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.     

Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells.

alpha-Galactosylceramide (alpha-GalCer) is a glycolipid with potent antitumor properties that binds to CD1d molecules and activates mouse Valpha14 and human Valpha24 NKT cells. Surprisingly, we found that, as early as 90 min after alpha-GalCer injection in vivo, NK cells also displayed considerable signs of activation, including IFN-gamma production and CD69 induction. NK activation was not observed in RAG- or CD1-deficient mice, and it was decreased by pretreatment with anti-IFN-gamma Abs, suggesting that, despite its rapid induction, it was a secondary event that depended on IFN-gamma release by NKT cells. At later time points, B cells and CD8 T cells also began to express CD69. These findings identify a high-speed communication network between the innate and adaptive immune systems in vivo that is initiated upon NKT cell activation. They also suggest that the antitumor effects of alpha-GalCer result from the sequential recruitment of distinct innate and adaptive effector lymphocytes.     

Cutting edge: CD1a+ antigen-presenting cells in human dermis respond rapidly to CCR7 ligands

Recent data from murine models have confirmed that Langerhans cells are not the only population of APCs in the skin involved in initiating immune responses. In healthy human skin, we identify CD1a(+) dermal APCs located close to the lymphatic vessels in the upper layers of the dermis that are unequivocally distinct from migrating Langerhans cells but exhibit both potent allostimulatory capacity and a chemotactic response to CCR7 ligands. In contrast, CD14(+) dermal APCs are distributed throughout the dermis and lack a chemotactic response to CCR7 ligands. CD1a(+) dermal APCs therefore represent an APC population distinct from Langerhans cells that are capable of migrating to lymph nodes and stimulating naive T cells. In humans, CD1a(+) dermal APCs may fulfill some of the roles previously ascribed to Langerhans cells.     

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.     

Cutting edge: regulation of uterine NKT cells by a fetal class I molecule other than CD1

The peri-implantation uterus contains an expanded population of NK1.1(+) V alpha 14(+) TCR(int) (NKT) lymphocytes. Although these cells bear the above features in common with other NKT cells populations in thymus, bone marrow, liver, and spleen, they differ from these other populations in terms of an altered V beta repertoire and absence of a CD4(+) component. In this study, we demonstrate that the uterine population also differs from other NKT cell populations because they recognize a class I/class I-like molecule other than CD1, whereas most previously described V alpha 14(+) NKT cells are CD1-restricted. Moreover, the class I/class I-like molecule leading to the uterine NKT cell expansion may be supplied by the fetus. These data demonstrate a novel mechanism whereby the fetus is capable of modulating the maternal immune system.     

Cutting edge: CD1d deficiency impairs murine host defense against the spirochete, Borrelia burgdorferi

CD1 molecules can present microbial lipid Ag to T cells, suggesting that they participate in host defense against pathogens. In this study, we examined the role of CD1d in resistance to infection with the Lyme disease spirochete, Borrelia burgdorferi (Bb), an organism with proinflammatory lipid Ag. Bb infection of CD1d-deficient (CD1d(-/-)) mouse strains normally resistant to this pathogen resulted in arthritis. Pathology correlated with an increased prevalence of spirochete DNA in tissues and enhanced production of Bb-specific IgG, including IgG to Ag rapidly down-modulated on spirochetes in vivo. CD1d(-/-) mice exhibited high-titer Bb-specific IgG2a, an isotype commonly induced in disease-susceptible mice but not in the disease-resistant control mice in this study. These results show that CD1d deficiency impairs host resistance to a spirochete pathogen, and are the first example of a mutation that imparts Bb-resistant mice with the Ab and disease profile of a susceptible mouse strain.     

Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals

Invariant NK T (iNKT) cells influence the response to viral infections, although the mechanisms are poorly defined. In this study we show that these innate-like lymphocytes secrete IFN-gamma upon culture with CpG oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but it does not require CD1d expression. iNKT cells also produce IFN-gamma in response to mouse CMV infection. Their mechanism of mouse CMV detection is quite similar to that of CpG, requiring both TLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently, iNKT cells have the ability to respond to a variety of microbes, including viruses, in an Ag-independent manner, suggesting they may play a broad role in antipathogen defenses despite their limited TCR repertoire.     

