Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.     

Donor bone marrow type II (non-Valpha14Jalpha18 CD1d-restricted) NKT cells suppress graft-versus-host disease by producing IFN-gamma and IL-4

NKT cells in donor bone marrow (BM) have been demonstrated to protect against graft-vs-host disease (GVHD) following BM transplantation. Murine NKT cells are divided into two distinct subsets based on the invariant Valpha14Jalpha18 TCR expression. However, details of the subset and mechanisms of the BM NKT cells involved in suppressing GVHD have not been clarified. Irradiated BALB/c or C3H/HeN mice administered B6 or Jalpha18(-/-) BM cells show attenuation of GVHD, whereas recipients given CD1d(-/-) BM cells did not show attenuation. Moreover, coinjection of BM non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cells and CD1d(-/-) BM cells suppressed GVHD, whereas coinjection of BM Valpha14Jalpha18 TCR (type I) NKT cells did not. These protective effects on GVHD depended upon IFN-gamma-producing type II NKT cells, which induced the apoptosis of donor T cells. The splenocytes of mice administered BM cells from B6.IL-4(-/-) or Jalpha18(-/-)IL-4(-/-) mice produced lower levels of IL-4 and IL-10 than the splenocytes of mice transplanted with BM cells from B6, B6.IFN-gamma(-/-), Jalpha18(-/-), or Jalpha18(-/-)IFN-gamma(-/-) mice. Taken together, our results show that IFN-gamma-producing BM type II NKT cells suppress GVHD by inducing the apoptosis of donor T cells, while IL-4-producing BM type II NKT cells protect against GVHD by deviating the immune system toward a Th2-type response.     

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Valpha14Jalpha18 TCR alpha-chain specific for glycolipid Ags presented by CD1d. Murine Valpha14Jalpha18(+) account for 30-50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Valpha24Vbeta11(+) NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3(+) cells) and blood (0.02%). In contrast to those in blood, most hepatic Valpha24(+) NKT cells express the Vbeta11 chain. They include CD4(+), CD8(+), and CD4(-)CD8(-) cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Valpha24(+) T cells are potent producers of IFN-gamma and TNF-alpha, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, alpha-galactosylceramide. Valpha24(+)Vbeta11(+) cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-gamma in response to alpha-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.     

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.     

DOCK2 is required in T cell precursors for development of Valpha14 NK T cells

Mouse CD1d-restricted Valpha14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in Valpha14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of Valpha14 NKT cells in the thymus, liver, and spleen. When alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of Valpha14 NKT cells. Supporting this idea, staining with CD1d/alpha-GalCer tetramers revealed that CD44- NK1.1- Valpha14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of Valpha14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of Valpha14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors

CD1d-restricted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel Valpha24-negative and 13 canonical Valpha24-positive/Vbeta11-positive human NKT cell clones generated using alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramers. The Valpha24-negative clones expressed Vbeta11 paired with Valpha10, Valpha2, or Valpha3. Strikingly, their Valpha-chains had highly conserved rearrangements to Jalpha18, resulting in CDR3alpha loop sequences that are nearly identical to those of canonical TCRs. Valpha24-positive and Valpha24-negative clones responded similarly to alpha-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3alpha loop is sufficient for recognition of alpha-GalCer despite CDR1alpha and CDR2alpha sequence variation. Unlike Valpha24-positive clones, the Valpha24-negative clones responded poorly to a glucose-linked glycolipid (alpha-glucosylceramide), which correlated with their lack of a conserved CDR1alpha amino acid motif, suggesting that fine specificity for alpha-linked glycosphingolipids is influenced by Valpha-encoded TCR regions. Valpha24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of Jalpha18-positive TCRs that can recognize alpha-GalCer. One alpha-GalCer-reactive, Valpha24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR beta-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR alpha-chain elements contribute to alpha-linked glycosphingolipid specificity, whereas TCR beta-chains can confer heterogeneous additional reactivities.     

Ordered and coordinated rearrangement of the TCR alpha locus: role of secondary rearrangement in thymic selection

The Ag receptor of the T lymphocyte is composed of an alphabeta heterodimer. Both alpha- and beta-chains are products of the somatic rearrangement of V(D)J segments encoded on the respective loci. During T cell development, beta-chain rearrangement precedes alpha-chain rearrangement. The mechanism of allelic exclusion ensures the expression of a single beta-chain in each T cell, whereas a large number of T cells express two functional alpha-chains. Here we demonstrate evidence that TCR alpha rearrangement is initiated by rearranging a 3' Valpha segment and a 5' Jalpha segment on both chromosomes. Rearrangement then proceeds by using upstream Valpha and downstream Jalpha segments until it is terminated by successful positive selection. This ordered and coordinated rearrangement allows a single thymocyte to sequentially express multiple TCRs with different specificities to optimize the efficiency of positive selection. Thus, the lack of allelic exclusion and TCR alpha secondary rearrangement play a key role in the formation of a functional T cell repertoire.     

