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

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3β, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3β loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3β in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist α-linked glycolipid antigen OCH and structurally different endogenous β-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3β sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3β for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3β dependent functional hierarchy of human iNKT cells.     

The Molecular Basis for Recognition of CD1d/α-Galactosylceramide by a Human Non-Vα24 T Cell Receptor

CD1d-mediated presentation of glycolipid antigens to T cells is capable of initiating powerful immune responses that can have a beneficial impact on many diseases. Molecular analyses have recently detailed the lipid antigen recognition strategies utilized by the invariant Vα24-Jα18 TCR rearrangements of iNKT cells, which comprise a subset of the human CD1d-restricted T cell population. In contrast, little is known about how lipid antigens are recognized by functionally distinct CD1d-restricted T cells bearing different TCRα chain rearrangements. Here we present crystallographic and biophysical analyses of α-galactosylceramide (α-GalCer) recognition by a human CD1d-restricted TCR that utilizes a Vα3.1-Jα18 rearrangement and displays a more restricted specificity for α-linked glycolipids than that of iNKT TCRs. Despite having sequence divergence in the CDR1α and CDR2α loops, this TCR employs a convergent recognition strategy to engage CD1d/αGalCer, with a binding affinity (∼2 µM) almost identical to that of an iNKT TCR used in this study. The CDR3α loop, similar in sequence to iNKT-TCRs, engages CD1d/αGalCer in a similar position as that seen with iNKT-TCRs, however fewer actual contacts are made. Instead, the CDR1α loop contributes important contacts to CD1d/αGalCer, with an emphasis on the 4′OH of the galactose headgroup. This is consistent with the inability of Vα24− T cells to respond to α-glucosylceramide, which differs from αGalCer in the position of the 4′OH. These data illustrate how fine specificity for a lipid containing α-linked galactose is achieved by a TCR structurally distinct from that of iNKT cells.     

The complementarity determining region 2 of BV8S2 (V beta 8.2) contributes to antigen recognition by rat invariant NKT cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.     

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.     

Nanomapping of CD1d–glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging

CD1d molecule, a monomorphic major histocompatibility complex class I‐like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d–glycolipid complex. THP1 cells were pulsed with three different glycolipids (α‐GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d–α‐GalCer and CD1d–C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm² (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm². Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d–glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Cutting edge: structural basis for the recognition of β-linked glycolipid antigens by invariant NKT cells

Invariant NKT (iNKT) cells expressing a semi-invariant Vα14 TCR recognize self and foreign lipid Ags when presented by the nonclassical MHCI homolog CD1d. Whereas the majority of known iNKT cell Ags are characterized by the presence of a single α-linked sugar, mammalian self Ags are β-linked glycosphingolipids, posing the interesting question of how the semi-invariant TCR can bind to such structurally distinct ligands. In this study, we show that the mouse iNKT TCR recognizes the complex β-linked Ag isoglobotrihexosylceramide (iGb3; Galα1-3-Galβ1-4-Glcβ1-1Cer) by forcing the proximal β-linked sugar of the trisaccharide head group to adopt the typical binding orientation of α-linked glycolipids. The squashed iGb3 orientation is stabilized by several interactions between the trisaccharide and CD1d residues. Finally, the formation of novel contacts between the proximal and second sugar of iGb3 and CDR2α residues of the TCR suggests an expanded recognition logic that can possibly distinguish foreign Ags from self Ags.     

iNKT cells in microbial immunity: recognition of microbial glycolipids

Natural killer T cells expressing an invariant T cell antigen receptor (iNKT cells) are cells of the innate immune system. After recognizing glycolipid antigens presented by CD1d molecules on antigen presenting cells (APCs), iNKT cells rapidly produce large quantities of cytokines, thereby stimulating many types of cells. Recent studies have described several mechanisms of iNKT cell activation and the contribution of these cells to antimicrobial responses. iNKT cells can be activated by endogenous antigens and/or inflammatory cytokines from APCs. However, iNKT cells also recognize certain microbial glycolipids by their invariant T cell antigen receptor (TCR), and they contribute to pathogen clearance in certain microbial infections. These findings indicate that the iNKT TCR is useful for detecting certain microbial pathogens. Moreover, recent studies suggest that iNKT cell glycolipid antigens may be useful in antimicrobial therapy and vaccines.     

Unique interplay between sugar and lipid in determining the antigenic potency of bacterial antigens for NKT cells

Invariant natural killer T (iNKT) cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.     