A cell-type specific CD1d expression program modulates invariant NKT cell development and function

Invariant NK T (iNKT) cells are a distinct subset of T cells that rapidly produce an array of immunoregulatory cytokines upon activation. Cytokines produced by iNKT cells subsequently transactivate other leukocytes and elicit their respective effector functions. In this way, iNKT cells play a central role in coordinating the development of immune responses in a variety of settings. However, the mechanisms governing the quality of the iNKT cell response elicited remain poorly defined. To address whether changes in the CD1d expression pattern could regulate iNKT cell function, we generated a transgenic (Tg) mouse model in which thymocytes and peripheral T cells express high levels of CD1d (Lck-CD1d Tg+ mice). The expression of CD1d by T cells was sufficient to rescue development of iNKT cells in mice deficient of endogenous CD1d. However, the relative proportions of iNKT cell subsets in Lck-CD1d Tg+ mice were distinctly different from those in wild-type mice, suggesting an altered developmental program. Additionally, iNKT cells were hyporesponsive to antigenic stimulation in vivo. Interestingly, Lck-CD1d Tg+ mice develop liver pathology in the absence of any exogenous manipulation. The results of these studies suggest that changes to the CD1d expression program modulate iNKT cell development and function.     

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.     

Cutting edge: Programmed death-1/programmed death ligand 1 interaction regulates the induction and maintenance of invariant NKT cell anergy

Invariant NKT (iNKT) cells are a distinct subset of T lymphocytes that recognize glycolipid Ags. Upon TCR stimulation, iNKT cells promptly secrete a wide range of cytokines and therefore have been investigated as a target for immunotherapy. However, after primary activation, iNKT cells become hyporesponsive toward their ligand (anergy). The further mechanism behind iNKT cell anergy is poorly understood. We found that a low level of programmed death-1 (PD-1) was constitutively expressed on iNKT cells and that PD-1 expression was increased after stimulation and lasted at least 2 mo. Moreover, not only did blocking of the PD-1/PD ligand 1 (PD-L1) pathway prevent the induction of anergy in iNKT cells, but anergic iNKT cells also recovered responsiveness and these "rescued" cells efficiently mediated antitumor immunity. Our findings suggest that the PD-1/PD-L1 interaction is essential for the induction and maintenance of iNKT cell anergy.     

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.     

Cutting edge: contribution of NK cells to the homing of thymic CD4+NKT cells to the liver

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.     

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.     

Evaluation of the function of human invariant NKT cells from cancer patients using alpha-galactosylceramide-loaded murine dendritic cells

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-gamma, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-gamma. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated alpha-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.     

Cutting edge: a naturally occurring mutation in CD1e impairs lipid antigen presentation

The human CD1a-d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM(6). CD1e is formed by the association of beta(2)-microglobulin with an alpha-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM(6) presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.     

Cutting edge: expression of chemokine receptor CXCR1 on human effector CD8+ T cells

IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2, thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate. Presently, we showed that CXCR1(+) cells were predominantly found among CD8(+) T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8(+) T cells. Indeed, human CMV-specific CD8(+) T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8(+) T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor. The results of a chemotaxis assay showed that the migration of CXCR1(+)CD8(+) T cells was induced by IL-8. These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8(+) T cells.     

Liver invariant NKT cells and listeriosis

The invariant (i) NKT cells represent unique T lymphocytes expressing TCRValpha14. Although iNKT cells have been regarded as T lymphocytes expressing NK1.1, they do not consistently express this marker. NK1.1 allows recognition of "missing-self" and thus controls inhibition/activation of iNKT cells. It is thus tempting to assume that iNKT cells participate in the regulation of host immune responses during microbial infection by controlling NK1.1 expression. These findings shed light on the unique role of iNKT cells in microbial infection and provide an evidence for unique aspects of the NK1.1 on these cells as a regulatory molecule.     

Human cytomegalovirus (HCMV) US2 protein interacts with human CD1d (hCD1d) and down-regulates invariant NKT (iNKT) cell activity

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-membrane glycoprotein US2, which interferes with antigen presenting mechanism of major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and α3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.     

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.     

Cutting edge: murine cytomegalovirus induces a polyfunctional CD4 T cell response

CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-A(b)-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-gamma, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-gamma production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen.