Innate-Like Control of Human iNKT Cell Autoreactivity via the Hypervariable CDR3β Loop

T-cell receptor variability gives rise to a functional hierarchy of human invariant Natural Killer T-cells through a powerful effect on CD1d binding affinity, which is independent of CD1d ligands.


Introduction
Invariant Natural Killer T (iNKT) cells are a conserved subset of highly potent and versatile T-cells which specifically recognize the non-polymorphic lipid-presenting molecule CD1d [UniprotKB P15813] [1]. iNKT cells co-express a unique T-Cell Receptor (iNKT TCR), which mediates recognition of CD1d, and the pan-NK receptor NKR-P1A (CD161). Human and mouse iNKT TCRs feature a homologous invariant TCRa chain, i.e. Va24-Ja18 in humans and Va14-Ja18 in mice. In addition, all human iNKT TCRs make use of a single TCR Vb family, Vb11, whereas mouse iNKT TCRs utilize several different TCR Vb families.
The current paradox of iNKT biology lies in the fact that, despite their apparent innate-like simplicity, they can exert directly conflicting functions. On the one hand, several in vivo studies have demonstrated an essential role for iNKT cells in the induction and maintenance of immunological tolerance [2,3]. Consistent with this, iNKT cells exert a protective role in animal models of spontaneous autoimmunity [4,5], and numerical and functional defects of iNKT cells are observed in different human autoimmune diseases [6].
In contrast to these tolerogenic functions, iNKT cells can exert potent cytotoxic functions and contribute to host defense against tumors and various infectious pathogens [7,8,9]. Whether different subsets of iNKTs are involved in these opposed roles or whether individual iNKT clones fulfill both of these functions under different conditions is unknown. Several mechanisms underpin iNKT activation during host defense, such as TLR [10,11,12] and PPAR-c activation [13], co-stimulatory molecule signaling [14], and inflammatory cytokines [15,16]. However, it is unknown how iNKT cells are induced to mediate their tolerogenic functions under non-inflammatory conditions. Some iNKT clones exhibit substantial activation in response to CD1d-expressing antigen-presenting cells in the absence of exogenous antigens. This autoreactive function is essential for both iNKT selection [17] and tolerogenic activity [18]. While iNKT TCR binding to CD1d is absolutely required [19], the mechanistic basis of iNKT cell autoreactivity is largely unresolved. In particular, the importance of specific CD1d-presented endogenous lipid antigens for the autoreactive interaction of the iNKT TCR with CD1d is contentious.
Studies in mice have suggested that the iNKT repertoire displays clonal heterogeneity with regard to recognition of weaker stimulatory lipid antigens, such as the a-galactosylceramide analogue OCH. These differences can be explained by the differential Vb family usage in mouse iNKT TCRs [20,21,22]. However, human iNKT TCRs use a single Vb family and so the short hypervariable complementarity determining region (CDR3b) loop in human iNKT TCRs is their only truly adaptive element. It is not known whether this is sufficient to endow the human iNKT TCR with meaningful ability to discriminate a diverse range of human CD1d-presented antigens.
Here we examined a large panel of human iNKT cell lines and clones for their binding to different CD1d-ligand tetramers and related this both to the affinity of their TCRs to different CD1d-ligand complexes and to their functional recognition of diverse antigens. The results presented here demonstrate that variations in the CDR3b loop have a profound, antigen-independent, impact on the iNKT TCR's affinity to CD1d and on iNKT cell autoreactive function.

OCH-CD1d Tetramers Reveal Broad Heterogeneity of K7-CD1d Tetramer Positive Human iNKT Cells
Previous studies have shown that the CDR3b loop is dispensable for the ability of human iNKT cells to strongly react to the a-galactosylceramide antigen KRN7000 (K7), a xenobiotic glycolipid which can be presented to iNKT cells by CD1d. In fact, K7-CD1d tetramer staining does not allow discrimination of different human iNKT cell subsets by flow cytometry. We hypothesized that CD1d-tetramers loaded with weaker antigens might be better able to reveal the existence of CDR3b-dependent variation among human iNKT cells.
Therefore, we first examined whether different human iNKT subsets could be segregated by their binding to CD1d tetramers that were loaded with the synthetic iNKT partial agonist antigen OCH. For this purpose, polyclonal iNKT lines, generated from healthy donors by in vitro stimulation with K7, were tested for their binding to both K7-and OCH-CD1d tetramers. In all of these lines, K7-CD1d tetramers stained a single, clearly distinct, homogeneous, and strongly fluorescent population of iNKT lymphocytes ( Figure 1A). In contrast, staining of the same lines with OCH-CD1d tetramers revealed a considerable degree of variation in fluorescence, suggesting the presence of distinct iNKT subpopulations ( Figure 1A). Importantly, similar qualitative differences between K7-and OCH-CD1d tetramer staining of iNKT cells could also be observed ex vivo ( Figure 1B), indicating that these differences were not due to an artifact of previous in vitro stimulation with K7. In order to examine whether the broadly heterogeneous OCH-CD1d tetramer staining of human iNKT cells resulted from stable clonal variation or from transient changes in TCR expression levels, we generated a large panel of ''K7/OCH-naïve'' human iNKT cell clones and lines. For this purpose, Va24+/Vb11+ T cells were directly sorted ex vivo from healthy human donors and expanded using the non-specific T cell mitogen phytohaemagglutinin. Ninety-seven different human Va24+/Vb11+ T cell lines and 256 Va24+/Vb11+ T cell clones Author Summary Our immune system uses randomly modified T-cell receptors (TCRs) to adapt its discriminative capacity to rapidly changing pathogens. The T-cell receptor (TCR) has six flexible, variable peptide loops that make contact with antigens presented to them on the surface of other cells. Invariant Natural Killer T-cells (iNKT) are regulatory T-cells with a unique type of TCR (iNKT-TCR) that recognizes lipid antigens presented by specific MHC-like molecules known as CD1d. In human iNKT-TCRs, only one of the six loops, CDR3beta, is variable. By comparing how different human iNKT clones bind and react to different CD1d-lipid complexes we uncover the existence of a hierarchical order of the human iNKT cell repertoire in which strongly CD1dbinding clones are autoreactive while weak CD1d-binding clones are non-autoreactive. Direct measurements of iNKT-TCR binding to CD1d using surface plasmon resonance recapitulated this hierarchy at the protein level. The data show that variation in the CDR3beta loop conveys dramatic differences in human iNKT TCR affinity that are independent of the CD1d bound ligand. Thus the CDR3beta loop provides the structural basis for the functional hierarchy of the human iNKT repertoire. We postulate that during the life-course, CDR3beta-dependent asymmetrical activation of different human iNKT clones leads to a bias in the iNKT repertoire, and this could result in age-dependent defects of iNKT-mediated immune regulation in later life. from 13 different healthy donors were established and analyzed by flow cytometry with K7-and OCH-CD1d tetramers.
Importantly, the differences in OCH-CD1d tetramer staining could not be explained by differences in either TCR or CD4 coreceptor expression. Whereas K7-CD1d tetramer binding significantly correlated with surface expression levels of the Va24 and Vb11 TCR chains, no such association was observed for OCH-CD1d tetramer staining ( Figure 2C). Furthermore, CD4 coreceptor usage was not related to the intensity of the iNKT clones' OCH or K7-CD1d tetramer staining (unpublished results).
The results of these experiments revealed that the human iNKT repertoire is broadly heterogeneous with regard to the ability of individual clones to bind OCH-CD1d tetramers, independent of either CD4 co-receptor or TCR expression levels.
Human OCH HIGH and OCH LOW iNKT Cells Exhibit Differential Binding to CD1d Molecules Presenting b-Glycosylceramide The above results indicated that clonally distributed qualitative differences in iNKT TCRs were responsible for the considerable variation in OCH-CD1d tetramer binding. However, differences in iNKT TCR mediated recognition of an unnatural compound like OCH would be physiologically irrelevant if they simply reflected random differences in OCH-specific antigen selectivity. To explore this possibility, 18 iNKT clones of broadly varying OCH-CD1d MFI were tested for their ability to bind CD1d tetramers loaded with the common mammalian glycolipid b-glycosylceramide (bGC). These 18 iNKT clones displayed significant variation, up to 50-fold, in bGC-CD1d tetramer staining ( Figure 3A). Importantly, a strong association was evident between OCH-CD1d tetramer staining and bGC-CD1d tetramer staining, while no correlation was seen between bGC-CD1d tetramer staining and Va24 TCR chain surface expression ( Figure 3B). These results demonstrated that the observed broad variation in OCH-CD1d tetramer binding between individual human iNKT clones was not simply due to their antigen selectivity but was a reflection of a general variability in human iNKT TCR binding to CD1d loaded with weak antigenic lipids. Furthermore, they indicated that OCH-CD1d tetramer binding can act as a surrogate marker for human iNKT cell recognition of endogenous CD1d antigens.
The Hypervariable CDR3b Loop Has a Strong Effect on the Affinity of Human iNKT TCRs to CD1d Presenting Either aor b-Anomeric Glycolipids Based on the above results we hypothesized that the observed substantial differences in tetramer staining between OCH HIGH and OCH LOW iNKT clones resulted from significant variations in TCR:CD1d binding affinity. As expected, sequencing of the TCR Va24 and Vb11 chains demonstrated the usage of the known invariant Va24-Ja18 rearrangement in all clones, while Vb11 in these clones was rearranged with several different Jb families, resulting in highly variable CDR3b sequences. This indicated that, in human iNKT TCRs, structural differences of the CDR3b loop have a substantial impact on iNKT TCR binding to CD1d. To test this in a cell-free system we cloned the extracellular domains of the TCR Vb11 chains from a panel of seven OCH HIGH and OCH LOW iNKT cell clones (Table 1), as well as the invariant TCR Va24 chain from one iNKT clone, and used them to generate soluble Va24/Vb11 iNKT TCRs. Binding of these recombinant iNKT TCRs to K7-, OCH-, as well as bGCand lactosylceramide (LacCer-) loaded recombinant human CD1d complexes was measured using surface plasmon resonance ( Figure 4A; Table 2).
The results of these experiments showed a striking variation, up to 40-fold, between the different iNKT TCRs in their binding affinity (K D ) to a given ligand-CD1d complex (for K7-CD1d, K D : 0.24-3.67 mM; for OCH-CD1d, K D : 2.17-38.3 mM; for bGC-CD1d, K D : 2.17-85 mM; for LacCer-CD1d, K D : 2.1-54 mM; see Table 2). These findings clearly showed that the CDR3b loop of human iNKT TCRs can strongly impact on their binding to ligand-CD1d complexes.
Importantly, the binding affinities of all seven recombinant iNKT TCRs to OCH-CD1d strongly correlated with the OCH-CD1d tetramer staining (MFI) of their corresponding original iNKT clones ( Figure 4B). Moreover, the binding affinity of a given iNKT TCR to OCH-CD1d also correlated closely with its affinity to either bGCor K7-CD1d ( Figure 4C). Therefore, the wide variation in affinity between our seven human iNKT TCRs contrasted to the lack of variation in antigen selectivity. In other words, the CDR3b loop of human iNKT TCRs modulated the overall binding affinity to different human ligand-CD1d complexes irrespective of the bound ligand.
Based on these findings we hypothesized that the TCRs of OCH HIGH iNKT clones could also mediate enhanced functional recognition of endogenous ligand-CD1d complexes. We tested this hypothesis by comparing autoreactive responses of OCH HIGH and OCH LOW iNKT clones to CD1d-expressing antigen-presenting cells.

Autoreactive Functions of Human iNKT Cells Correlate with Their OCH-CD1d Binding
We directly compared the extent of proliferation, cytokine secretion, and cytotoxicity of human OCH HIGH and OCH LOW iNKT cells in response to CD1d expressing human cell lines presenting either endogenous or specific exogenous (''pulsed'') glycolipids. Because functional responses of iNKT cells might change during long term in vitro culture, we compared different donor-matched pairs of OCH HIGH and OCH LOW iNKT cell clones with identical in vitro history, i.e. each pair was sorted from a given donor 3 wk prior to the experiment and kept under identical cell culture conditions until the day of the experiment. The selected clones were all CD4+ and were additionally matched for TCR expression levels. For all pairs, OCH HIGH iNKT clones exhibited significantly greater proliferation than OCH LOW iNKT clones in response to either unpulsed or OCH-pulsed T2-CD1d lymphoblasts. In contrast, when T2-CD1d were pulsed with the strong agonist ligand K7, both OCH HIGH and OCH LOW iNKT clones proliferated vigorously, and to similar extent ( Figure 5A).
Next, we measured CD1d-dependent secretion of a panel of cytokines by OCH HIGH and OCH LOW iNKT clones. The OCH HIGH iNKT clones secreted considerably greater quantities of cytokines than their OCH LOW counterparts in response to Figure 2. Clonal variation in OCH-CD1d tetramer binding by human iNKT cells is not related to TCR expression levels. Flow cytometric analysis of one representative CD4+ human Va24+/Vb11+ iNKT line (A) and three representative CD4+ human Va24+/Vb11+ iNKT clones from different donors (B) demonstrates clonal variation in binding to OCH-CD1d (upper row), but not K7-CD1d (lower row) tetramers. (C) K7-and OCH-CD1d tetramer staining in pure human iNKT lines (n = 68) and clones (n = 256) was related to expression levels of iNKT TCR Va24 and Vb11. The intensity (MFI) of K7-but not OCH-CD1d tetramer staining was strongly associated with Va24 and Vb11 expression, as determined by Pearson correlation analysis, but not with CD4+ (blue markers) or CD42CD82 double negative (red markers) phenotype. doi:10.1371/journal.pbio.1000402.g002 either unpulsed or OCH-pulsed T2-CD1d cells ( Figure 5B, C), while no significant differences in cytokine secretion were observed between OCH HIGH and OCH LOW iNKT clones upon stimulation with K7-pulsed T2-CD1d cells. A general Th0-type cytokine secretion pattern was observed in response to stimulation with either K7 or OCH, while a Th1 pattern was often produced by autoreactive stimulation of OCH HIGH iNKT ( Figure 5C). Although most OCH LOW iNKT clones did not exhibit autoreactive cytokine release, two OCH LOW iNKT clones reproducibly secreted significant amounts of IL-13 and either IL-4 or IL-5, but no IFNc or TNF-a, while one OCH LOW iNKT clone secreted measurable amounts of IFNc and TNF-a, but no Th2 cytokines.
None of the tested iNKT clones secreted detectable amounts of cytokines in response to CD1d-deficient T2-lymphoblasts, and blocking of surface CD1d molecules on T2-CD1d by the monoclonal antibody CD1d42 effectively prevented autoreactive secretion of cytokines by OCH HIGH or OCH LOW iNKT cells (unpublished data). Therefore, autoreactive cytokine secretion by these iNKT clones was wholly dependent on their recognition of surface CD1d.
Finally, in Cr 51 release assays, OCH-pulsed T2-CD1d were much more efficiently killed by OCH HIGH iNKT clones than their corresponding OCH LOW iNKT clones ( Figure 6D). In contrast, K7-pulsed T2-CD1d were efficiently lysed by both OCH HIGH and    Table 2). (B) The affinity of the seven recombinant iNKT TCRs to OCH-CD1d, as determined by SPR, was linearly related to the staining intensity (MFI) of the original iNKT clone with OCH-CD1d tetramers. (C) The seven recombinant human iNKT TCRs followed a strict hierarchy of binding to ligand-CD1d complex, which was not affected by the specific CD1d-bound ligand. These iNKT TCRs differed only with regard to their CDR3beta sequence ( Together, these results demonstrated that OCH-CD1d tetramer staining allows for identification of distinct human OCH HIGH and OCH LOW iNKT clones, which exhibit differential functional ability to respond to endogenous ligand-CD1d complexes. The above results indicated that the autoreactive potential of human iNKT clones is governed by the affinity of their iNKT TCR to CD1d, and therefore the structure of their CDR3b loop.

TCRs from OCH HIGH but not OCH LOW Human iNKT Subsets Bind to Endogenous CD1d-Ligand Complexes
In order to test our hypothesis that OCH HIGH and OCH LOW iNKT TCRs differed in their binding to endogenous ligand-CD1d complexes, we generated soluble fluorescent iNKT TCR-tetramers derived from an autoreactive OCH HIGH iNKT clone and a non-autoreactive OCH LOW iNKT clone. As shown in Figure 6, both iNKT TCR tetramers bound well to K7-pulsed T2-CD1d. In contrast, only the OCH HIGH -derived iNKT TCR tetramer was able to effectively stain unpulsed T2-CD1d. These results further substantiated our hypothesis that autoreactive recognition of CD1d by human iNKT cells is primarily determined by the structure of their iNKT TCRs' CDR3b loop.
All together, these studies demonstrated that the human iNKT cell repertoire exhibits considerable clonally distributed CDR3bdependent differences in overall TCR affinity to CD1d, irrespective of the bound ligand, and that these inherent structural differences control iNKT autoreactive activation.

Discussion
iNKT cells are a conserved subset of highly potent regulatory T cells at the innate-adaptive interface. The hallmark of human iNKT cells is their unique TCR, which is composed of an invariant TCR Va24-Ja18 alpha chain and a semi-invariant TCR Vb11 chain. The only variable, and therefore potentially adaptive, element in human iNKT TCRs is their hypervariable CDR3b loop. The results of the present study demonstrate for the first time, to our knowledge, that the structure of the hypervariable CDR3b loop in human iNKT TCRs exerts a strong impact on CD1d binding and is a key determinant of iNKT cell autoreactivity. The magnitude of the effect of CDR3b variations on human iNKT TCR:CD1d binding observed here was unexpected as previous studies with mouse iNKT TCRs have reported only minor effects of CDR3b mutations on CD1d binding. Furthermore, they strongly suggest that CDR3b loops in autoreactive iNKT TCRs make functionally important direct protein-protein contacts with human CD1d, rather than contacts with CD1d-bound ligands, thereby affecting overall affinity rather than antigen specificity.
