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Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex–mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73+ B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.

Systemic lupus erythematosus (SLE) is the most common form of lupus; however, only one new drug was approved for SLE in the last 60 years. Inhibition of glycolysis via 2-deoxyglucose (2DG), a competitive inhibitor of glucose-6-phosphate, has recently shown efficacy in attenuating tumor growth and reducing autoimmune pathology; however, the mechanism of this efficacy is somewhat unclear. To elucidate how 2DG longitudinally modulates pathways within the context of systemic lupus erythematosus (SLE), BXSB.Yaa IL15−/−,CD8−/− mice, an SLE model, were treated orally with 2DG for 96-hours or 4-weeks, after which the transcriptional profiles of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) were analyzed using unsupervised clustering and weighted gene correlation network analysis. Principal component analysis showed mice clustered by tissue. Tissues correlated to B cells and T follicular helper cells grouped together according to their summary gene expression for modules treatment or treatment, in a time dependent manner, and three pathway groups, transcription, immune, and glucose, emerged. However, within each tissue cluster, pathways for each module were uniquely altered by treatment or treatment, in a time dependent manner, according to ANOVA. Furthermore, while most pathways identified were grouped into either immune or metabolic, there was little overlap of specific pathways identified between tissues. These results show that 2DG has a systemic impact altering immune and metabolic pathways, but in a tissue-specific manner. Ongoing work includes identifying genes that are potentially central to pathway alterations for follow-up analysis. Supported by the AAI Intersect Fellowship

Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4+ T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4+ T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4+ T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4+ T cells were introduced into Rag1−/− mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4+ T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4+ T cells in a nonredundant manner with myeloid/stromal cells.

Inhibition of glycolysis via 2-deoxyglucose (2DG), a competitive inhibitor of glucose-6-phosphate, has recently shown efficacy in attenuating tumor growth and reducing autoimmune pathology; however, the mechanism of this efficacy is somewhat unclear. To elucidate how 2DG longitudinally modulates pathways without the influence of immunity in diseased status, wild-type C57BL/6J mice were treated orally with 2DG for 96-hours or 4-weeks, after which the transcriptional profiles of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) were analyzed using unsupervised clustering and weighted gene correlation network analysis. Principal component analysis showed mice clustered by tissue. Tissues grouped together according to their summary gene expression for modules correlated to time, treatment and/or tissue, and two pathway groups, immune and metabolic, emerged. However, within each tissue cluster, pathways for each module were uniquely altered by time, treatment and/or tissue, according to ANOVA. Furthermore, while most pathways identified were grouped into either immune or metabolic, there was little overlap of specific pathways identified between tissues. These results show that 2DG has a systemic impact altering immune and metabolic pathways, but in a tissue-specific manner. Ongoing work includes identifying genes that are potentially central to pathway alterations for follow-up analysis.

Eight years have passed since the approval of belimumab, a monoclonal antibody directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus. erythematous (SLE). While well tolerated, the efficacy of belimumab remains limited and is not recommended for patients suffering severe nephritis, a life threatening complication of SLE. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB model is a long established model of human SLE developing elevated antinuclear antibodies and immune complex nephritis along with other manifestations of autoimmune disease. We used CRISPR-Cas9 to inactivate Activation Induced Cytidine Deaminase (Aicda, AID) directly in the BXSB background and found greatly improved survival. While mice continued to develop plasma cells, follicular structure was restored and renal pathology was reduced. While antinuclear antibody concentrations were reduced in knockout mice, renal immune complex deposition was grossly the same as wild type while complement deposition was very limited in the knockout mice. Mice develop expanded germinal center B cell populations as in other models of AID deficiency without concurrent expansion of follicular T cells. The prolonged survival in these mice appears to be attributed to the reduced renal pathology warranting further exploration as current therapeutics targeting lupus nephritis are limited and thus in great demand.

The pleiotropic cytokine IL-21 is implicated in the pathogenesis of human systemic lupus erythematosus by polymorphisms in the molecule and its receptor (IL-21R). The systemic lupus erythematosus-like autoimmune disease of BXSB.Yaa mice is critically dependent on IL-21 signaling, providing a model for understanding IL-21/IL-21R signaling in lupus pathogenesis. In this study, we generated BXSB.Yaa mice selectively deficient in IL-21R on B cells, on all T cells, or on CD8+ T cells alone and examined the effects on disease. We found that IL-21 signaling to B cells is essential for the development of all classical disease manifestations, but that IL-21 signaling also supports the expansion of central memory, CD8+ suppressor cells and broadly represses the cytokine activity of CD4+ T cells. These results indicate that IL-21 has both disease-promoting and disease-suppressive effects in the autoimmune disease of BXSB.Yaa mice.

Dysregulation of the T cell-dependent Ab response can lead to numerous immunological disorders, ranging from systemic lupus erythematosus to B cell lymphomas. Cellular processes governed by MHC class II proteins play a major role in this response and its dysregulation. The extent to which processes controlled by the diverse family of MHC class I proteins impact such autoimmune and neoplastic disorders, however, is less clear. In this study, we genetically dissect the contributions of individual MHC class I family members and the pathological processes under their control in the systemic lupus erythematosus-like disease of BXSB.Yaa mice and B cell lymphomagenesis of SJL mice. This study reveals a powerful repressive regulatory axis comprised of MHC class I-dependent CD8+ T cells and NK cells. These results indicate that the predominant role of the MHC class I protein family in such immunological disorders is to protect from more aggressive diseases.

Cytoplasmic Ags derived from viruses, cytosolic bacteria, tumors, and allografts are presented to T cells by MHC class I or class II molecules. In the case of class II-restricted Ags, professional APCs acquire them during uptake of dead class II-negative cells and present them via a process called indirect presentation. It is generally assumed that the cytosolic Ag-processing machinery, which supplies peptides for presentation by class I molecules, plays very little role in indirect presentation of class II-restricted cytoplasmic Ags. Remarkably, upon testing this assumption, we found that proteasomes, TAP, and endoplasmic reticulum-associated aminopeptidase associated with Ag processing, but not tapasin, partially destroyed or removed cytoplasmic class II-restricted Ags, such that their inhibition or deficiency led to dramatically increased Th cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags, both of which are indirectly presented. This effect was neither due to enhanced endoplasmic reticulum-associated degradation nor competition for Ag between class I and class II molecules. From these findings, a novel model emerged in which the cytosolic Ag-processing machinery regulates the quantity of cytoplasmic peptides available for presentation by class II molecules and, hence, modulates Th cell responses.

The FcγRs found on macrophages (Mϕs) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Mϕs and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8+ T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4+ T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Mϕs and DCs. The presentation of phagocytosed OVA-ICs to CD4+ T cells was considerably enhanced on wild-type versus FcRn-deficient Mϕs, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG–FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to

The role of NKT cells during immune responses is diverse, ranging from antiviral and antitumor activity to the regulation of autoimmune diseases; however, the regulatory function of CD1d-dependent NKT cells in rejection responses against allogeneic graft is uncertain. In this study, we demonstrated the direct regulatory effects of CD1d-dependent NKT cells using an allogeneic skin transplantation model. H-Y-mismatched skin graft survival was shortened in CD1d−/− recipients compared with wild-type recipients. Adoptive transfer of syngeneic NKT cells via splenocytes or hepatic mononuclear cells into CD1d−/− recipients restored graft survival times to those of wild-type recipients. α-Galactosylceramide, a specific activator of NKT cells, further prolonged graft survival. Although CD1d-dependent NKT cells did not extend skin graft survival in either major or complete minor histocompatibility-mismatched models, these cells affected graft survival in minor Ag mismatch models according to the magnitude of the antigenic difference. The afferent arm of NKT cell activation during transplantation required CD1d molecules expressed on host APCs and the migration of CD1d-dependent NKT cells into grafts. Moreover, the regulatory effects of CD1d-dependent NKT cells against alloantigen were primarily IL-10 dependent. Taken together, we concluded that CD1d-dependent NKT cells may directly affect the outcome of allogeneic skin graft through an IL-10-dependent regulatory mechanism.

