cells with cognate Ags are of low affinity (0.1–50 μM) and relatively fast dissociation half-life (t1/2 = 10–50 s) (22, 23, 24, 25). Va14Ja18 TCR of an iNKT cell hybridoma has been demonstrated to interact with αGalCer-loaded CD1d1 with relatively high affinity (0.2 μM) and very long half-life (t1/2 = 175 s) (26). The high-avidity interaction of Va14Ja18 TCR with CD1d1-αGalCer dimer appears to be influenced by TCR β-chain repertoire (27). Recent studies have implicated both optimal dwell time (28) and affinity (29) of TCR-Ag interaction as critical determinants of T cell sensitivity and activation. Furthermore, interactions of conventional TCR with Ag are thought to be stabilized by CD4 and CD8 coreceptors (30, 31, 32). Long dwell time of Va14Ja18 TCR/CD1d1-αGalCer interaction (26) appears counterintuitive to the optimal dwell-time requirements for T cell activation. Because iNKT cells might not use coreceptors during Ag engagement, this interaction might require intrinsically high affinity and long dwell time for activation of this T cell subset.

Ag recognition by conventional T cells entails self-nonself discrimination. Thus, T cells are tuned to extraordinarily sensitive recognition of foreign Ags (on the order of 20–100 molecules per cell) and base activation decisions on affinity and dwell time (23, 24, 28, 29). Considering that a large body of evidence indicates that iNKT cells recognize self-Ag, a paradox ensues. How is it that iNKT cells are not continually activated by very small amounts of self-Ag presented on APCs in vivo? Self-Ag recognition must be finely tuned to prevent iNKT cell activation during physiological conditions, but to rapidly respond to disturbances in cellular physiology. In other words, iNKT cells need to be very sensitive to modest changes in self-Ag concentration. In biological systems, this kind of fine-tuning is often achieved by cooperative ligand engagement. Cooperativity itself is defined as a positive or negative change in multimeric receptor affinity for ligand following primary and subsequent subunit binding events. Thus, positive cooperativity permits disproportionately sensitive ligand engagement by multimeric receptors, resulting in an almost digital off-on response (33). A form of cooperativity in conventional T cell Ag recognition is afforded by coreceptor-mediated stabilization of TCR-Ag interaction in the immunological synapse. How iNKT cells substitute for coreceptor usage and yet remain unresponsive to low levels of self-Ag remains unknown. Initial report of CD1d1-αGalCer tetramers demonstrated that an iNKT cell hybridoma engages Ag with a Hill coefficient of 4.5, which was interpreted to signify the tetravalency of CD1d1-αGalCer tetramers. Hill coefficient depends on valency but is always lower than the number of binding sites of the multimer (i.e., lower than four for any tetrameric molecule) (34). Because the Hill equation used to calculate the Hill coefficient was not provided in the first (35), how the value of 4.5 was obtained remains elusive.

Several questions regarding CD1d1-lipid/TCR interactions remain: Are there affinity and concentration thresholds for the induction of distinct iNKT cell responses? Does the TCR β-chain repertoire impact iNKT cell Ag reactivity in vivo? Does cooperativity play a role in iNKT cell receptor-Ag interactions? In studies relevant to these questions, we demonstrate that the avidity thresholds for iNKT cell receptor determine sensitivity for glycolipid Ag recognition. Despite the invariant nature of the TCR α-chain, TCR β-chain usage by iNKT cells critically impacts the specificity and the avidity for glycolipid Ags. Furthermore, when responding to a suboptimal affinity ligand, high relative avidity iNKT cells are selected. Interestingly, iNKT cell receptor appears to have structure and/or organization distinct from other αβ TCR and engages Ag cooperatively. Taken together, these features of iNKT cell receptor permit sensitive self-Ag recognition and determine their functional outcomes.

Experiments with B6-Ja180/0 (36) (a gift from M. Taniguchi (University of Chiba, Chiba, Japan)), B6.129-CD1d10/0 (37), and C57BL/6 (The Jackson Laboratory, Bar Harbor, ME) were in compliance with the regulations of the Institutional Animal Care and Use Committee of Vanderbilt University.

CTL clones and hybridomas (generously provided by A. Bendelac (Princeton University, Princeton, NJ) and K. Hayakawa (Fox Chase Cancer Center, Philadelphia, PA)) have been described (38, 39, 40, 41).

C57BL/6 thymocytes and splenocytes reacted with anti-CD161-PE were separated with anti-PE magnetic microbeads using an automated sorter (Miltenyi Biotec, Auburn, CA). Samples were typically >95% CD161+.

Glycolipids (12, 19) and peptides (38, 39) have been described. Kirin Brewery (Gumma, Japan) generously provided αGalCer. SV40 T Ag-derived epitope IV (VVYDFLKL), H28 (ILENFPRL), and H60 (LTFNYRNL) peptides were synthesized by F-moc chemistry at the Macromolecular Core Facility (Pennsylvania State University).

Preparation of CD1d1-glycolipid (15) and H2Kb-peptide tetramers (27, 28) has been described. Dimers of CD1d1 (custom order; BD PharMingen, San Diego, CA) and H2Kb (DimerX; BD PharMingen) are dimeric owing to their fusion to IgG1 H chains. To obviate the potential for artifacts induced by detection mediated via a fluorochrome-conjugated secondary Ab, the dimers were Alexa Fluor 647- or PE-conjugated via Fab specific for the Fc portion of IgG1 (anti-mouse IgG1 Alexa Fluor 647 and PE Zenon kits; Molecular Probes, Eugene, OR). Every batch of tetramer generated was tested for complete loading of αGalCer and its analogues by glycolipid titration loading and testing by reaction with the best characterized iNKT hybridoma N38-2C12.

All Abs were from BD PharMingen unless otherwise stated. Tetramer-stained Va14Ja18 iNKT hybridomas (N37-1H5a, Vb8.2Jb2.6; N38-2C12, Vb8.2Jb2.5; N38-3C3, Vb8.2Jb2.2; and DN32.D3, Vb8.2Jb2.4) were also labeled with anti-TCR Cβ-PE (H57-597), NKT cell-enriched thymocytes and splenocytes with anti-NK1.1-PE (PK136), and anti-TCR Cβ-FITC. CTL clones (SV40 epitope IV-specific 2168T as well as minor histocompatibility (mH) Ag-specific SPH60, BH60, and SPH28) reacted with tetramers were also stained with anti-CD8α-FITC. Samples were analyzed using FACSCalibur and CellQuest, version 3.0 (BD Biosciences, Franklin Lakes, NJ) as well as FlowJo 4.2 (Tree Star, San Carlos, CA).

Equilibrium (>2 h) binding experiments were performed using increasing tetramer concentrations in 100 μl of PBS containing 2% FCS (Invitrogen, Carlsbad, CA) and 0.05% NaN3 at 4°C, to prevent capping and internalization of the TCR. Kav was calculated from specific mean fluorescence intensity (MFI; difference between total MFI at a defined tetramer concentration and background MFI derived from ligand-free tetramer binding to the same cells) using nonlinear regression analysis fitted to classical Michaelis-Menten kinetics (Prism 3.02; GraphPad Software, San Diego, CA). MFI (% maximum) shown in the relevant figures is based on Vmax calculated from nonlinear regression analysis of the data for adequate graphical representation. This permits easy and reliable comparison of data generated in different experiments. Nonlinear Michaelis-Menten regression analysis was preferred, because Scatchard transformation, which uses linear regression, amplifies any variation of the data from the linear curve. That notwithstanding, the results from the Michaelis-Menten kinetics were confirmed by using classical Scatchard transformations to derive the Kav (42).

T cells were labeled with 50 μg/ml H2Kb-peptide or 10 μg/ml CD1d-glycolipid tetramers, respectively, incubated at 4°C for 3 h, and washed extensively. Cells were also stained with 10 μg/ml anti-TCR Cβ-FITC to monitor TCR levels. Following initial tetramer binding, 106 cells were chased in 3 ml of buffer with rocking at 4 or 37°C for the indicated time periods and analyzed by flow cytometry.

Mice were injected i.v. with the indicated concentrations of glycolipids diluted in PBS from a 220 μg/ml stock solution in vehicle (0.5% v/v polysorbate and 0.9% w/v NaCl). Controls were injected with corresponding dose of vehicle. After 90 min, IL-2, IL-4, IL-13, CSF-2, IFN-γ, and TNF-α in control and immune sera were measured by ELISA using Abs and methods that we have described previously (43).