The versatility of the αβ T‐cell antigen receptor

The T‐cell antigen receptor is a heterodimeric αβ protein (TCR) expressed on the surface of T‐lymphocytes, with each chain of the TCR comprising three complementarity‐determining regions (CDRs) that collectively form the antigen‐binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR‐pMHC interaction is crucially important in cell‐mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR‐pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T‐cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC‐related molecules that instead of presenting peptides are ideally suited to bind lipid‐based antigens. Structural studies within the CD1‐lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1‐lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC‐like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival.     

The T cell receptor beta-chain second complementarity determining region loop (CDR2beta governs T cell activation and Vbeta specificity by bacterial superantigens

Superantigens (SAgs) are microbial toxins defined by their ability to activate T lymphocytes in a T cell receptor (TCR) β-chain variable domain (Vβ)-specific manner. Although existing structural information indicates that diverse bacterial SAgs all uniformly engage the Vβ second complementarity determining region (CDR2β) loop, the molecular rules that dictate SAg-mediated T cell activation and Vβ specificity are not fully understood. Herein we report the crystal structure of human Vβ2.1 (hVβ2.1) in complex with the toxic shock syndrome toxin-1 (TSST-1) SAg, and mutagenesis of hVβ2.1 indicates that the non-canonical length of CDR2β is a critical determinant for recognition by TSST-1 as well as the distantly related SAg streptococcal pyrogenic exotoxin C. Frame work (FR) region 3 is uniquely critical for TSST-1 function explaining the fine Vβ-specificity exhibited by this SAg. Furthermore, domain swapping experiments with SAgs, which use distinct domains to engage both CDR2β and FR3/4β revealed that the CDR2β contacts dictate T lymphocyte Vβ-specificity. These findings demonstrate that the TCR CDR2β loop is the critical determinant for functional recognition and Vβ-specificity by diverse bacterial SAgs.     

Structural basis for the recognition of C20:2-αGalCer by the invariant natural killer T cell receptor-like antibody L363

Natural killer T (NKT) cells express a semi-invariant Vα14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to α- and β-anomeric glycosphingolipids, as well as microbial α-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen αGalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. Here, we report the 3.1 Å resolution crystal structure of the Fab of one of these antibodies, L363, bound to mCD1d complexed with the αGalCer analog C20:2, revealing that L363 is an iNKT TCR-like antibody that binds CD1d-presented αGalCer in a manner similar to the TCR. The structure reveals that L363 depends on both the L and H chains for binding to the glycolipid-mCD1d complex, although only the L chain is involved in contacts with the glycolipid antigen. The H chain of L363 features residue Trp-104, which mimics the TCR CDR3α residue Leu-99, which is crucial for CD1d binding. We characterized the antigen-specificity of L363 toward several different glycolipids, demonstrating that whereas the TCR can induce structural changes in both antigen and CD1d to recognize disparate lipid antigens, the antibody L363 can only induce the F′ roof formation in CD1d but fails to reorient the glycolipid headgroup necessary for binding. In summary, L363 is a powerful tool to study mechanism of iNKT cell activation for structural analogs of KRN7000, and our study can aid in the design of antibodies with altered antigen specificity.     

CD1d-independent activation of mouse and human iNKT cells by bacterial superantigens

Invariant NKT (iNKT) cells are infrequent but important immunomodulatory lymphocytes that exhibit CD1d-restricted reactivity with glycolipid Ags. iNKT cells express a unique T-cell receptor (TCR) composed of an invariant α-chain, paired with a limited range of β-chains. Superantigens (SAgs) are microbial toxins defined by their ability to activate conventional T cells in a TCR β-chain variable domain (Vβ)-specific manner. However, whether iNKT cells are directly activated by bacterial SAgs remains an open question. Herein, we explored the responsiveness of mouse and human iNKT cells to a panel of staphylococcal and streptococcal SAgs and examined the contribution of major histocompatibility complex (MHC) class II and CD1d to these responses. Bacterial SAgs that target mouse Vβ8, such as staphylococcal enterotoxin B (SEB), were able to activate mouse hybridoma and primary hepatic iNKT cells in the presence of mouse APCs expressing human leukocyte antigen (HLA)-DR4. iNKT cell-mediated cytokine secretion in SEB-challenged HLA-DR4-transgenic mice was CD1d-independent and accompanied by a high interferon-γ:interleukin-4 ratio consistent with an in vivo Th1 bias. Furthermore, iNKT cells from SEB-injected HLA-DR4-transgenic mice, and iNKT cells from SEB-treated human PBMCs, showed early activation by intracellular cytokine staining and CD69 expression. Unlike iNKT cell stimulation by α-galactosylceramide, stimulation by SEB did not induce TCR downregulation of either mouse or human iNKT cells. We conclude that Vβ8-targeting bacterial SAgs can activate iNKT cells by utilizing a novel pathway that requires MHC class II interactions, but not CD1d. Therefore, iNKT cells fulfill important effector functions in response to bacterial SAgs and may provide attractive targets in the management of SAg-induced illnesses.     