The role of the hypervariable CDR3b loop in human iNKT TCRs is currently unresolved. It is dispensable for binding to CD1d molecules that are loaded with the strong agonist ligand K7, and hence K7-CD1d tetramers do not support subset differentiation of human iNKT cells. Consistent with this, the recently solved structures of one human and two mouse iNKT TCR:K7-CD1d co-crystals have found no relevant contacts between CDR3b and the K7-CD1d complex [20,23]. In contrast, recent mutagenesis studies have indicated that the CDR3b loop of mouse iNKT TCRs may exert some impact on the affinity to CD1d, particularly when CD1d was loaded with weaker antigens [24,25,26].
We found that human iNKT cells were surprisingly heterogeneous in their binding to CD1d tetramers loaded with the partial agonist ligand OCH, which is a synthetic analogue of K7. Up to 200-fold differences in OCH-CD1d tetramer staining were observed between individual iNKT clones, independent of variations in TCR expression. The same clones exhibited only modest differences in K7-CD1d tetramer staining, which could largely be explained simply by variations in TCR expression. Importantly, we found that the clonal variation in OCH-CD1d tetramer binding was directly related to OCH-CD1d dependent exhibit cytotoxicity in response to lipid-pulsed or endogenous lipid presenting CD1d-positive antigen presenting cells. (A) Proliferation of three representative pairs of OCH HIGH and OCH LOW iNKT clones from different healthy donors in response to K7-, OCH-, or vehicle-pulsed human CD1dexpressing T2 cells (T2-CD1d) or to K7-pulsed CD1d negative T2 cells (T2-) is shown. OCH HIGH clones consistently displayed greater proliferation than OCH LOW clones in response to OCH or vehicle pulsed T2-CD1d. cpm, counts per minute. Mean values 6 s.e.m. are shown. (B) Cytokine secretion profiles of a representative pair of matched OCH HIGH and OCH LOW iNKT clones in response to the strong agonist ligand K7 and the partial agonist ligand OCH, presented by T2-CD1d, are shown. OCH HIGH iNKT clones exhibited much stronger cytokine secretion than OCH LOW iNKT cells in response to OCH-pulsed T2-CD1d, while cytokine secretion was similar for both in response to K7-pulsed T2-CD1d. (C) Autoreactive cytokine release in response to T2-CD1d in the absence of added exogenous ligands is shown for four matched pairs of OCH HIGH and OCH LOW iNKT clones. OCH HIGH but not OCH LOW iNKT clones consistently exhibited substantial autoreactive cytokine secretion. (D) Specific lysis of K7-(filled markers) and OCH-(unfilled markers) pulsed T2-CD1d targets is shown for three matched pairs of OCH HIGH and OCH LOW iNKT clones from different donors. doi:10.1371/journal.pbio.1000402.g005 Figure 6. Differential binding of OCH HIGH and OCH LOW iNKT clone derived TCR tetramers to endogenous lipid presenting CD1d molecules. PE-conjugated recombinant iNKT TCR tetramers derived from OCH HIGH (4C1369; red lines) and OCH LOW (4C12; blue lines) iNKT clones, at increasing concentrations, were used to stain T2-CD1d lymphoblasts. Clear staining of vehicle-pulsed T2-CD1d (unfilled markers) was only seen with the OCH HIGH TCR tetramer, whereas both iNKT TCR tetramers strongly bound to K7-pulsed T2-CD1d (filled markers). The black bar shows background staining of T2-cells with iNKT TCR tetramers. doi:10.1371/journal.pbio.1000402.g006 functional responses, while no such linkage was observed between K7-CD1d tetramer staining and K7-dependent functional iNKT activation. These data underpinned the notion that the five germline encoded CDR loops in human iNKT TCRs, i.e. CDR1a-3a and CDR1b-2b, are sufficient for effective iNKT cell interaction with K7-CD1d [26]. Importantly, they strongly indicated that productive iNKT TCR interactions with OCH-CD1d require additional binding energy provided by certain CDR3b loop structures. We tested this hypothesis by directly measuring the binding of K7-and OCH-CD1d complexes to a panel of seven recombinant human iNKT TCRs, which were derived from selected OCH HIGH and OCH LOW iNKT clones. These recombinant iNKT TCRs differed only in their CDR3b structure. The results of these experiments demonstrated that the broad clonal heterogeneity in OCH-CD1d tetramer staining is indeed directly determined by the iNKT clones' TCRs binding affinities to OCH-CD1d, and hence the structure of the CDR3b loop. Conversely, while all tested recombinant iNKT TCRs bound approximately 10-fold better to K7-CD1d than to OCH-CD1d, the fold-differences in affinity between the strongest and the weakest binding iNKT TCRs were similar for binding to either OCH-or K7-CD1d. Together with the above discussed tetramerbased and functional studies, this indicates that the synthetic CD1d ligand K7 pushes the interaction between human CD1d and iNKT TCRs beyond a physiological range. This is consistent with numerous in vivo and in vitro studies which showed that K7 induces concurrent massive iNKT cell secretion of TH1-, TH2-, and TH17-type cytokines, whereas OCH causes a clearly TH2biased cytokine secretion pattern [27]. Also, addition of K7 to mouse fetal thymic organ cultures leads to effective deletion of iNKT cells [28], and K7 stimulation induces a prolonged anergy in iNKT cells [29], which supports the view that K7 is not a physiological ligand for iNKT cells. Hence, a full understanding of the biological role of CDR3b loop polymorphism will require more studies with weaker agonistic antigens, and the results of this study suggest that OCH is a good surrogate for endogenous weak agonist antigens.