Bulk C57BL/6 splenocytes were incubated for the indicated amounts of time with increasing concentrations of glycolipid Ags. Following stimulation, iNKT cell receptor level was determined by flow-cytometric analysis following staining with CD1d1-αGalCer tetramer, anti-TCRβ Ab, within electronically gated B220 and CD8-negative lymphocytes. In other experiments, Ags were first equilibrium-loaded overnight onto B6.129-Tcra0/0 splenocytes, and then mixed with C57BL/6 splenocytes magnetically depleted of MHC class II-positive cells. To directly evaluate iNKT cell division during culture, splenocytes were labeled with 2 μM CFSE (Molecular Probes) in PBS for 8 min at room temperature, followed by quenching with cold FCS and washing with ice-cold RPMI 1640 supplemented with 10% FCS before culture. Evaluation of iNKT cell proliferation was performed by multiplying the percentage of iNKT cells determined by flow cytometry with the total cell number.

Soluble mouse CD1d1 was Ni-affinity purified, as described (44), and bound to ELISA plates at a concentration of 10 μg/ml. Following binding at 4°C for 18 h and blocking of unbound sites with 2% FCS, plate-bound soluble mouse CD1d1 was loaded with 0.1 μM lipids for 12 h at 37°C. After removing excess lipids, the Ag was allowed to dissociate for the indicated times at 37°C. The wells were washed again, and ∼5 × 104 hybridoma cells were added to each well. Controls included wells bound with 2 μg/ml anti-CD3ε (positive) or with 5 μg/ml BSA (negative) loaded with 1 μg/ml αGalCer. IL-2 secreted upon activation was monitored by ELISA. Data are presented as the percentage of maximum activation.

Hill coefficient was determined from epitope titration experiments. Briefly, CD1d1 and H2Kb-H28 tetramers or dimers were loaded with increasing amounts of glycolipid ligand or peptide epitopes, respectively. Note that H2Kb tetramers were initially folded with H28-derived epitope, a peptide with low affinity for H2Kb, allowing rapid and efficient ligand exchange (Y. Yoshimura and S. Joyce, unpublished data). Glycolipid and peptide loading occurred at 37°C and room temperature, respectively, for 16–18 h.

Hill curve was derived from data transformation; fractional saturation (Ys) of the receptor was determined as the ratio of specific MFI to maximum MFI (Vmax) at a defined ligand concentration and plotted against the concentration of added ligand (glycolipid or peptide). Linear graph of logarithmic roots of the values for the x- and y-axes were used to determine the slope of the Hill curve revealing the Hill coefficient (34).

There are a number of different methods available to assess the kinetics and extent of ligand-receptor interactions. Biophysical methods using purified recombinant molecules have been extremely useful in the study of a variety of immunological receptors (45, 46, 47). That notwithstanding, methods that examine molecules on living cells are particularly powerful (24, 26, 28, 48, 49). To gain insight into the parameters that govern the binding interactions of the CD1d1 ligand to the specialized TCR of iNKT cells required for their activation, we first determined the Kav (measured affinity of tetrameric Ags for the cognate TCR) between tetrameric CD1d1-glycolipid Ag and its receptor on live cells. For comparison, the Kav of peptide Ags for the TCR expressed by recently activated CD8+ T lymphocyte (CTL) clones specific for two H2Kb-restricted mH (H60 and H28 (27)) Ags and a viral (SV-40 T Ag-derived epitope IV (39)) Ag was measured. The comparison with class I-restricted Ags was used, because iNKT cells reflect memory/activated T lymphocyte phenotype similar to CTL clones.

CD1d1-αGalCer tetramer binds Va14Ja18+ but not Va14-negative NKT hybridomas (Fig. 1,A). Similarly, H2Kb-peptide tetramers specifically bind their cognate, but not irrelevant, CTL clones (data not shown and Refs. 38 and 39). Nonspecific binding was <5% in all cases (Fig. 1 A and data not shown). From the binding isotherms, the Kav of Ag-TCR interaction was calculated (see Materials and Methods).

FIGURE 1.

Biochemical features of glycolipid Ag/Va14Ja18 TCR and peptide Ag/TCR interactions. A, Ag-free and αGalCer-loaded CD1d1 tetramer were reacted with Va14-negative or iNKT hybridomas to determine the specificity of the reagent. B–D, Saturation binding isotherms were generated by reacting the indicated concentrations of CD1d1-αGalCer tetramers with four iNKT hybridomas (N37-1H5a, Vb8.2Jb2.6; N38-2C12, Vb8.2Jb2.5; N38-3C3, Vb8.2Jb2.2; and DN32.D3, Vb8.2Jb2.4) (B) or NKT cell-enriched thymocytes and splenocytes (D). Similar isotherms were generated using the indicated concentrations of H2Kb-peptide tetramers in a reaction with specific CTL clones (SV40 epitope IV-specific 2168T as well as mH Ag-specific SPH60, BH60 and SPH28) (C). All binding reactions were performed at 4°C in the presence of sodium azide to prevent ligand-induced capping and TCR internalization. Specific MFI in B–D measured by flow cytometry are represented as a fraction of maximum binding. Kav was calculated using the Michaelis-Menten equation (see Materials and Methods). E, To determine the t1/2 of Ag-receptor binding, the indicated T cells were reacted with specific tetrameric Ag. After extensive washes, the dissociation of Ag from cells during the chase was monitored by flow cytometry. Specific MFI is represented as a fraction of that detected at the beginning of chase.

FIGURE 1.

Biochemical features of glycolipid Ag/Va14Ja18 TCR and peptide Ag/TCR interactions. A, Ag-free and αGalCer-loaded CD1d1 tetramer were reacted with Va14-negative or iNKT hybridomas to determine the specificity of the reagent. B–D, Saturation binding isotherms were generated by reacting the indicated concentrations of CD1d1-αGalCer tetramers with four iNKT hybridomas (N37-1H5a, Vb8.2Jb2.6; N38-2C12, Vb8.2Jb2.5; N38-3C3, Vb8.2Jb2.2; and DN32.D3, Vb8.2Jb2.4) (B) or NKT cell-enriched thymocytes and splenocytes (D). Similar isotherms were generated using the indicated concentrations of H2Kb-peptide tetramers in a reaction with specific CTL clones (SV40 epitope IV-specific 2168T as well as mH Ag-specific SPH60, BH60 and SPH28) (C). All binding reactions were performed at 4°C in the presence of sodium azide to prevent ligand-induced capping and TCR internalization. Specific MFI in B–D measured by flow cytometry are represented as a fraction of maximum binding. Kav was calculated using the Michaelis-Menten equation (see Materials and Methods). E, To determine the t1/2 of Ag-receptor binding, the indicated T cells were reacted with specific tetrameric Ag. After extensive washes, the dissociation of Ag from cells during the chase was monitored by flow cytometry. Specific MFI is represented as a fraction of that detected at the beginning of chase.

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Va14Ja18 TCR binds CD1d1-αGalCer with a Kav ranging from 7 to 17 nM (Fig. 1,B and Table I). TCR of conventional T cells bind H2Kb-peptide tetramers with a wide range of Kav, ranging from ∼20 to 220 nM (Fig. 1,C and Table I). Note that, in this study, the saturation binding isotherms at equilibrium were derived at 4°C. Whereas the Kav values obtained at 4°C may not be the same as those at 37°C, the relationship of the Kav between different Ag-TCR interactions remains unaltered (50). Consistent with that reported (50), we also found that the Kav determined for two iNKT hybridomas at 4°C (N37-1H5a, 10.5 nM; N38-2C12, 18.8 nM) maintained their avidity relationship at 37°C (N37-1H5a, 39.3 nM; N38-2C12, 65.2 nM).

Table I.