Single MHC mutation eliminates enthalpy associated with T cell receptor binding

The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition. From the range of responses observed, three representative HLA-A2 mutants, T163A, W167A, and K66A, were selected for further study. Binding constants and co-crystal structures of the AHIII TCR and the three mutants were determined. K66 in HLA-A2 makes contacts with both peptide and TCR, and has been identified as a critical residue for recognition by numerous TCR. The K66A mutation resulted in the lowest AHIII T cell response and the lowest binding affinity, which suggests that the T cell response may correlate with affinity. Importantly, the K66A mutation does not affect the conformation of the peptide. The change in affinity appears to be due to a loss in hydrogen bonds in the interface as a result of a conformational change in the TCR complementarity-determining region 3 (CDR3) loop. Isothermal titration calorimetry confirmed the loss of hydrogen bonding by a large loss in enthalpy. Our findings are inconsistent with the notion that the CDR1 and CDR2 loops of the TCR are responsible for MHC restriction, while the CDR3 loops interact solely with the peptide. Instead, we present here an MHC mutation that does not change the conformation of the peptide, yet results in an altered conformation of a CDR3.     

Tight regulation of diacylglycerol-mediated signaling is critical for proper invariant NKT cell development

Type I NKT cells, or invariant NKT (iNKT) cells, express a semi-invariant TCR characterized by its unique Vα14-Jα18 usage (iVα14TCR). Upon interaction with glycolipid/CD1d complexes, the iVα14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the protein kinase C-IκB kinase (IKK)α/β-NF-κB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. In this article, we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NF-κB and Ras-Erk1/2 activation. Moreover, constitutive IKKβ and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development.     

Human invariant NKT cells display alloreactivity instructed by invariant TCR-CD1d interaction and killer Ig receptors

Invariant NKT (iNKT) cells are a subset of highly conserved immunoregulatory T cells that modify a variety of immune responses, including alloreactivity. Central to their function is the interaction of the invariant TCR with glycosphingolipid (GSL) ligands presented by the nonpolymorphic MHC class I molecule CD1d and their ability to secrete rapidly large amounts of immunomodulatory cytokines when activated. Whether iNKT cells, like NK and conventional T cells, can directly display alloreactivity is not known. We show in this study that human iNKT cells and APC can establish a direct cross-talk leading to preferential maturation of allogeneic APC and a considerably higher reactivity of iNKT cells cultured with allogeneic rather that autologous APC. Although the allogeneic activation of iNKT cells is invariant TCR-CD1d interaction-dependent, GSL profiling suggests it does not involve the recognition of disparate CD1d/GSL complexes. Instead, we show that contrary to previous reports, iNKT cells, like NK and T cells, express killer Ig receptors at a frequency similar to that of conventional T cells and that iNKT cell allogeneic activation requires up-regulation and function of activating killer Ig receptors. Thus, iNKT cells can display alloreactivity, for which they use mechanisms characteristic of both NK and conventional T cells.     

Disparate degrees of hypervariable loop flexibility control T-cell receptor cross-reactivity, specificity, and binding mechanism

αβ T-cell receptors (TCRs) recognize multiple antigenic peptides bound and presented by major histocompatibility complex molecules. TCR cross-reactivity has been attributed in part to the flexibility of TCR complementarity-determining region (CDR) loops, yet there have been limited direct studies of loop dynamics to determine the extent of its role. Here we studied the flexibility of the binding loops of the αβ TCR A6 using crystallographic, spectroscopic, and computational methods. A significant role for flexibility in binding and cross-reactivity was indicated only for the CDR3α and CDR3β hypervariable loops. Examination of the energy landscapes of these two loops indicated that CDR3β possesses a broad, smooth energy landscape, leading to rapid sampling in the free TCR of a range of conformations compatible with different ligands. The landscape for CDR3α is more rugged, resulting in more limited conformational sampling that leads to specificity for a reduced set of peptides as well as the major histocompatibility complex protein. In addition to informing on the mechanisms of cross-reactivity and specificity, the energy landscapes of the two loops indicate a complex mechanism for TCR binding, incorporating elements of both conformational selection and induced fit in a manner that blends features of popular models for TCR recognition.     