IL-21 is a type I cytokine whose receptor is expressed on T, B, and NK cells. Within the B cell lineage, IL-21 regulates IgG1 production and cooperates with IL-4 for the production of multiple Ab classes in vivo. Using IL-21-transgenic mice and hydrodynamics-based gene delivery of IL-21 plasmid DNA into wild-type mice as well as in vitro studies, we demonstrate that although IL-21 induces death of resting B cells, it promotes differentiation of B cells into postswitch and plasma cells. Thus, IL-21 differentially influences B cell fate depending on the signaling context, explaining how IL-21 can be proapoptotic for B cells in vitro yet critical for Ag-specific Ig production in vivo. Moreover, we demonstrate that IL-21 unexpectedly induces expression of both Blimp-1 and Bcl-6, indicating mechanisms as to how IL-21 can serve as a complex regulator of B cell maturation and terminal differentiation. Finally, BXSB-Yaa mice, which develop a systemic lupus erythematosus-like disease, have greatly elevated IL-21, suggesting a role for IL-21 in the development of autoimmune disease.

Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2Kb-restricted epitope defining the mouse H4b minor H Ag. H4b is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4b but not the H4a epitope. Further, ex vivo CD8+ T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t1/2 compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.

The mouse H13 minor histocompatibility (H) Ag, originally detected as a barrier to allograft transplants, is remarkable in that rejection is a consequence of an extremely subtle interchange, P4Val/Ile, in a nonamer H2-Db-bound peptide. Moreover, H13 peptides lack the canonical P5Asn central anchor residue normally considered important for forming a peptide/MHC complex. To understand how these noncanonical peptide pMHC complexes form physiologically active TCR ligands, crystal structures of allelic H13 pDb complexes and a P5Asn anchored pDb analog were solved to high resolution. The structures show that the basis of TCRs to distinguish self from nonself H13 peptides is their ability to distinguish a single solvent-exposed methyl group. In addition, the structures demonstrate that there is no need for H13 peptides to derive any stabilization from interactions within the central C pocket to generate fully functional pMHC complexes. These results provide a structural explanation for a classical non-MHC-encoded H Ag, and they call into question the requirement for contact between anchor residues and the major MHC binding pockets in vaccine design.

Systemic lupus erythematosus (SLE) is an autoimmune disease mediated mainly by autoantibodies reactive to nuclear acids and proteins. These autoantibodies are mostly of IgG isotype, indicating that class-switch recombination (CSR) may play an important role in the development of SLE. CSR is initiated by the enzyme, activation-induced cytidine deaminase (AID) which induces double strand DNA breaks then repaired by homologous recombination using the RAD51 complex. We hypothesize that therapeutic targeting of CSR will retard the development of SLE by inducing cell death in AID-expressed B cells and reducing pathogenic IgG antibodies. The BXSB/MpJ mouse is a well characterized model of human SLE, exhibiting a robust germinal center response and copious production of immunoglobulins. 4,4’-diisothiocyanatostilbene-2-2’-disulfonic acid (DIDS) is a molecule which impairs homologous recombination by inhibiting the binding of RAD51 complex to DNA. In this study, male BXSB/MpJ mice were injected with DIDS or control. Treated mice showed a reduction in the germinal center B cell population and a corresponding decrease in follicular T helper cells. T1 B cell and follicular B cell populations were significantly decreased in DIDS treated mice but no changes were found in marginal zone B cells. Sera were analyzed for the immunoglobulin levels and antinuclear antibodies. These findings demonstrate that targeting CSR by DIDS reduces potentially pathogenic B lymphocyte populations and may be a viable novel therapeutic option in the treatment of SLE.

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include β2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3aa Ag by using them as targets in a cell-mediated lympholysis assay with H3aa-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.

T follicular helper cells (TFH) localize to B cell follicles and secrete Interleukin 21 (IL21), a cytokine that is vital for driving the proliferation of antigen-stimulated B cells within germinal centers and their subsequent differentiation into memory B cells and antibody-secreting plasma cells. Much of the current understanding of TFH is limited to studies following immunization of adult mice. Therefore, the ontological processes by which TFH naturally develop and express IL21 are poorly understood. Here we take advantage of a novel IL21-venus fluorescent protein reporter mouse to show that a major fraction of the earliest activated CD4 T cells that develop spontaneously in the thymi and the peripheries of young naive mice express IL21 within 2 weeks of birth. This population is rivaled in frequency only by FoxP3 natural T regulatory cells (nTREG) cells. Results from RNAseq profiling, their partial developmental dependence on IL6 and IL21 signaling and durability after adoptive transfers are all consistent with the generation of a novel, natural TFH (nTFH) population. This nTFH population is functionally defined by IL21 and is able to persist in that differentiation state in naive mice. We further show that thymic development of nTFH requires AIRE, while FoxP3 nTREG act in the periphery to restrain nTFH. We conclude that the precocious and robust presentation of nTFH in naive mice is caused by inherent self-reactivity that is developmentally driven by encounter with promiscuous self-antigens through AIRE. TREG are therefore the primary means by which nTFH are prevented from exercising their pathogenic potential in the periphery.