Kinetic parameters of Ag-TCR interactions

T CellReactivityaKd (nM)b ± SEM (n)ct1/2 (min at 37°C)b ± SEM (n)Hill Coefficient (h)b ± SEM (n)
TetramerDimer
iNKT hybridomas/CD1d1-αGalCer      
 N37-1H5a CD1d1 + self lipid 7.5 ± 0.3 (3) 19.1 ± 1.3 (5) 2.8 ± 0.4 (3)  
 N38-2C12 CD1d1 + self lipid 8.5 ± 2.0 (4) 42.1 ± 4.3 (5) 2.8 ± 0.3 (3) 1.8 ± 0.2 (2) 
 DN32.D3 CD1d1 + self lipid 16.8 ± 0.9 (3) 37.3 ± 2.6 (2) 2.6 (1)  
 N38-3C3 CD1d1 + self lipid 17.6 ± 3.4 (3) 16.4 ± 1.0 (2) 2.4 (1)  
C57BL/6 iNKT cells/CD1d1-αGalCer      
 Thymic CD1d1 + self lipid 10.7 ± 3.0 (4) 9.8 ± 0.9 (5) 2.5 ± 0.2 (3)  
 Splenic CD1d1 + self lipid 8.8 ± 0.7 (4) 11.4 ± 0.6 (3) 2.7 ± 0.2 (3)  
Conventional CD8+ T lymphocytes/H2Kb-peptide      
 2168T H2Kb + epilV 23.9 ± 2.7 (3) ND 1.1 ± 0.1 (4)  
 SPH60 H2Kb + H60 20.4 ± 2.9 (3) 9.4 ± 0.03 (2) 1.0 ± 0.1 (3) 1.0 ± 0.2 (2) 
 BH60 H2Kb + H60 33.4 ± 1.7 (3) 3.9 ± 0.01 (2) 0.9 ± 0.04 (3)  
 SPH28 H2Kb + H28 216.7 ± 9.2 (4) 2.1 ± 0.9 (2) ND  
T CellReactivityaKd (nM)b ± SEM (n)ct1/2 (min at 37°C)b ± SEM (n)Hill Coefficient (h)b ± SEM (n)
TetramerDimer
iNKT hybridomas/CD1d1-αGalCer      
 N37-1H5a CD1d1 + self lipid 7.5 ± 0.3 (3) 19.1 ± 1.3 (5) 2.8 ± 0.4 (3)  
 N38-2C12 CD1d1 + self lipid 8.5 ± 2.0 (4) 42.1 ± 4.3 (5) 2.8 ± 0.3 (3) 1.8 ± 0.2 (2) 
 DN32.D3 CD1d1 + self lipid 16.8 ± 0.9 (3) 37.3 ± 2.6 (2) 2.6 (1)  
 N38-3C3 CD1d1 + self lipid 17.6 ± 3.4 (3) 16.4 ± 1.0 (2) 2.4 (1)  
C57BL/6 iNKT cells/CD1d1-αGalCer      
 Thymic CD1d1 + self lipid 10.7 ± 3.0 (4) 9.8 ± 0.9 (5) 2.5 ± 0.2 (3)  
 Splenic CD1d1 + self lipid 8.8 ± 0.7 (4) 11.4 ± 0.6 (3) 2.7 ± 0.2 (3)  
Conventional CD8+ T lymphocytes/H2Kb-peptide      
 2168T H2Kb + epilV 23.9 ± 2.7 (3) ND 1.1 ± 0.1 (4)  
 SPH60 H2Kb + H60 20.4 ± 2.9 (3) 9.4 ± 0.03 (2) 1.0 ± 0.1 (3) 1.0 ± 0.2 (2) 
 BH60 H2Kb + H60 33.4 ± 1.7 (3) 3.9 ± 0.01 (2) 0.9 ± 0.04 (3)  
 SPH28 H2Kb + H28 216.7 ± 9.2 (4) 2.1 ± 0.9 (2) ND  
a

The reactivities of all of the T lymphocytes have been described (see Results for references).

b

Calculations of the kinetic parameters are described in Materials and Methods (also see Results for details).

c

n, Number of experimental values.

To obtain a more physiological estimate of the Kav between CD1d1-αGalCer and Va14Ja18 TCR, the above binding analysis was extended to NKT cell-enriched thymocytes and splenocytes. The Kav of CD1d1-αGalCer for Va14Ja18 TCR of live NKT cells (Fig. 1,D and Table I) is similar to that observed with iNKT hybridomas (B and Table I). Taken together, the Kav of the CD1d1-αGalCer-Va14Ja18 TCR engagement is similar to or higher than that of immunodominant peptide Ag-TCR interaction.

Functional T cell responses following Ag recognition have been correlated with the dwell time, measured as the t1/2 of ligand engagement by its receptor (28, 49, 51, 52). We found that the t1/2 of glycolipid Ag/Va14Ja18 TCR is long, lasting between 10 and 40 min (Fig. 1,E, top two panels; Table I), which is longer than the t1/2 observed for peptide Ag/TCR interactions investigated (Fig. 1,E, bottom panel; Table I). Hence, the off-rate of CD1d1-αGalCer/TCR interaction on the surface of intact NKT cells appears quantitatively distinct from that of conventional T lymphocytes.

Our data are consistent with previously published reports (28, 53) for studies of peptide-Ag-specific T cells. Due to the manner in which our experiments were performed and analyzed, the data may appear inconsistent with recent dwell-time measurements between CD1d1-αGalCer and iNKT cell receptors (26). Specifically, we did not use anti-CD1d1 or anti-MHC Abs in our experiments, and CD1d1-lipid or MHC-peptide levels detected postchase were not normalized to prechase reacted anti-TCRβ levels. Abs to MHC molecules added during the chase period prevent the dissociating monomers from reassociating with their receptor (49). In our experiments, t1/2 of H60 tetramer for cognate TCR determined in the absence (9 ± 0.01 min; see Table I) or presence of an H2Kb-reactive Ab, EH144 (2–10 min; n = 5), was similar. Likewise, t1/2 of CD1d1-αGalCer tetramers for cognate iNKT cell receptor determined in the absence (9.8 ± 0.9 min; n = 5) or presence of 100 μg/ml CD1d1-reactive Ab 1B1 (17.6 ± 7.4 min; n = 2) was similar. It should also be noted that, unlike MHC class II, which is not expressed by mouse T cells, MHC class I and CD1d1 are expressed by T lymphocytes. The use of anti-class I or anti-CD1d1 has the potential to cross-link the dissociating tetramer to the T cells, thereby skewing the data toward increased dwell time. Hence, the comparative off-rate measurements were performed in the absence of Abs.

We also found that the TCR levels on iNKT cells and CTL at time zero and at 120 min of chase were similar when they were stained with anti-TCRβ Ab postchase (data not shown). In contrast, prestaining with anti-TCRβ resulted in a significant loss of TCRβ staining during chase, most likely due to the t1/2 of anti-TCRβ Ab and cell surface TCR interaction. Thus, we chose not to normalize the remaining CD1d1-αGalCer tetramer bound postchase to prechase reacted anti-TCRβ staining, as described in published reports (28, 53). Nevertheless, our results are consistent with the conclusion that iNKT cell receptor interaction with CD1d1-presenting glycolipid Ag exhibits longer dwell time than that of CTL receptor interaction with peptidic Ags (26).

Altered glycolipid ligands derived from αGalCer elicit distinct functional responses from iNKT cells in vivo and in vitro (19). Recently, the TCR β-chain repertoire of iNKT cells was implicated in high-affinity dimeric CD1d1-αGalCer binding; the Vb8.2+ iNKT cells have higher affinity for Ag than those that express Vb7 (27). Differences in TCR β-chain repertoire and/or the affinity for altered glycolipid ligands could explain the differential Ag specificity and functional outcomes. Tetramers of CD1d1-αGalCer and its analogues OCH, 3,4D, and NH were generated concurrently under saturating conditions. Tetramers of CD1d1-αGalCer, -OCH, and -3,4D have exquisite specificity for iNKT cells (Fig. 2,A). However, CD1d1–3,4D (an analogue lacking the two hydroxyl groups at C atoms 3 and 4 of the long-chain base) and especially CD1d1-NH (C atom 2′ amine-modified αGalCer) bind poorly or not at all, respectively, to iNKT hybridomas (Fig. 2 A).

FIGURE 2.

CD1d1-OCH is recognized with lower Kav but similar dwell time compared with CD1d1-αGalCer by a Vb8.1,8.2-skewed iNKT cell repertoire. A, Equimolar quantities (30 nM) of CD1d1-glycolipid tetramers were reacted with a Va14-negative (N37-1A12) or two iNKT (N37-1H5a and N38-3C3) hybridomas, and NKT cell-enriched thymocytes. B, Expression of TCRβ, Vb8.1,8.2, or Vb7 on CD1d1-αGalCer and -OCH tetramer-positive, electronically gated HSAlowCD8low thymocytes. C, Expression of TCRβ, Vb8.1,8.2, or Vb7 on CD1d1-αGalCer and -OCH tetramer-positive, magnetically sorted NK1.1+ thymocytes. D and E, Saturation binding isotherms were generated using iNKT hybridomas and NKT cell-enriched thymocytes and splenocytes reacted with the indicated concentrations of CD1d1-αGalCer or -OCH tetramers. Similar binding isotherms were generated using Vb8.1,8.2+ and Vb7+ splenic iNKT cells. From the binding isotherms, Kav was calculated as described in Fig. 1. F, t1/2 of CD1d1-αGalCer and CD1d1-OCH binding to thymic iNKT cells was determined as described in Fig. 1. Binding reactions in A–E were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

FIGURE 2.

CD1d1-OCH is recognized with lower Kav but similar dwell time compared with CD1d1-αGalCer by a Vb8.1,8.2-skewed iNKT cell repertoire. A, Equimolar quantities (30 nM) of CD1d1-glycolipid tetramers were reacted with a Va14-negative (N37-1A12) or two iNKT (N37-1H5a and N38-3C3) hybridomas, and NKT cell-enriched thymocytes. B, Expression of TCRβ, Vb8.1,8.2, or Vb7 on CD1d1-αGalCer and -OCH tetramer-positive, electronically gated HSAlowCD8low thymocytes. C, Expression of TCRβ, Vb8.1,8.2, or Vb7 on CD1d1-αGalCer and -OCH tetramer-positive, magnetically sorted NK1.1+ thymocytes. D and E, Saturation binding isotherms were generated using iNKT hybridomas and NKT cell-enriched thymocytes and splenocytes reacted with the indicated concentrations of CD1d1-αGalCer or -OCH tetramers. Similar binding isotherms were generated using Vb8.1,8.2+ and Vb7+ splenic iNKT cells. From the binding isotherms, Kav was calculated as described in Fig. 1. F, t1/2 of CD1d1-αGalCer and CD1d1-OCH binding to thymic iNKT cells was determined as described in Fig. 1. Binding reactions in A–E were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

Close modal

To determine the TCR β-chain repertoire of iNKT cells recognizing OCH, bulk (Fig. 2,B) and sorted NK1.1+ (C) thymocytes were reacted with CD1d1-αGalCer or CD1d1-OCH tetramers and TCRβ-, Vb8.1,8.2-, or Vb7-specific Abs. Surprisingly, CD1d1-OCH tetramer detected ∼30% fewer total iNKT cells compared with CD1d1-αGalCer tetramer (Fig. 2, B and C). Interestingly, a large majority of CD1d1-OCH tetramer-positive cells expressed Vb8.1,8.2; they reflected 85–90% of CD1d1-αGalCer tetramer-reactive cells (Fig. 2, B and C). Furthermore, Vb8.1,8.2-negative and Vb7+ iNKT cells inefficiently reacted with CD1d1-OCH tetramer (25–50% of CD1d1-αGalCer tetramer-positive cells). However, TCR β-chain usage for CD1d1–3,4D-reactive cells could not be determined, because the MFI of this interaction was very low, and hence, it was difficult to resolve positive from negative staining. These results for the first time directly demonstrate that TCR β-chain repertoire of iNKT cells in vivo impacts their Ag binding. This difference could be a result of differing avidities of the altered lipids for their TCR. Therefore, CD1d1-αGalCer, -OCH, and -3,4D tetramers were used to determine their Kav for the TCR. CD1d1-OCH binds iNKT TCR with about 3- to 4-fold lower Kav compared with CD1d1-αGalCer, whereas CD1d1–3,4D had a 6-fold lower Kav (Fig. 2,D and/or Table II).

Table II.

Kinetic parameters of glycolipid Ag analogue/TCR interactions

T CellKd (nM) ± SEM (n)at1/2 (min at 37°C)b ± SEM (n)Hill Coefficient (h) ± SEM (n)
αGalCercOCH3,4DαGalCercOCHαGalCercOCH
iNKT hybridomas        
N37-1H5a 7.5 ± 0.3 (3) 29.9 ± 0.3 (2) ND 10.7 ± 3.0 (4) ND 2.8 ± 0.4 (3) 2.2 ± 0.4 (2) 
N38-2C12 8.5 ± 2.0 (4) 22.1 ± 1.7 (2) ND 42.1 ± 4.3 (5) ND 2.8 ± 0.3 (3) 2.3 ± 0.4 (2) 
C57BL/6 iNKT cells        
Thymic        
TCRβ+ 10.7 ± 3.0 (4) 31.2 ± 0.4 (2) 59.5 ± 3.1 (2) 9.8 ± 0.9 (5) 13.0 ± 1.9 (2) 2.5 ± 0.2 (3) ND 
Vb8.1,8.2+ 10.5 ± 0.1 (2) 35.7 ± 1.9 (2)      
Vb7+ 16.0 ± 0.8 (2) 46.6 ± 1.8 (2)      
Splenic 8.8 ± 0.7 (4) 27.4 ± 10.9 (2) ND ND ND 2.7 ± 0.2 (3) ND 
Splenic C57BL/6 iNKT cells expanded with glycolipid Ag stimulation (96 h)        
αGalCer 9.1 ± 0.4 (2) 44.2 ± 2.9 (2)      
OCH 9.1 ± 0.6 (2) 39.7 ± 7.2 (2)      
3,4D 6.4 ± 0.5 (2) 25.1 ± 1.9 (2)      
T CellKd (nM) ± SEM (n)at1/2 (min at 37°C)b ± SEM (n)Hill Coefficient (h) ± SEM (n)
αGalCercOCH3,4DαGalCercOCHαGalCercOCH
iNKT hybridomas        
N37-1H5a 7.5 ± 0.3 (3) 29.9 ± 0.3 (2) ND 10.7 ± 3.0 (4) ND 2.8 ± 0.4 (3) 2.2 ± 0.4 (2) 
N38-2C12 8.5 ± 2.0 (4) 22.1 ± 1.7 (2) ND 42.1 ± 4.3 (5) ND 2.8 ± 0.3 (3) 2.3 ± 0.4 (2) 
C57BL/6 iNKT cells        
Thymic        
TCRβ+ 10.7 ± 3.0 (4) 31.2 ± 0.4 (2) 59.5 ± 3.1 (2) 9.8 ± 0.9 (5) 13.0 ± 1.9 (2) 2.5 ± 0.2 (3) ND 
Vb8.1,8.2+ 10.5 ± 0.1 (2) 35.7 ± 1.9 (2)      
Vb7+ 16.0 ± 0.8 (2) 46.6 ± 1.8 (2)      
Splenic 8.8 ± 0.7 (4) 27.4 ± 10.9 (2) ND ND ND 2.7 ± 0.2 (3) ND 
Splenic C57BL/6 iNKT cells expanded with glycolipid Ag stimulation (96 h)        
αGalCer 9.1 ± 0.4 (2) 44.2 ± 2.9 (2)      
OCH 9.1 ± 0.6 (2) 39.7 ± 7.2 (2)      
3,4D 6.4 ± 0.5 (2) 25.1 ± 1.9 (2)      
a

n, Number of experimental values.

b

Calculations of the kinetic parameters are described in Materials and Methods (also see Results for details).

c

αGalCer data is the same as presented in Table 1.

Considering that the TCR β-chain repertoire of cells recognizing OCH was Vb8.1,8.2 skewed, we hypothesized that TCR β-chain of the iNKT cell receptor impacts Kav for Ag. We found that Vb7+ iNKT cells have 50% lower Kav for both CD1d1-αGalCer and -OCH compared with Vb8.1,8.2+ iNKT cells (Fig. 2,E and Table II). Note that Kav determination was performed with Vb7+ cells that detectably bound CD1d1-OCH tetramer, which represented only ∼50% of total CD1d1-αGalCer tetramer-reactive Vb7+ iNKT cells. Therefore, the results potentially represent a higher Kav than that of the entire Vb7+ iNKT population. Because the dwell time of TCR and Ag interaction correlates with the capacity for T cell activation, the t1/2 of CD1d1-αGalCer, and CD1d1-OCH from iNKT cell receptor was determined as described above. The results indicate that both glycolipid Ags have similar dwell times for their cognate receptors (Fig. 2,F and Table II). Taken together, the data suggest that the TCR β-chain repertoire and the Kav of Ag-receptor interaction, but not the dwell time, might govern distinct functional outcomes from iNKT cells.

A number of in vitro studies have indicated that iNKT cells recognize CD1d1-αGalCer with nanomolar sensitivity (10, 11, 12, 14, 35). Ags with different binding affinity for their TCR activate T cells with distinct activation thresholds (54, 55, 56, 57, 58). To determine the sensitivity of effector responses by iNKT cells in vivo, C57BL/6 mice were injected i.v. with αGalCer and OCH, and serum cytokine response was measured after 90 min. CD1d1-restricted NKT cell (B6.129-CD1d10/0)- and iNKT cell (B6-Ja180/0)-deficient mice do not respond to these glycolipids (Fig. 3 A), nor do C57BL/6 mice injected with the vehicle used to dissolve the glycolipid Ags (data not shown).

FIGURE 3.

Kinetics of CD1d1 loading with αGalCer and OCH explain similar early iNKT cell response in vivo to the two glycolipids. A, C57BL/6 mice or control B6.129-CD1d10/0 and B6-Ja180/0 mice were injected i.v. with the indicated concentrations of αGalCer, OCH, or vehicle. After 90 min, serum cytokines were monitored. Background cytokine level (<3%) elicited by vehicle-treated mice was subtracted from the Ag-treated response. The data represent cytokine responses (±SE) elicited by four individual mice in two identical experiments. B, Va14Ja18 TCR down-regulation was monitored at the indicated time points following addition of αGalCer or OCH to C57BL/6 splenocytes. iNKT cell receptor level was determined by flow-cytometric analysis following reaction with CD1d1-αGalCer tetramer and anti-TCRβ Ab, within electronically gated B220 and CD8-negative lymphocytes. C, Va14Ja18 TCR down-regulation was monitored following reaction of C57BL/6 splenocytes magnetically depleted of MHC class II-positive cells with B6.129-Tcra0/0 splenocytes equilibrium loaded with Ag overnight. D, Cytokines elicited by C57BL/6 splenocytes were monitored 48 h following addition of indicated quantities of αGalCer or OCH in vitro by sandwich ELISA. E, The dissociation of αGalCer and OCH from plate-bound soluble CD1d1 was monitored after removing excess glycolipids, and chasing the Ag for 4, 12, 24, and 36 h at 37°C, using an iNKT cell hybridoma, N38-2C12, as a probe. Activation-induced IL-2 was determined and plotted as percentage of maximum, a value obtained at start of chase.

FIGURE 3.

Kinetics of CD1d1 loading with αGalCer and OCH explain similar early iNKT cell response in vivo to the two glycolipids. A, C57BL/6 mice or control B6.129-CD1d10/0 and B6-Ja180/0 mice were injected i.v. with the indicated concentrations of αGalCer, OCH, or vehicle. After 90 min, serum cytokines were monitored. Background cytokine level (<3%) elicited by vehicle-treated mice was subtracted from the Ag-treated response. The data represent cytokine responses (±SE) elicited by four individual mice in two identical experiments. B, Va14Ja18 TCR down-regulation was monitored at the indicated time points following addition of αGalCer or OCH to C57BL/6 splenocytes. iNKT cell receptor level was determined by flow-cytometric analysis following reaction with CD1d1-αGalCer tetramer and anti-TCRβ Ab, within electronically gated B220 and CD8-negative lymphocytes. C, Va14Ja18 TCR down-regulation was monitored following reaction of C57BL/6 splenocytes magnetically depleted of MHC class II-positive cells with B6.129-Tcra0/0 splenocytes equilibrium loaded with Ag overnight. D, Cytokines elicited by C57BL/6 splenocytes were monitored 48 h following addition of indicated quantities of αGalCer or OCH in vitro by sandwich ELISA. E, The dissociation of αGalCer and OCH from plate-bound soluble CD1d1 was monitored after removing excess glycolipids, and chasing the Ag for 4, 12, 24, and 36 h at 37°C, using an iNKT cell hybridoma, N38-2C12, as a probe. Activation-induced IL-2 was determined and plotted as percentage of maximum, a value obtained at start of chase.

Close modal

Mice administered 0.5 μg of OCH elicited substantial amounts of IL-2 and IL-4; TNF-α, IL-13, and CSF-2 (GM-CSF) were also detectable within 90 min (Fig. 3,A). Administration of 1.0 μg of αGalCer or OCH elicited a robust cytokine response including TNF-α, IL-13, and CSF-2 (Fig. 3 A). Note that the observed IFN-γ response is at the very low end of maximum at this early time point. Furthermore, the previously reported differential IFN-γ response to OCH and αGalCer are strikingly apparent only at or after 6 h (19), because that is the time point at which IFN-γ peaks (59). Thus, at early time, αGalCer and OCH are recognized with similar sensitivity in vivo.

Activation of T cells is an effect of Ag-TCR engagement and consequent intracellular signaling. T cell activation correlates with the extent of receptor down-regulation due to signal-dependent altered intracellular TCR trafficking (60, 61, 62). Surprisingly, iNKT cells respond to αGalCer and OCH with similar early sensitivity (Fig. 3,A), despite different equilibrium binding properties of TCR and specific Ag (Fig. 2, D–F, and Table II). To determine the cellular basis of αGalCer and OCH sensitivity, the kinetics and extent of TCR down-regulation following addition of increasing concentrations of αGalCer and OCH to splenocytes in vitro were evaluated. Both αGalCer and OCH down-regulated similar levels of surface TCR within 4–12 h of Ag stimulation (Fig. 3 B, top three panels). However, αGalCer was ∼10-fold more potent at inducing surface TCR down-regulation after 24 h of stimulation. Thus, the kinetics of TCR down-regulation reflected the early induced iNKT cell response in vivo.

Two plausible mechanisms can explain the difference observed in early and late iNKT cell responses to αGalCer and OCH. αGalCer, because of its higher Kav for Va14Ja18 TCR compared with OCH, is a more potent iNKT cell ligand resulting in more sustained TCR down-regulation and activation. Alternatively, OCH, because of its shortened sphingosine and acyl chains, binds CD1d1 faster than αGalCer, and hence compensates for its low Kav and elicits an early iNKT cell response. To distinguish between the two possibilities, B6.129-Tcra0/0 splenocytes, which lack T and iNKT cells, were incubated with increasing quantities of αGalCer and OCH for 24 h. They were then used to stimulate C57BL/6 splenocytes depleted of MHC class II-positive cells, after which iNKT cell receptor down-regulation was evaluated. OCH was 10- to 20-fold less efficient in TCR down-regulation compared with αGalCer at all time points tested (Fig. 3 C). This result is consistent with the hypothesis that αGalCer and OCH have different kinetics of CD1d1 loading, and that the similar early iNKT cell response to the two Ags in vivo reflects rapid on-rate of OCH compared with αGalCer.

To determine the concentration threshold required for the elicitation of distinct cytokines from iNKT cells by αGalCer and its analogue OCH, C57BL/6 splenocytes were stimulated with increasing concentrations of these glycolipids. The results revealed that IL-2 and IFN-γ response after 48 h (Fig. 3,D) required at least 50% iNKT cell receptor down-regulation measured at 24 h (B, bottom panel) and medium Ag concentration threshold of αGalCer and OCH (D). In contrast, secretion of CSF-2 and IL-4 was more sensitive to low concentrations of glycolipid Ags and, hence, responded to low levels of TCR down-modulation (Fig. 3, B and D). In support of our previous report (19), OCH preferentially induced an IL-4 response, whereas 50-fold higher concentration of OCH was required to produce an IFN-γ response similar to that induced by αGalCer (Fig. 3 D). Thus, the secretion of cytokines by iNKT cells follows a hierarchical Ag response pattern, with higher avidity and higher concentrations required for secretion of IFN-γ and IL-2 compared with both low avidity and low concentration for CSF-2 and IL-4.