Invariant V alpha 14+ NKT cells participate in the early response to enteric Listeria monocytogenes infection

Invariant Valpha14(+) NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Valpha14(+) NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid alpha-galactosylceramide (CD1d/alphaGC), we found that splenic and hepatic Valpha14(+) NKT cells in C57BL/6 mice were early producers of IFN-gamma (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Valpha14(+) NKT cells derived from TCRalpha degrees Valpha14-Jalpha18 transgenic (TCRalpha degrees Valpha14Tg) mice into alymphoid Rag(null) gamma(c)(null) mice demonstrated that Valpha14(+) NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-gamma and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jalpha18null mice, but not TCRalpha(null) or TCRalpha(null) Valpha14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Valpha14(+) NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-gamma production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.     

Valpha24-JalphaQ-independent,CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.     

A novel mouse model for invariant NKT cell study

We have generated a novel mouse model harboring the in-frame rearranged TCRValpha specific for invariant NKT (iNKT) cells (Valpha14-Jalpha18) on one allele by crossing the mouse cloned from NKT cells with wild-type mice. This genomic configuration would ensure further rearrangement and expression of TCRValpha14-Jalpha18 under the endogenous promoters and enhancers. Mice harboring such an in-frame rearranged TCRValpha (Valpha14-Jalpha18 mouse) possessed an increase in iNKT cells in the thymus, liver, spleen, and bone marrow. Intriguingly, both Th1- and Th2-type cytokines were produced upon stimulation with alphaGalactosylceramide, an agonist of iNKT cells, and the IgE level in the serum remained unaffected in the Valpha14-Jalpha18 mouse. These features markedly distinguish the nature of iNKT cells present in the Valpha14-Jalpha18 mouse from that of iNKT cells found in the Valpha14-Jalpha18 transgenic mouse. Besides these, the expression of TCRVgammadelta cells remained intact, and the use of the TCRVbeta repertoire in iNKT cells was highly biased to TCRVbeta8 in the Valpha14-Jalpha18 mouse. Furthermore, alphaGalactosylceramide-CD1d dimer-reactive immature iNKT cells expressed less Rag2 as compared with the conventional immature T cells at the positive selection stage. Cell cycle analysis on the thymocytes revealed that no particular subset proliferated more vigorously than the others. Crossing the Valpha14-Jalpha18 mouse with the CD1d knockout mouse revealed a novel population of iNKT cells whose coreceptor expression profile was similar to that assigned to iNKT precursor cells. These mice will be useful for the study on the development of iNKT cells as well as on their functions in the immune system.     

TCR beta chain influences but does not solely control autoreactivity of V alpha 14J281T cells.

CD1d-dependent accumulation of alphabeta T cells bearing a canonical Valpha14Jalpha281 alpha-chain (Valpha14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Valpha14+ T cells do not exhibit significant autoreactivity despite high reactivity to alpha-galactosylceramide presented by CD1d (alpha-GalCer/CD1d). To assess the role of TCRbeta chain in determining the alpha-GalCer/CD1d vs autoreactive specificity of Valpha14+ T cells, we conducted TCRalpha or TCRbeta chain transduction experiments. In this study we demonstrate, by combining different TCRbeta chains with the Valpha14 alpha-chain in retrovirally transduced T cell lines, that the Valpha14 alpha-chain plays a primary role, necessary but not sufficient for imparting alpha-GalCer/CD1d recognition. beta-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Valpha14+ T cells, since transduction of TCRalphabeta chains from a high CD1d autoreactive Valpha14+ T cell line conferred the alpha-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Valpha14+ T cell reactivity is due to both beta-chain diversity and control mechanism(s) beyond primary TCR structure.     