There are two competing models to explain how differences in CDR3b loop structure could translate into variations of weak antigen recognition. In an ''antigen-dependent'' or ''adaptive'' model, the CDR3b loop bestows upon iNKT cells a degree of lipid selectivity by controlling iNKT TCR affinity to CD1d in a lipid antigen-specific manner. Alternatively, in an ''antigenindependent'' or ''innate-like'' model, the CDR3b loop structure modulates iNKT TCR binding affinity to CD1d via proteinprotein interactions. This model would allow higher, but not lower, affinity TCR structures to recognize CD1d molecules presenting weaker lipid antigens but, crucially, without differential patterns of lipid antigen selectivity between iNKT TCRs of similar CD1d affinity. In other words, this model predicts that the inherent CDR3b sequence in a given human iNKT clone would determine its iNKT TCR's general ability to bind to diverse ligand-CD1d complexes. An important corollary of this would be a fixed hierarchy of high and low affinity iNKT clones. A prediction arising from this model would be that iNKT cells lack the ability to develop immunological memory to specific pathogens, which is a hallmark of adaptive immunity. Although iNKT TCRs clearly belong to the broader family of rearranged, and therefore ''adaptive,'' TCRs and BCRs, their limited structural diversity and lack of antigen-selectivity, as proposed by this model, are strongly reminiscent of innate immune receptors.
In order to test which of the two above models best explains the observed CDR3b-dependent variation in iNKT TCR binding to OCH-CD1d, we examined recognition of two b-linked glucosylceramides, bGC and LacCer, by a panel of iNKT TCRs. K7 and OCH are a-linked monosaccharide glycosylceramides and are not expressed in mammals, whereas bGC and LacCer are natural blinked glycosylceramides of mammalian cell membranes. The different configurations of aand b-anomeric glycolipids enforce substantial differences in the orientation of their glycosyl headgroups when presented by CD1d [30,31]. Therefore, if the substantial variation in iNKT TCR affinity to OCH-CD1d observed in our study was mainly a function of clonal variation in lipid antigen specificity, as predicted by the ''adaptive'' model, there should be no association between an individual iNKT TCR's affinity to OCH-CD1d and its affinity to either bGC-CD1d or LacCer-CD1d. However, the results of the present study strongly support the ''innate'' model: bGC-CD1d tetramer binding to human iNKT clones correlated in a linear fashion with OCH-CD1d tetramer binding, and our binding studies with several different soluble iNKT TCRs demonstrated that the CDR3b loop of human iNKT TCRs strongly modulated the overall binding affinity to different human ligand-CD1d complexes, independent of the bound ligand.
CDR3b loop hypervariability of human iNKT TCRs therefore strongly impacts on overall affinity to CD1d but does not exert a relevant effect on antigen selectivity. The powerful effect of natural CDR3b variations on human iNKT TCR:CD1d affinity observed in our study was unexpected as previous iNKT TCR mutagenesis studies in mice have suggested only a weak impact of CDR3b structure on iNKT TCR binding affinity [24,25,26]. Indeed, hybridomata expressing mouse iNKT TCRs with randomized CDR3b regions only displayed moderate variability in binding to K7-CD1d tetramers, and only very few TCRs were capable of interacting with CD1d presenting endogenous lipids [25].Furthermore, previously published iNKT TCR:CD1d co-crystal structures showed a negligible contribution of the CDR3b to the interaction [20,23]. The apparent discrepancies between these studies and the current findings could indicate relevant species differences, as the mutagenesis studies have concentrated on mouse iNKT binding or else might reflect differences in study design: the only crystal structure study of human iNKT TCR:CD1d binding was limited to a single iNKT TCR of unknown weak antigen-CD1d affinity while the current study systematically screened a large panel of naturally occurring human iNKT clones. Interestingly, while the iNKT TCR used for the human co-crystal structure study displayed very limited contacts between its CDR3b loop and CD1d, a modeling exercise of TCR Vb11 docking onto CD1d in the same study [23] pointed to a significant degree of plasticity of the CDR3b conformation. In particular, the CDR3b loop of one of our previously published CD1d-restricted Va242 Vb11+ TCRs, TCR 5E [32], could make significant contacts with the alpha-2 helix of human CD1d [23]. Consistent with this, a refolded hybrid TCR of the 5E Vb11 chain and the invariant Va24-Ja18 chain binds with high affinity to both CD1d/OCH and CD1d/bGC (unpublished data). Therefore, certain CDR3b loop structures can potentially facilitate the recognition of human CD1d loaded with weak ligands by providing additional binding energy to the TCR-CD1d interaction.
Sequence analysis of the CDR3b loops studied did not reveal any obvious correlations between CD1d binding affinity and either physicochemical properties of the loop as a whole or the position of specific residues within the sequence. This is not surprising, given the high degree of conformational flexibility of CDR loops.
The above described considerable binding affinities of some human iNKT TCRs to naturally occurring beta-anomeric glycolipids, i.e. bGC and LacCer, have important implications for the clonal distribution of iNKT autoreactivity. CD1ddependent autoreactivity of iNKT cells, i.e. their CD1dmediated activation in the absence of exogenous antigens, is likely to play important biological roles, but the molecular mechanisms determining iNKT autoreactivity have been unresolved. CD1d-dependent autoreactivity is observed in approximately 30% of mouse iNKT hybridomas [19], and studies in iNKT deficient and autoimmune prone mice have shown that autoreactive CD1d-recognition is required for iNKT selection and also iNKT-mediated immunological tolerance [15,18,33,34]. However, much less is known about the role of CD1d-dependent iNKT autoreactivity in humans. Neonatal human iNKT cells exhibit an activated memory phenotype, indicating their in vivo recognition of CD1d molecules in the absence of exogenous ligands [35].
An ''adaptive'' model has been proposed to explain autoreactive activation of iNKT cells in mouse models of bacterial infection, and it was postulated that autoreactive murine iNKT cells specifically recognize de novo synthesized antigens, such as isogloboside 3 [36]. Consistent with this model, mouse CD1d requires endosomal trafficking to elicit autoreactive activation of murine iNKT cells, which suggests that processing of the ligand-CD1d complex is essential [37]. However, in contrast to mouse iNKT cells, human iNKT cell autoreactivity is not dependent on CD1d trafficking or endosomal acidification [38], again suggesting important species differences between mouse and human iNKT cell activation.
The antigen-independent ''innate-like'' model discussed above offers a simpler explanation for the clonally distributed iNKT autoreactivity. iNKT clones with higher overall iNKT TCR:CD1d affinity would have an intrinsically greater autoreactive potential than low affinity clones, and these differences in autoreactive potential would be independent of de novo synthesized CD1d-bound ligands. Autoreactive activation of iNKT clones in this model would still be controlled by local conditions, such as TLR signaling [12], CD1d expression [16], or cytokine expression [39]. High affinity iNKT clones would be capable of exerting autoreactive functions under physiological conditions, while low affinity iNKT clones would only be recruited under more proinflammatory conditions, e.g. during bacterial infections.
Our functional analyses of autoreactive activation of OCH HIGH and OCH LOW iNKT clones support the ''innate-like'' model. Firstly, autoreactive activation of several matched pairs of human iNKT clones was closely associated with their OCH-CD1d tetramer binding characteristics. Secondly, only iNKT TCRtetramers generated from OCH HIGH iNKT clones were able to bind to CD1d-expressing antigen-presenting cells in the absence of exogenous lipid. The above data therefore underpin the ''innate-like'' model, whereby the hypervariable CDR3b loop balances TCR binding affinity to CD1d protein, and hence the autoreactive potential of an iNKT clone, independent of the bound ligand.
The different activation thresholds of ex vivo sorted human OCH HIGH and OCH LOW iNKT clones shown herein suggest different in vivo functions of these subsets. For example, OCH HIGH and OCH LOW iNKT cells might differ in their ability to drive the formation of immature DCs and consequently in their capability to constitutively promote peripheral tolerance. Finally, it is intriguing to speculate that CDR3b-dependent asymmetrical activation of the human iNKT repertoire could, over time, skew the balance between OCH HIGH and OCH LOW iNKT clones, with ensuing consequences for iNKT-dependent functions in both host defense and immunological tolerance.

Generation of Soluble Heterodimeric TCRs
Soluble TCR heterodimers were generated as previously described [41]. Briefly, the extracellular region of each TCR chain was individually cloned in the bacterial expression vector pGMT7 and expressed in Escherichia coli BL21-DE3 (pLysS). Residues Thr48 and Ser57, respectively, of the aand b-chain TCR constant region domains were both mutated to cysteine. Expression, refolding, and purification of the resultant disulfidelinked iNKT TCR ab heterodimers was carried out as previously described [32].

Surface Plasmon Resonance
Streptavidin (,5,000 RU) was linked to a Biacore CM-5 chip (BIAcore AB, UK) using the amino-coupling kit according to manufacturer's instructions, and lipid-CD1d complexes or control proteins (bGC-CD1b and HLA-A2*01-NY-Eso-1(157-165) complex) were flowed over individual flow cells at ,50 mg/ml until the response measured ,1,000 RU. Serial dilutions of recombinant iNKT TCRs were then flowed over the relevant flow cells at a rate of 5 ml/min (for equilibrium binding measurements) or 50 ml/min (for kinetic measurements). Responses were recorded in real time on a Biacore 3000 machine at 25uC, and data were analyzed using BIAevaluation software (Biacore, Sweden). Equilibrium dissociation constants (KD values) were determined assuming a 1:1 interaction (A+B « AB) by plotting specific equilibrium binding responses against protein concentrations followed by non-linear least squares fitting of the Langmuir binding equation, AB = B6AB max /(K D +B), and were confirmed by linear Scatchard plot analysis using Origin 6.0 software (Microcal, USA). Kinetic binding parameters (k on and k off ) were determined using BIAevaluation software.

Proliferation and Cytokine Secretion Assays
T2 lymphoblast cells (T2-) and CD1d-expressing T2 lymphoblast cells (T2-CD1d) were used as antigen presenting cells (APC). 5610 4 iNKT cells were plated in a 96-well round-bottom plate in triplicates with either medium alone, with 2.5610 4 T2-CD1d, or with T2 lymphoblasts. Before use, T2-CD1d and T2 lymphoblasts were treated with 0.1 mg/ml mitomycin C for 1 h at 37uC and extensively washed with PBS. Lipid antigens (K7, OCH, and bGC) were added at a final concentration of 100 ng/ml. Lipids were solubilized at 200 mg/ml by sonication in vehicle (0.5% Tween-20), which was also used as a negative control. IL-2 was added to the culture medium at a final concentration of 10 IU/ml. Proliferation was measured during the last 18 h of a 96 h incubation by addition of 1 mCi [ 3 H]-methyl-thymidine (1 Ci = 37 GBq, Amersham Pharmacia), followed by harvesting and scintillation counting (Perkin Elmer beta counter).

Cytotoxicity Assays
T2 lymphoblasts and T2-CD1d were cultured for 16 h either in the presence of lipid antigens at 100 ng/ml concentration or an equivalent quantity of vehicle. They were then labeled with 100 mCi of 51 Cr (GE Healthcare, UK) for 1 h at 37uC and washed 3 times with warm RPMI 1640 supplemented with 1% FBS.
iNKT cells were added in duplicates at different effector-totarget cell ratios and cultured for 4 h. Maximal 51 Cr release was determined from target cells lysed by hydrochloric acid. The percentage of specific lysis was calculated by the following formula: [(experimental cpm 2 spontaneous release cpm)/(maximum release cpm 2 spontaneous release cpm)] 6100%. Percentage of unspecific lysis was always ,20%.

Generation of Fluorescent iNKT TCR Tetramers
Soluble iNKT-TCR heterodimers were biotinylated via an engineered BirA motif on the C-terminus of their TCR b-chain and then conjugated to PE-streptavidin (Molecular Probes, USA). Multimeric complexes were purified by FPLC (Pharmacia, Sweden) on an SD200 column (Pharmacia, Sweden) and concentrated to 1 mg/ml using Vivaspin20 concentrators (Vivascience, UK).