To fully understand the properties of CD1d1-OCH interaction, we used a cell-free Ag presentation assay to determine its dissociation kinetics. Plate-bound soluble CD1d1 was loaded with equimolar quantities of αGalCer or OCH. After removing unbound lipid, the complexes were allowed to dissociate for varying time periods at 37°C. The t1/2 of Ag-CD1d1 complex was monitored by its ability to activate iNKT cell hybridomas. OCH interaction with CD1d1 was more labile, because it dissociated faster than αGalCer from CD1 (Fig. 3 E). Thus, similar early sensitivity of iNKT cells to αGalCer and OCH in vivo reflects the differences in the kinetics of their interaction with CD1d1 and also the differences in their equilibrium parameters of TCR engagement.

The altered lipid ligand, 3,4D, engages the iNKT cell receptor, albeit with low Kav compared with αGalCer and OCH (Fig. 2,B and Table II), and elicits a weak cytokine response in vivo (19). To elucidate the biochemical basis of this weak response, the proliferative capacity of iNKT cells to Ag engagement was determined in vitro by CFSE dye dilution assay. After stimulation of splenocytes with Ags for 96 h, iNKT cells were costained with CD1d1-αGalCer tetramer and TCRβ-specific Ab. At high concentration (575 nM), αGalCer, OCH, and 3,4D induced extensive iNKT cell proliferation (Fig. 4,A). In contrast, at a lower concentration (2.9 nM) of these same Ags, αGalCer induced a strong proliferative response; OCH induced a partial proliferative response, whereas 3,4D and NH elicited a very weak or no response, respectively (Fig. 4,A). Furthermore, quantitation of the proliferative response revealed that αGalCer induced maximum proliferation at 5.75 nM, and OCH at 57.5 nM, whereas maximum expansion was not reached even with 575 nM of 3,4D (Fig. 4,B). We also noted that stimulation with supraoptimal Ag concentrations does not result in increased proliferation, but actually reduces total iNKT cell expansion (Fig. 4 B).

FIGURE 4.

Activation of iNKT cells by suboptimal avidity Ag 3,4D in vitro causes selective expansion of high-avidity clones. A, C57BL/6 splenocytes were stimulated with the indicated concentrations of glycolipid Ags for 96 h. iNKT cell number and cell division history were determined using tetramers and CFSE (see Materials and Methods). B, Total iNKT cell number was determined following 96 h of Ag-stimulated culture. C, TCR β-chain repertoire of CD1d1-αGalCer- and -OCH-reactive cells following 96 h of in vitro iNKT cell stimulation with indicated concentrations of glycolipid Ags. D, C57BL/6 splenocytes were stimulated with the indicated glycolipid. The resulting iNKT cell population was reacted with the indicated concentration of CD1d1-αGalCer or CD1d1-OCH tetramers. From the binding isotherms, Kav was determined as described in Fig. 1. Binding reactions were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

FIGURE 4.

Activation of iNKT cells by suboptimal avidity Ag 3,4D in vitro causes selective expansion of high-avidity clones. A, C57BL/6 splenocytes were stimulated with the indicated concentrations of glycolipid Ags for 96 h. iNKT cell number and cell division history were determined using tetramers and CFSE (see Materials and Methods). B, Total iNKT cell number was determined following 96 h of Ag-stimulated culture. C, TCR β-chain repertoire of CD1d1-αGalCer- and -OCH-reactive cells following 96 h of in vitro iNKT cell stimulation with indicated concentrations of glycolipid Ags. D, C57BL/6 splenocytes were stimulated with the indicated glycolipid. The resulting iNKT cell population was reacted with the indicated concentration of CD1d1-αGalCer or CD1d1-OCH tetramers. From the binding isotherms, Kav was determined as described in Fig. 1. Binding reactions were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

Close modal

Together, the data reveal that, despite differences in Kav and the TCR β-chain repertoire, the altered lipid ligands induce proliferative response (Figs. 2, B–F, and 4, A and B). Therefore, the β-chain repertoire and the Kav of Ag-expanded iNKT cells were determined. TCR β-chain repertoire of iNKT cells following αGalCer, OCH, and 3,4D stimulation remains largely unaltered at Ag concentrations inducing a maximum proliferative response, although a slight decrease in the percentage of Vb8-negative iNKT cells was noted (∼35% of expanded iNKT (Fig. 4,C) compared with ∼45% for naive iNKT cells (Fig. 2,B)). Additionally, very little if any difference was observed in the Vb repertoire of iNKT cells expanded with different suboptimal doses of αGalCer and OCH (data not shown). Interestingly, iNKT cell activation by 3,4D, but not αGalCer or OCH, resulted in the expansion of iNKT cells responding to Ag with higher Kav for αGalCer and OCH (Fig. 4,D and Table II). Thus, high-avidity iNKT cells preferentially expand to suboptimal TCR engagement.

Self-Ag recognition must be finely tuned to prevent iNKT cell activation during physiological conditions, but respond rapidly to disturbances in cellular physiology. In other words, iNKT cells need to be very sensitive to modest changes in Ag concentration. In biological systems, this kind of fine-tuning is often achieved by using cooperative ligand-receptor interactions (33, 63). To determine whether cooperativity participates in sensitive glycolipid Ag recognition, this mode of interaction was determined by calculating the Hill coefficient (see Materials and Methods). The Hill coefficient of the interaction between the tetrameric Ag and the iNKT cell receptor was >2 (Fig. 5,A and Table I). In stark contrast, all MHC class I-restricted TCR had a calculated Hill coefficient of ∼1 (Fig. 5,B and Table I), indicating a lack of cooperativity. Peptide binding to each H2Kb monomer of the tetrameric molecule is an independent event. Saturation binding of the tetramer to the TCR with increasing concentration of added peptide indicates occupancy of all four sites (Fig. 5 B). Furthermore, an analysis of the stoichiometry of class I H chain, β2-microglobulin, and peptide following ligand exchange by Edman sequence determination (64) revealed a 1:1:1 ratio of the three components (data not shown). Thus, a Hill coefficient of 1 is not due to incomplete loading of the class I tetramer.

FIGURE 5.

Cooperative engagement of multimeric CD1d1 by the Va14Ja18 TCR. A constant concentration of multimer (CD1d1 tetramer (A and C); H2Kb tetramer (B); CD1d1 and H2Kb dimers and tetramers (D)) was loaded with the indicated concentrations of Ag (αGalCer or OCH (A and C); peptide (B)) and reacted with freshly isolated NKT cell-enriched thymocytes (A), NKT hybridomas (A and C), or CTL clones (B). Binding was monitored as described in Fig. 1. From the binding curve, Hill plots and Hill coefficients (h), represented beneath the Ag-binding curves, were derived (see Materials and Methods). Tetramers of CD1d1 and H2Kb were generated by fluorochrome-conjugated streptavidin-mediated tetramerization of biotinylated monomers. Binding of dimers of CD1d1 and H2Kb, owing to their fusion to IgG1 H chains, were detected with Alexa Flour 647- or PE-conjugated Fab specific for the Fc portion. The high regression coefficient (>0.90) for all Hill plots indicates that the calculated h values are significant. All binding reactions were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

FIGURE 5.

Cooperative engagement of multimeric CD1d1 by the Va14Ja18 TCR. A constant concentration of multimer (CD1d1 tetramer (A and C); H2Kb tetramer (B); CD1d1 and H2Kb dimers and tetramers (D)) was loaded with the indicated concentrations of Ag (αGalCer or OCH (A and C); peptide (B)) and reacted with freshly isolated NKT cell-enriched thymocytes (A), NKT hybridomas (A and C), or CTL clones (B). Binding was monitored as described in Fig. 1. From the binding curve, Hill plots and Hill coefficients (h), represented beneath the Ag-binding curves, were derived (see Materials and Methods). Tetramers of CD1d1 and H2Kb were generated by fluorochrome-conjugated streptavidin-mediated tetramerization of biotinylated monomers. Binding of dimers of CD1d1 and H2Kb, owing to their fusion to IgG1 H chains, were detected with Alexa Flour 647- or PE-conjugated Fab specific for the Fc portion. The high regression coefficient (>0.90) for all Hill plots indicates that the calculated h values are significant. All binding reactions were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

Close modal

OCH is a structurally different Ag, particularly in the hydrophobic component thought to interact with CD1d1. Also, OCH interaction with CD1d1 has distinct kinetic parameters compared with αGalCer (Figs. 2,F and 3,E). Thus, to exclude the possibility that the biochemical or structural properties of αGalCer loading onto CD1d1 account for the observed cooperative response, Hill coefficient was measured for the binding of CD1d1-OCH to the Va14Ja18 TCR. As expected, we found that the Hill coefficient for CD1d1-OCH and CD1d1-αGalCer for the same Va14Ja18 TCR are very similar (Fig. 5,C and Table II). Thus, Hill coefficient measurement does not reflect the loading properties of glycolipid Ags, but rather, it is the property of the Ag receptors with which it interacts.

