Long-term retention of mature NK1.1+ NKT cells in the thymus

The NKT cell pool in the thymus contains immature (NK1.1(-)) and mature (NK1.1(+)) subsets that represent distinct linear stages of a linear developmental pathway. An unexplained paradox is why immature NK1.1(-) NKT cells are mainly exported to the periphery instead of the more mature and more abundant NK1.1(+) NKT cells. In this study we have determined that mature NK1.1(+) NKT cells are retained by the thymus to form an extremely long-lived resident population capable of rapid and prolonged production of IFN-gamma and IL-4. The retention of mature NKT cells provides an explanation for why the periphery is mainly seeded by immature NK1.1(-) cells despite mature NK1.1(+) NKT cells being more abundant in the thymus. This is the first study to identify a mature T cell subset retained within the thymus and is additional evidence of the distinct developmental pathways of mainstream T cells and NKT cells.     

Expression of human CD1d molecules protects target cells from NK cell-mediated cytolysis

The cytotoxic activity of NK cells can be inhibited by classical and nonclassical MHC molecules. The CD1 system is formed by a family of glycoproteins that are related to classical MHC. CD1a, b, and c molecules present lipids or glycolipids to T cells and are involved in defense against microbial infections, especially mycobacteria. It has been shown recently that these molecules can inhibit target cell lysis by human NK cells. It has also been shown that mouse CD1d molecules can protect cells from NK cell-mediated cytotoxicity. In the present study, we describe how human CD1d, orthologous to murine CD1 molecules, can inhibit NK cell-mediated cytolysis. We have expressed CD1d in the HLA class I-deficient cell lines L721.221 and C1R. The inhibitory effect is observed when effector NK cells from different donors are used, as well as in different cell lines with NK activity. The inhibitory effect was reversed by incubating the target cells with a mAb specific for human CD1d. Incubation of target cells with the ligands for CD1d, alpha-galactosylceramide (alpha-GalCer), and beta-GalCer abolishes the protective effect of CD1d in our in vitro killing assays. Staining the effector cells using CD1d tetramers loaded with alpha-GalCer was negative, suggesting that the putative inhibitory receptor does not recognize CD1d molecules loaded with alpha-GalCer.     

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.     

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

Expression of CD1d molecules by human schwann cells and potential interactions with immunoregulatory invariant NK T cells

CD1d-restricted NKT cells expressing invariant TCR alpha-chains (iNKT cells) produce both proinflammatory and anti-inflammatory cytokines rapidly upon activation, and are believed to play an important role in both host defense and immunoregulation. To address the potential implications of iNKT cell responses for infectious or inflammatory diseases of the nervous system, we investigated the expression of CD1d in human peripheral nerve. We found that CD1d was expressed on the surface of Schwann cells in situ and on primary or immortalized Schwann cell lines in culture. Schwann cells activated iNKT cells in a CD1d-dependent manner in the presence of alpha-galactosylceramide. Surprisingly, the cytokine production of iNKT cells stimulated by alpha-galactosylceramide presented by CD1d+ Schwann cells showed a predominance of Th2-associated cytokines such as IL-5 and IL-13 with a marked deficiency of proinflammatory Th1 cytokines such as IFN-gamma or TNF-alpha. Our findings suggest a mechanism by which iNKT cells may restrain inflammatory responses in peripheral nerves, and raise the possibility that the expression of CD1d by Schwann cells could be relevant in the pathogenesis of infectious and inflammatory diseases of the peripheral nervous system.     

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.     

Cutting edge: activation of NK T cells by CD1d and alpha-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype.

NK T cells recognize glycolipid Ags such as alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like molecule CD1d. In this paper we have studied the in vivo effects of alpha-GalCer on the generation of adaptive immune responses. Treatment of mice with alpha-GalCer resulted in rapid activation of NK T cells and production of the cytokines IL-4 and IFN-gamma. However, after this initial stimulation, NK T cells became polarized for the production of IL-4. Further, as soon as 6 days after alpha-GalCer injection, a marked increase in serum IgE levels was observed. Administration of alpha-GalCer at the time of priming of mice with protein Ag resulted in the generation of Ag-specific Th2 cells and a profound increase in the production of IgE. Collectively, these findings indicate that alpha-GalCer may be useful for modulating immune responses toward a Th2 phenotype during prophylaxis and therapy.     

CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion

Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.     

Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.     

Interview: glycolipid antigen presentation by CD1d and the therapeutic potential of NKT cell activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d--the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.     

Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules.

In mice, the NK1.1 alloantigen is expressed on all NK cells and it initiates transmembrane signals that activate cytotoxicity. NK1.1 has been mapped to a chromosomal region that encodes two families of receptor-like molecules, the NKR-P1 family and the Ly-49 family. Both of these gene families encode type II integral membrane proteins whose extracellular domains are homologous with known C-type lectins. We have isolated and expressed a member of the NKR-P1 gene family (mNKR-P1.9) and have demonstrated both by immunofluorescence and by immunoprecipitation that this cDNA encodes NK1.1. These findings, which demonstrate the receptor-like structure of NK1.1, will facilitate studies regarding the role of NK1.1 in natural killing and regarding the identification of possible NK1.1 ligands.     

IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1- MHC class I-like/CD1-dependent CD4+ T cells.

NK T cells are an unusual subset of T lymphocytes. They express NK1. 1 Ag, are CD1 restricted, and highly skewed toward Vbeta8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-gamma immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-gamma by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-alphabeta is markedly increased. In contrast, IFN-gamma production remains essentially unchanged. In beta2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L-CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1- cells. This NK1.1-IL-4-producing subset shares three important characteristics with NK T cells: 1) Vbeta8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1- cells, and that these two subsets are biased toward IL-4 production by IL-7.     

Mutation of a positively charged cytoplasmic motif within CD1d results in multiple defects in antigen presentation to NKT cells

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.     

CD1d/NKT在抗HBV和HCV中的作用

CD1d是人类白细胞表面的抗原分子,它可以将脂质抗原递呈给天然杀伤T细胞（NKT）,使其激活.而NKT是近年来发现的一类特殊的T细胞,它既可以表达T细胞表面标志,又可以表达天然杀伤细胞（NK）表面标志,能识别由CD1d递呈的抗原,广泛参与自身免疫调节.近年来,许多研究发现,CD1d/NKT可以有效抑制肝炎病毒的复制.     

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.     

Murine CD160, Ig-like receptor on NK cells and NKT cells, recognizes classical and nonclassical MHC class I and regulates NK cell activation

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-gamma production induced by phorbol ester plus ionomycin, whereas it enhances IFN-gamma production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.     

Prevention of type 1 diabetes by invariant NKT cells is independent of peripheral CD1d expression

Invariant NKT (iNKT) cells can prevent diabetes by inhibiting the differentiation of anti-islet T cells. We recently showed that neither iNKT cell protection against diabetes nor iNKT cell inhibition of T cell differentiation in vitro requires cytokines such as IL-4, IL-10, IL-13, and TGF-beta. In contrast, cell-cell contacts were required for iNKT cell inhibition of T cell differentiation in vitro. The present study was designed to determine whether the CD1d molecule is involved in the inhibitory function of iNKT cells. Experiments were performed in vitro and in vivo, using cells lacking CD1d expression. The in vivo experiments used CD1d-deficient mice that were either reconstituted with iNKT cells or expressed a CD1d transgene exclusively in the thymus. Both mouse models had functional iNKT cells in the periphery, even though CD1d was not expressed in peripheral tissues. Surprisingly, both in vitro inhibition of T cell differentiation by iNKT cells and mouse protection against diabetes by iNKT cells were CD1d-independent. These results reveal that iNKT cells can exert critical immunoregulatory effects in the absence of CD1d recognition and that different molecular interactions are involved in iNKT cell functions.