IL-21 is a member of the IL-2 cytokine family that exerts potent biological effects on multiple hemopoietic cell types. It is produced by activated CD4 T cells, while its receptor, IL-21R, is found on cells including NK cells, dendritic, T cells and B cells. To investigate the role of IL-21 signaling in autoimmune disease, we addressed whether this cytokine contributes to the severe systemic lupus erythematosus (SLE)-like syndrome developed by BXSB-Yaa mice. We show that BXSB-Yaa CD4 T cells produce IL-21 but lack discriminative T follicular helper cell markers. We also show that BXSB-Yaa mice completely lacking the IL-21R or Igh6 are remarkably resistant to multiple aspects of the prototypic BXSB-Yaa autoimmune disease. Finally, we show that mixed bone marrow chimeric BXSB-Yaa mice lacking IL-21R specifically in the B-cell compartment are similarly resistant. These results suggest that IL-21 produced by a novel population of BXSB-Yaa CD4 T-cells acts through B-cells to evoke multiple features characteristic of severe SLE. The results indicate that IL-21 signaling through B-cells is an essential step in the pathogenesis of this SLE-like autoimmune disease and potentially others. Funded by an Arthritis Foundation grant to JB.

Mouse models of SLE are considered pivotal in understanding the genetics of SLE along with identifying targets for therapy. Male mice of the BXSB/MpJ recombinant inbred strain, first described by Murphy and Roths in 1978, develop a spontaneous and severe SLE-like syndrome resulting in death averaging ~6 months of age, while females show only a very chronic syndrome. A mutant locus found on the Y chromosome is, therefore, appropriately named the Y-linked autoimmune accelerator (Yaa). Previous analysis have suggested that Yaa fails to induce an SLE-like disease in B6 mice, the caveat to this being that the SLE criteria analyzed were limited to serological analysis, histopathology, and mortality on mice up to 8–12 months of age. We report here that when mice of a significantly older age, 15–24 months, are compared to age-matched B6 controls, the B6.SB-Yaa/J mice clearly developed an SLE-like disease. This is characterized by significant splenomegaly, monocytosis, immune cells with an activation phenotype, the presence of homogeneous anti-nuclear antibody staining, glomerulonephritis, decreased weight and survival as well as a gene expression signature similar to BXSB/MpJ mice. These results suggest that Yaa alone is sufficient to induce an SLE-like syndrome in aged B6 mice. Funded by NIH R21DK074463 and DK56597 to DCR, Arthritis Foundation to JAB.

IgG antibodies modified for high binding affinity for their receptor FcRn have emerged as a major treatment for a wide array of both neoplastic and autoimmune diseases. The goal of this study was to analyze the effect of human IgG1 antibodies with enhanced in vitro binding to FcRn on their in vivo half-life in mice deficient in mouse FcRn and expressing human FcRn. Two mutants (N434A and T307A/E380A/N434A) of the humanized monoclonal antibody Hu4D5, directed against epidermal growth factor receptor 2 (p185 HER2), show increased pH-dependent binding to human FcRn in vitro. They also have an extended half-life in vivo compared to wild-type antibody in FcRn transgenic mice but not in mice expressing endogenous mouse FcRn. When injected into FcRn-humanized mice at a concentration sufficient to partially saturate human FcRn, the mutants with an extended serum half-life were most effective in reducing the half-life of a tracer antibody. Finally, we use a serum-transfer model of arthritis and FcRn-humanized mice to test whether mutant antibodies could have enhanced therapeutic potential by promoting accelerated clearance of arthritogenic antibodies. Results indicate that the engineered mutant T307A/E380A/N434A is most effective in vivo in ameliorating the inflammatory arthritic lesions. In summary, FcRn-humanized mice are a promising animal model for human IgG therapeutic development.

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.