To independently demonstrate cooperative Ag engagement by iNKT cells with multimeric Ags other than soluble, biotinylated monomers of CD1d1 and H2Kb prepared in-house, we determined the Hill coefficients with commercially obtained dimeric IgG1-CD1d1 and IgG1-H2Kb fusion molecules loaded with αGalCer and H60 peptide, respectively, for iNKT cells and H60-specific SPH60 CTL clone. iNKT cells demonstrated cooperative engagement of both dimeric and tetrameric Ag by the Va14Ja18 TCR (Fig. 5,D and Table I). As expected, neither dimeric nor tetrameric H2Kb cooperatively engaged their cognate TCR (Fig. 5,D and Table I). Thus, we conclude that, in contrast to conventional T lymphocytes, glycolipid Ag recognition by iNKT cells involves cooperativity.

A plausible model for cooperative tetrameric Ag engagement by Va14Ja18 TCR is receptor partitioning and oligomerization within lipid rafts (50). To test this model, Hill coefficients for Ag-receptor interactions were determined for two representative iNKT hybridomas (N38-2C12 and N37-1H5a), NKT cell-enriched thymocytes, and two CTL clones (SPH60 and BH60), following disruption of their lipid rafts. Lipid rafts were disrupted by cholesterol depletion with methyl-β-cyclodextrin (65) or alternatively by filipin-mediated intercalation of this membrane microdomain (66). Disruption of lipid rafts did not alter the Hill coefficient for any of the interactions tested (data not shown), suggesting that these membrane microdomains are not critical for cooperative Ag engagement by iNKT cell receptor.

To further examine the structural properties of iNKT cell Ag receptor, we used fluorescence resonance energy transfer (FRET) measurements between CD1d1 and H2Kb multimers and Abs specific for components of the TCR complex. In the course of our studies, we observed that costaining of iNKT cells ex vivo by allophycocyanin- or PE-conjugated CD1d1 tetramers and PE- or allophycocyanin-TCRβ (clone H57-597) or anti-CD3ε (clone 145-2C11) Abs resulted in large and repeatable increase in FL3 channel fluorescence in a properly compensated flow-cytometric experiment (Fig. 6, A (iNKT cell hybridoma N38-2C12) and B (thymic iNKT cells)). Such large FRET shift was not observed with high-intensity staining Abs specific for cell surface molecules not within the TCR complex (e.g., anti-CD44, clone IM7; Fig. 6). As PE and allophycocyanin have overlapping fluorescence emission and absorption spectra, respectively, it was likely that this result was a consequence of nonradiative FRET. This hypothesis was tested by running samples on the flow cytometer with the red diode laser (emission, 635 nm) and its FL4 filter switched off. Indeed, we still observed FL3 fluorescence only when costaining with CD1d1 multimers and TCR complex-specific Abs. The large FRET observed upon tetramer-anti-TCRβ/anti-CD3ε binding to iNKT cells presented an opportunity to test the hypothesis that the structural orientation and/or organization of Va14Ja18 TCR are distinct from αβ TCR of conventional CTL. When H60-specific CTL were costained in a manner identical with that of iNKT cells, and analysis was restricted to equivalent MFI of anti-TCRβ or anti-CD3ε and H2Kb multimer, very little FRET was detected (Fig. 6,A). Similarly, we observed FRET using PE-conjugated CD1d1 tetramers and allophycocyanin-conjugated anti-TCRβ or anti-CD3ε Abs (data not shown). FRET between CD1d1-αGalCer tetramers and TCRβ on iNKT cells directly correlated with the staining intensity, even at relatively low concentrations (∼2.5 nM; Fig. 6,C). In contrast, no FRET between H2Kb tetramers and TCRβ or CD3ε was observed, even with saturating concentrations of H2Kb tetramers (∼250 nM; Fig. 6 C). FRET is exquisitely sensitive to small changes in donor and acceptor fluorochrome distances (FRET, ∼r6). Thus, these results strongly suggest that iNKT cell receptor has a distinct structure and/or organization, resulting in shorter distance between donor and acceptor fluorochromes used.

FIGURE 6.

iNKT cell receptor has distinct structure and/or organization. CTL clone SPH60 (A), iNKT hybridoma N38-2C12 (A), and thymic iNKT cells (B) were reacted with allophycocyanin-conjugated H2Kb-H60 and CD1d1-αGalCer tetramers, respectively. They were also reacted with PE-conjugated Abs against TCRβ (H57-597; specific for TCRβ chain FG loop), CD3ε, or CD44. FRET was measured as the fluorescence in the FL3 channel, with the red diode laser off. CD1d1- and H2Kb-tetramer concentrations were adjusted to obtain equal MFI of tetramer staining. Considering that the 488-nm emission of the argon-ion laser cannot excite allophycocyanin, fluorescence detected in the FL3 channel is due to FRET-mediated allophycocyanin excitation. FRET-induced fluorescence intensity is indicated as a function of CD1d1-αGalCer tetramer concentration (C). All binding reactions were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

FIGURE 6.

iNKT cell receptor has distinct structure and/or organization. CTL clone SPH60 (A), iNKT hybridoma N38-2C12 (A), and thymic iNKT cells (B) were reacted with allophycocyanin-conjugated H2Kb-H60 and CD1d1-αGalCer tetramers, respectively. They were also reacted with PE-conjugated Abs against TCRβ (H57-597; specific for TCRβ chain FG loop), CD3ε, or CD44. FRET was measured as the fluorescence in the FL3 channel, with the red diode laser off. CD1d1- and H2Kb-tetramer concentrations were adjusted to obtain equal MFI of tetramer staining. Considering that the 488-nm emission of the argon-ion laser cannot excite allophycocyanin, fluorescence detected in the FL3 channel is due to FRET-mediated allophycocyanin excitation. FRET-induced fluorescence intensity is indicated as a function of CD1d1-αGalCer tetramer concentration (C). All binding reactions were performed at 4°C in the presence of sodium azide to prevent capping and internalization.

Close modal

In summary, our findings demonstrate that iNKT cell receptors recognize glycolipid Ags with avidities similar to, if not higher than, those of immunodominant, high-affinity αβ TCR of conventional T cells. In contrast to CTL, which recognize Ag over a large avidity range (20–220 nM), iNKT cells efficiently recognize Ag within a narrow window of avidity (10–40 nM). Interestingly, although the TCR-Ag dwell time for αGalCer and OCH are very similar, TCR down-regulation as well as the proliferative and cytokine response of iNKT cells to these Ags directly correlated with avidity for Ag. Strikingly, both αGalCer- and OCH-bound CD1d1 tetramers and dimers display cooperative engagement of the iNKT cell receptor, a property that CTL clones tested in this study lack. Additional data revealed FRET between specific combinations of fluorochromes conjugated to CD1d1 tetramers or dimers (data not shown) and TCR β-chain or CD3ε-specific Abs. These findings suggest that the iNKT cell receptor structure and/or organization may be distinct from conventional αβ TCR.

Conventional T cells recognize peptide Ags with a wide range of avidities and dwell times (23, 24, 25, 28, 29). In contrast, strong recognition of αGalCer and OCH, poor recognition of 3,4D, and no recognition of NH (19), by the Va14Ja18 TCR with distinct Kav points toward a narrow kinetic window for iNKT cell activation. We demonstrate that both optimal Ag concentration and relative avidity are essential to elicit a strong proliferative response by iNKT cells. Interestingly, as observed with conventional T cell effector functions (56), iNKT cells exhibit hierarchical functional consequences to Ag quality and concentration. In support of our previous study (19), we also find a dissociation from a clear avidity-concentration dependence in IL-4 secretion following OCH compared with αGalCer stimulation. Both low Ag concentration and low Kav are sufficient for selective IL-4 secretion and iNKT cell proliferation. In contrast, higher Kav and Ag concentration are required for IFN-γ response. Consistent with this conclusion is the finding that dendritic cells presenting a high concentration of the high Kav Ag αGalCer induce sustained IFN-γ response from iNKT cells (67). In this regard, iNKT cell response closely follows the principle of Ag concentration threshold set for IFN-γ and IL-4 responses elicited by conventional T cells (68).

