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 Vα24-Jα18 alpha chain and a semi-invariant TCR Vβ11 chain. The only variable, and therefore potentially adaptive, element in human iNKT TCRs is their hypervariable CDR3β loop. The results of the present study demonstrate for the first time, to our knowledge, that the structure of the hypervariable CDR3β 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 CDR3β variations on human iNKT TCR:CD1d binding observed here was unexpected as previous studies with mouse iNKT TCRs have reported only minor effects of CDR3β mutations on CD1d binding. Furthermore, they strongly suggest that CDR3β 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 CDR3β 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 CDR3β and the K7-CD1d complex 
. In contrast, recent mutagenesis studies have indicated that the CDR3β loop of mouse iNKT TCRs may exert some impact on the affinity to CD1d, particularly when CD1d was loaded with weaker antigens 
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 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. CDR1α-3α and CDR1β-2β, are sufficient for effective iNKT cell interaction with K7-CD1d 
. Importantly, they strongly indicated that productive iNKT TCR interactions with OCH-CD1d require additional binding energy provided by certain CDR3β 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 OCHHIGH
iNKT clones. These recombinant iNKT TCRs differed only in their CDR3β 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 CDR3β 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 tetramer-based 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 TH2-biased cytokine secretion pattern 
. Also, addition of K7 to mouse fetal thymic organ cultures leads to effective deletion of iNKT cells 
, and K7 stimulation induces a prolonged anergy in iNKT cells 
, which supports the view that K7 is not a physiological ligand for iNKT cells. Hence, a full understanding of the biological role of CDR3β 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 CDR3β loop structure could translate into variations of weak antigen recognition. In an “antigen-dependent” or “adaptive” model, the CDR3β 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 “antigen-independent” or “innate-like” model, the CDR3β loop structure modulates iNKT TCR binding affinity to CD1d via protein-protein 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 CDR3β 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 CDR3β-dependent variation in iNKT TCR binding to OCH-CD1d, we examined recognition of two β-linked glucosylceramides, βGC and LacCer, by a panel of iNKT TCRs. K7 and OCH are α-linked monosaccharide glycosylceramides and are not expressed in mammals, whereas βGC and LacCer are natural β-linked glycosylceramides of mammalian cell membranes. The different configurations of α- and β-anomeric glycolipids enforce substantial differences in the orientation of their glycosyl headgroups when presented by CD1d 
. 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 βGC-CD1d or LacCer-CD1d. However, the results of the present study strongly support the “innate” model: βGC-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 CDR3β loop of human iNKT TCRs strongly modulated the overall binding affinity to different human ligand-CD1d complexes, independent of the bound ligand.
CDR3β 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 CDR3β 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 CDR3β structure on iNKT TCR binding affinity 
. Indeed, hybridomata expressing mouse iNKT TCRs with randomized CDR3β 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 
.Furthermore, previously published iNKT TCR:CD1d co-crystal structures showed a negligible contribution of the CDR3β to the interaction 
. 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 CDR3β loop and CD1d, a modeling exercise of TCR Vβ11 docking onto CD1d in the same study 
pointed to a significant degree of plasticity of the CDR3β conformation. In particular, the CDR3β loop of one of our previously published CD1d-restricted Vα24− Vβ11+ TCRs, TCR 5E 
, could make significant contacts with the alpha-2 helix of human CD1d 
. Consistent with this, a refolded hybrid TCR of the 5E Vβ11 chain and the invariant Vα24-Jα18 chain binds with high affinity to both CD1d/OCH and CD1d/βGC (unpublished data). Therefore, certain CDR3β 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 CDR3β 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. βGC and LacCer, have important implications for the clonal distribution of iNKT autoreactivity. CD1d-dependent autoreactivity of iNKT cells, i.e. their CD1d-mediated 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
, 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 
. 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 
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 
. 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 
. However, in contrast to mouse iNKT cells, human iNKT cell autoreactivity is not dependent on CD1d trafficking or endosomal acidification 
, 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 
, CD1d expression 
, or cytokine expression 
. 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 pro-inflammatory conditions, e.g. during bacterial infections.
Our functional analyses of autoreactive activation of OCHHIGH and OCHLOW 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 TCR-tetramers generated from OCHHIGH 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 CDR3β 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 OCHHIGH and OCHLOW iNKT clones shown herein suggest different in vivo functions of these subsets. For example, OCHHIGH and OCHLOW 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 CDR3β-dependent asymmetrical activation of the human iNKT repertoire could, over time, skew the balance between OCHHIGH and OCHLOW iNKT clones, with ensuing consequences for iNKT-dependent functions in both host defense and immunological tolerance.