NKT cells play a limited role in the neutrophilic inflammatory responses and host defense to pulmonary infection with Pseudomonas aeruginosa

CD1d-restricted NKT cells are reported to play a critical role in the host defense to pulmonary infection with Pseudomonas aeruginosa. However, the contribution of a major subset expressing a Valpha14-Jalpha18 gene segment remains unclear. In the present study, we re-evaluated the role of NKT cells in the neutrophilic inflammatory responses and host defense to this infection using mice genetically lacking Jalpha18 or CD1d (Jalpha18KO or CD1dKO mice). These mice cleared the bacteria in lungs at a comparable level to wild-type (WT) mice. There was no significant difference in the local neutrophilic responses, as shown by neutrophil counts and synthesis of MIP-2 and TNF-alpha, in either KO mice from those in WT mice. Administration of alpha-galactosylceramide, a specific activator of Valpha14+ NKT cells, failed to promote the bacterial clearance and neutrophilic responses, although the same treatment increased the synthesis of IFN-gamma, suggesting the involvement of this cytokine downstream of NKT cells. In agreement against this notion, these responses were not further enhanced by administration of recombinant IFN-gamma in the infected Jalpha18KO mice. Our data indicate that NKT cells play a limited role in the development of neutrophilic inflammatory responses and host defense to pulmonary infection with P. aeruginosa.     

Prevention of diabetes in nonobese diabetic mice mediated by CD1d-restricted nonclassical NKT cells

A role for regulatory lymphocytes has been demonstrated in the pathogenesis of type 1 diabetes in the NOD mouse but the nature of these cells is debated. CD1d-restricted NKT lymphocytes have been implicated in this process. Previous reports of reduced diabetes incidence in NOD mice in which the numbers of NKT cells are artificially increased have been attributed to the enhanced production of IL-4 by these cells and a role for classical NKT cells, using the Valpha14-Jalpha18 rearrangement. We now show that overexpression in NOD mice of CD1d-restricted TCR Valpha3.2(+)Vbeta9(+) NKT cells producing high levels of IFN-gamma but low amounts of IL-4 leads to prevention of type 1 diabetes, demonstrating a role for nonclassical CD1d-restricted NKT cells in the regulation of autoimmune diabetes.     

Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jalpha18(-/-) mice, which are exclusively deficient in the invariant Valpha14(+) (iValpha14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iValpha14 NKT cells fully reconstitutes the capacity of Jalpha18(-/-) mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69(+)) iValpha14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iValpha14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iValpha14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.     

Role of interferon-gamma in Valpha14+ natural killer T cell-mediated host defense against Streptococcus pneumoniae infection in murine lungs

Previously, we demonstrated that Valpha14+ NKT cells and IFN-gamma are important upstream components in neutrophil-mediated host defense against infection with Streptococcus pneumoniae. In the present study, we extended these findings by elucidating the role of IFN-gamma in this Valpha14+ NKT cell-promoted process. Administration of recombinant IFN-gamma to Jalpha18KO mice prolonged the shortened survival, promoted the attenuated clearance of bacteria and improved the reduced accumulation of neutrophils and synthesis of MIP-2 and TNF-alpha in the lungs, in comparison to wild-type (WT) mice. In addition, intravenous transfer of liver mononuclear cells (LMNC) from WT mice into Jalpha18KO mice resulted in complete recovery of the depleted responses listed above, whereas such effects were not detected when LMNC were obtained from IFN-gammaKO or Jalpha18KO mice. Activation of Valpha14+ NKT cells by alpha-galactosylceramide (alpha-GalCer) significantly enhanced the clearance of bacteria, accumulation of neutrophils and synthesis of MIP-2 and TNF-alpha in the infected lungs; this effect was significantly inhibited by a neutralizing anti-IFN-gamma antibody. Finally, in a flow cytometric analysis, TNF-alpha synthesis was detected largely by CD11b(bright+) cells in the infected lungs. Our results demonstrated that IFN-gamma plays an important role in the neutrophil-mediated host protective responses against pneumococcal infection promoted by Valpha14+ NKT cells.     

Restoration of NK T cell development in fyn-mutant mice by a TCR reveals a requirement for Fyn during early NK T cell ontogeny

NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1(-) NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn(-/-) mice exhibit a similar turnover rate as wild-type cells. The fyn(-/-) NK T cells respond to alpha-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR alpha-chain Valpha14Jalpha18 leads to a complete restoration of NK T cell numbers in fyn(-/-) mice. Together, these results suggest that Fyn may have a role before alpha-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the Valpha14Jalpha18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn(-/-) lymphoid populations to respond.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.     

Recycling CD1d1 molecules present endogenous antigens processed in an endocytic compartment to NKT cells

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Valpha14Jalpha281(+) NKT cells without affecting recognition by Valpha14(-) NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Valpha14(+) (but not Valpha14(-)) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.