Due to their potent immunoregulatory properties, therapeutic modulation of iNKT cell number and functional responses has been proposed for prevention of autoimmunity as well as for the enhancement of immune responses to tumors and vaccines. In the nonobese diabetic mouse model of autoimmune type I diabetes, iNKT number and function are low (43, 69, 70). Increasing the iNKT cell number (71, 72, 73) or the αGalCer treatment-induced Th2 bias (17, 18, 74) effectively reduces the incidence of type I diabetes in nonobese diabetic mice. Our data demonstrate that distinct glycolipid administration regimens may be required to induce tolerizing activity compared with IFN-γ-dependent antitumor and adjuvant properties of iNKT cells.

The natural self-Ag recognized by iNKT cells and its structural relationship to αGalCer remain unknown. However, we recently discovered that a cell line deficient in β-glucosylceramide (βGlcCer) is defective in the presentation of a self-Ag to iNKT cell hybridomas (15). Together with the evidence that the defect was not due to altered folding, intracellular traffic of CD1d1, or recognition of βGlcCer itself, these results suggest that βGlcCer is either a precursor or an essential factor in the synthesis and/or loading of a natural Ag. Thus, it is possible that the elusive self-Ag may be αGlcCer or a similar compound. Further support for the hypothesis that a self-Ag similar to αGalCer is recognized is the finding that transgenic overexpression of CD1d1 results in preferential deletion of Vb8.1,8.2+ iNKT cells (75). This result is fully consistent with our finding that Vb8.1,8.2+ iNKT cells have a higher Kav for αGalCer and OCH than Vb8.1,8.2-negative iNKT cells. Furthermore, the high Kav binding of CD1d1-αGalCer with Vb8.1,8.2+ Va14Ja18 TCR is consistent with the high Kav binding of dimeric Ag to similar TCR (27). Importantly, for the first time, we demonstrate that the repertoire and Ag Kav of iNKT cell receptors are regulated during proliferation and result in selection of high-avidity iNKT cells under conditions of suboptimal stimulation. These data, taken together, suggest that the narrow kinetic window of recognition of αGalCer and its analogues is reflective of the parameters of natural self-Ag recognition.

The 2C transgenic TCR exhibits differing peptide Ag binding modes on naive and effector cells, suggesting cooperativity (50). The existence of two TCR αβ molecules within a single CD3 complex was evoked to explain this result (50). However, recent data suggest that the stoichiometry of TCR αβ assembly with CD3 complex is 1:1 (76). Whether this stoichiometry changes during CTL activation remains to be established. Using tetramers of CD1d1 and H2Kb, we demonstrate cooperative Ag engagement of glycolipid Ags by iNKT cell receptors but not that of peptidic Ags by conventional αβ TCR. The binding mode of 2C transgenic TCR was investigated using an IgG1-H2Kb dimer, and evidence for TCR αβ dimerization was obtained by data deconvolution. To confirm the cooperative engagement of glycolipid Ag, we also used IgG1-CD1d1 and IgG1-H2Kb dimer similar to that used to investigate the 2C TCR (50). The results supported cooperative Ag engagement by iNKT cell, but not CTL receptors. Thus, the relationship of our findings with those previously reported is unclear.

Because H2Kb and CD1d1 tetramers were built upon the same batches of streptavidin-PE/allophycocyanin, cooperativity in one and not the other precludes conformational change in streptavidin or the fluorochrome. Furthermore, because of the wide separation between monomeric subunits of tetrameric CD1d1, it is extremely unlikely that a conformational change within CD1d1 itself is responsible for the observed Hill coefficient. This is further emphasized by the fact that CD1d1 dimers made in a manner distinct from tetramers also show cooperative binding. Cooperativity is independent of the parameters of glycolipid binding to CD1d1, because OCH, which interacts with CD1d1 with differing properties than αGalCer, had essentially the same Hill coefficient as αGalCer. Thus, the change in Hill coefficient does not reflect a change in the structure of CD1d1 tetramer, but rather a different organization and/or orientation of the TCR engaging such Ags.

How iNKT cells respond to self-Ag and yet remain quiescent in physiological situations remains unclear. In this study, we demonstrate that iNKT cell receptors exhibit cooperative engagement of glycolipid Ag. Cooperativity in biological systems is a common mechanism for achieving sensitivity to relatively modest changes in the strength of the signal (33, 63). In other words, a relatively small change in ligand concentration will result in full binding/activation of an enzyme/receptor. It is possible that iNKT cells use cooperativity to induce sensitive response to a small change in the concentration of self-Ag. In support of this hypothesis, self-Ag recognition of ex vivo-isolated iNKT cells is dependent on high levels of CD1d1 expression by target cells (2), and conversely, iNKT cell hybridomas recognizing physiologic levels of CD1d1 on target thymocytes or dendritic cells have high levels of Va14Ja18 TCR expression (6). Thus, the finding of cooperativity in iNKT cell Ag engagement, but not among CTL recognizing peptidic Ags may be one mechanism by which iNKT cells recognize self-Ag(s).

Our data indicate that the structure and/or organization of the iNKT cell receptor may be distinct from αβ TCR of CTL. FG loop within the Cb domain is a large, evolutionarily conserved structure, which forms a wall at the region where Cb and Vb domains of the TCR β-chain join to form a cavity (77). Ab mapping studies revealed that the FG loop is in close proximity to one of the CD3ε subunits (78). Transgenic mice expressing the TCR β-chain mutant lacking the FG loop have no gross deficiencies in the development and function of conventional CD4 and CD8 T cells (79), implying that αβ TCR pairing and surface expression are not grossly impaired. However, a careful analysis in a single specificity TCR transgenic system revealed that thymocytes lacking the FG loop had impaired negative selection (80), but TCR αβ pairing and expression were unhindered. In contrast, however, Va14Ja18 TCR α-chain was found not to pair at all with a Vb8.2-FG loop mutant, and hence, the mutant mice were impaired in iNKT cell development (81). Interestingly, the anti-TCRβ Ab H57-597, which exhibits strong FRET in conjunction with the CD1d1-αGalCer tetramer specifically binds the FG loop (77, 79). However, FRET was not observed in conjunction with H2Kb tetramers or dimers. FRET is observed between CD8a of 2C-transgenic CTL and H2Kb, suggesting the engagement of CD8a by monomeric H2Kb (82). Taken together, the data strongly suggest that the structure and/or the organization of the Va14Ja18 TCR complex are distinct from αβ TCR of conventional T cells, which might potentially account for the cooperative engagement of glycolipid Ags.

In conclusion, our findings demonstrate that iNKT cell functions are controlled by narrow avidity thresholds for glycolipid Ags and demonstrate novel properties of their Ag receptor that may have an important role in iNKT cell activation. These findings have important implications for the therapeutic use of iNKT cells.

We are greatly indebted to D. H. Margulies, D. Kranz, and S. Jameson for critical evaluation of the binding data and helpful comments on the manuscript, as well as to S. Roopenian for generous supplies of CTL clones. We thank M. Stanic for assistance and expertise in Hill constant determinations. We thank M. Wilson for CFSE labeling and in vitro expansion protocols. We thank Kirin Brewery for synthetic αGalCer; M. Taniguchi for B6-Ja180/0 mice; A. Bendelac and K. Hayakawa for NKT hybridomas; O. Naidenko and M. Kronenberg for helpful protocols for CD1 tetramer preparation; and A. J. Joyce for technical assistance.

1

This work was supported by National Institutes of Health (AI42284 and HL54977), Juvenile Diabetes Research Foundation, and Human Frontiers in Science Programme grants (to S.J.) and Ministry of Health, Labour, and Welfare (Japan) (to T.Y.).

3

Abbreviations used in this paper: iNKT, Va14Ja18 natural T; αGalCer, α-galactosylceramide; βGlcCer, β-glucosylceramide; mH, minor histocompatibility; MFI, mean fluorescence intensity; FRET, fluorescence resonance energy transfer.

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