Fine specificity of TCR complementarity-determining region residues and lipid antigen hydrophilic moieties in the recognition of a CD1-lipid complex

alphabeta TCR can recognize peptides presented by MHC molecules or lipids and glycolipids presented by CD1 proteins. Whereas the structural basis for peptide/MHC recognition is now clearly understood, it is not known how the TCR can interact with such disparate molecules as lipids. Recently, we demonstrated that the alphabeta TCR confers specificity for both the lipid Ag and CD1 isoform restriction, indicating that the TCR is likely to recognize a lipid/CD1 complex. We hypothesized that lipids may bind to CD1 via their hydrophobic alkyl and acyl chains, exposing the hydrophilic sugar, phosphate, and other polar functions for interaction with the TCR complementarity-determining regions (CDRs). To test this model, we mutated the residues in the CDR3 region of the DN1 TCR beta-chain that were predicted to project between the CD1b alpha helices in a model of the TCR/CD1 complex. In addition, we tested the requirement for the negatively charged and polar functions of mycolic acid for Ag recognition. Our findings indicate that the CDR loops of the TCR form the Ag recognition domain of CD1-restricted TCRs and suggest that the hydrophilic domains of a lipid Ag can form a combinatorial epitope recognized by the TCR.     

Advanced omics techniques shed light on CD1d-mediated lipid antigen presentation to iNKT cells

Invariant natural killer T cells (iNKT cells) can be activated through binding antigenic lipid/CD1d complexes to their TCR. Antigenic lipids are processed, loaded, and displayed in complex with CD1d by lipid antigen presenting cells (LAPCs). The mechanism of lipid antigen presentation via CD1d is highly conserved with recent work showing adipocytes are LAPCs that, besides having a role in lipid storage, can activate iNKT cells and play an important role in systemic metabolic disease. Recent studies shed light on parameters potentially dictating cytokine output and how obesity-associated metabolic disease may affect such parameters. By following a lipid antigen's journey, we identify five key areas which may dictate cytokine skew: co-stimulation, structural properties of the lipid antigen, stability of lipid antigen/CD1d complexes, intracellular and extracellular pH, and intracellular and extracellular lipid environment. Recent publications indicate that the combination of advanced omics-type approaches and machine learning may be a fruitful way to interconnect these 5 areas, with the ultimate goal to provide new insights for therapeutic exploration.     

Increased positive selection pressure within the complementarity determining regions of the T‐cell receptor β gene in New World monkeys

Because of the long‐term co‐evolution of TCR and MHC molecules, numerous nucleotide substitutions have accumulated within the domains of TCRβ genes. We previously found that nonsynonymous nucleotide substitutions occurred more frequently in complementarity determining region (CDR)β than in CDRα, even though only a limited number of common marmoset (Callithrix jacchus) and human T‐cell receptor β variable (TRBV) sequences were compared. This interesting finding raised the question of whether the increased selective pressure within CDRβ was species‐specific. In this study, we identified 21 TRBV region sequences from the common marmoset and performed comparative sequence analyses of the T‐cell receptor α variable (TRAV) and TRBV regions from human, chimpanzee, rhesus monkey, cotton‐top tamarin, Ma's night monkey, and common marmoset. The ratios of the number of nonsynonymous nucleotide substitutions per site (d_N) to the d_S values (d_N/d_S) were less than 1 within the framework regions (FRs) of TRAV and TRBV region sequences, suggesting that purifying selection is largely dominant within the FRs. In contrast, the d_N values were statistically significantly greater for CDRβ than for CDRα only in New World monkeys. Also, increased d_N/d_S ratios (d_N/d_S>1) were observed within CDRβ between humans and New World monkeys and, interestingly, between New World monkeys, which share a relatively recent common ancestor. Moreover, phylogenetic analysis by maximum likelihood analysis provided firm evidence to support that positive selection occurred within CDRβ along New World monkey lineages. These results suggest that increased positive selection pressure within CDRβ is common in New World monkeys rather than being species‐specific. This study provides an intriguing insight into the co‐evolution of TCR and MHC molecules within primates. Am. J. Primatol. 73:1082–1092, 2011. © 2011 Wiley‐Liss, Inc.     

Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.