Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

Prostaglandin D2 inhibits the production of IFN-gamma by invariant NK T cells: consequences in the control of B16 melanoma

Invariant NK T (iNKT) cells are a subset of innate/memory lymphocytes that recognize lipid Ags presented by CD1d-expressing APCs such as dendritic cells (DCs). Upon primary stimulation through their TCR, iNKT cells promptly produce large amounts of IFN-gamma and/or IL-4 that play critical roles in the regulation of innate and adaptive immune responses. To date, the role of environmental factors on iNKT cell functions has been poorly investigated. In this study, we addressed the question of whether PGD2, a potent eicosanoid lipid mediator involved in immune responses and inflammation, could be important in DC/iNKT cell cross-talk. We show that PGD2 dramatically reduced the production of IFN-gamma, but not IL-4, by iNKT cells in response to the superagonist alpha-galactosylceramide (alpha-GalCer) both in vitro and in vivo. This effect is mediated by the D prostanoid receptor 1 (DP1) expressed by DCs and iNKT cells and requires protein kinase A activation. We also report that PGD2 and BW245C (a selective DP1 agonist) reduce the protective effects of alpha-GalCer in B16F10-induced melanoma metastasis, an effect that depends on IFN-gamma production by iNKT cells. As a whole, these data reveal novel pathways regulating iNKT cell biologic functions and confirm the immunoregulatory roles of PGD2 on the innate response.     

Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents

CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

Natural killer dendritic cells have both antigen presenting and lytic function and in response to CpG produce IFN-gamma via autocrine IL-12

We have isolated rare cells bearing the NK cell surface marker NK1.1, as well as the dendritic cell (DC) marker CD11c, from the spleen, liver, lymph nodes, and thymus of normal mice. These cells possess both NK cell and DC function because they can lyse tumor cells and subsequently present Ags to naive Ag-specific T cells. Interestingly, in response to IL-4 plus either IL-2 or CpG, NKDC produce more IFN-gamma than do DC, or even NK cells. We determined that CpG, but not IL-2, induces NKDC to secrete IFN-gamma via the autocrine effects of IL-12. In vivo, CpG dramatically increases the number of NKDC. Furthermore, NKDC induce greater Ag-specific T cell activation than do DC after adoptive transfer. Their unique ability to lyse tumor cells, present Ags, and secrete inflammatory cytokines suggests that NKDC may play a crucial role in linking innate and adaptive immunity.     

Efficient activation of Valpha14 invariant NKT cells by foreign lipid antigen is associated with concurrent dendritic cell-specific self recognition

A burst release of cytokines by Valpha14 invariant NKT (iNKT) cells upon their TCR engagement critically regulates innate and adaptive immune responses. However, it remains unclear in vivo why iNKT cells respond efficiently to microbial or intracellular lipid Ags that are at low levels or that possess suboptimal antigenicity. We found that dendritic cells (DCs) potentiated iNKT cells to respond to a minimal amount of ligand alpha-galactosylceramide (alphaGalCer) through CD1d-dependent autoreactive responses that require endosomal processing and CD1d trafficking. The ability of potentiation of NKT cells was DC specific and did not depend on costimulatory signals and IL-12 production by DCs. However, DCs that failed to synthesize a major endogenous lipid Ag isoglobotrihexosylceramide were unable to potentiate NKT cells for efficient activation. Further analysis showed that differences in the level and pattern of endogenous lipid Ag presentation differentiate DCs and B cells for effective potentiation and subsequent activation of iNKT cells in the presence of an exogenous Ag. Thus, CD1d-dependent potentiation by DCs may be crucial for iNKT cell-mediated immunity against infectious agents.     

Dendritic cells mediate NK cell help for Th1 and CTL responses: two-signal requirement for the induction of NK cell helper function

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other's functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized "effector/memory" DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-gamma, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, "helper" function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.     

Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3

The steroid hormone 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is crucial in calcium homeostasis. Calcium plays a central role in T, B, and NK cell functions, and calcitriol is a known inhibitor of T cell proliferation and immunoglobulin production. We have analyzed here the immunoregulatory effects of calcitriol on NK cell function. We show that calcitriol specifically specifically inhibits, in a time- and dose-dependent fashion, the generation of cytotoxic activity from cultured CD16+ peripheral blood NK cells. It also suppresses, at similar molar concentrations (1-10 nM), interleukin 2 (IL-2) production by PHA-activated peripheral blood lymphocytes. Calcitriol does not interfere with the cytotoxic function of NK cells, whether fresh or generated in vitro, placing the inhibition at the level of NK cell activation. Interestingly enough, exogenous IL-2 can completely reverse the suppressive effect. These findings suggest that modulation of NK cell activation by control of the internal level of IL-2 may reflect an additional paracrine calcitriol-dependent circuit with immunoregulatory consequences.     

TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.     

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.     

Circulating myeloid dendritic cells of advanced cancer patients result in reduced activation and a biased cytokine profile in invariant NKT cells

CD1d-restricted invariant NKT (iNKT) cells play important regulatory roles in various immune responses, including antitumor immune responses. Previous studies have demonstrated quantitative and qualitative defects in iNKT cells of cancer patients, and these defects are clinically relevant as they are associated with poor prognosis. In this study we demonstrate that defects in the iNKT cell population can, at least in part, be attributed to defective interactions between iNKT cells and CD1d-expressing circulating myeloid dendritic cells (mDC), as mDC of patients with advanced melanoma and renal cell cancer reduced the activation and Th1 cytokine production of healthy donor-derived iNKT cells. Interestingly, this reduced activation of iNKT cells was restricted to patients with low circulating iNKT cell numbers and could be reversed by IL-12 and in part by the neutralization of TGF-beta, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-beta and IL-10 on human iNKT cells, because TGF-beta suppressed iNKT cell activation and proliferation and IFN-gamma production while IL-10 was identified as a cytokine involved in stimulating the activation and expansion of iNKT cells that could subsequently suppress NK cell and T cell responses.     

Activation of invariant NKT cells by the helminth parasite schistosoma mansoni

Mouse CD1d-restricted NKT cells, including invariant (i)NKT cells, are innate cells activated by glycolipid Ags and play important roles in the initiation and regulation of immune responses. Through their ability to promptly produce large amounts of Th1 and/or Th2 cytokines upon TCR engagement, iNKT cells exert crucial functions in the immune/inflammatory system during bacterial, protozoan, fungal, and viral infections. However, their roles during metazoan parasite infection, which are generally associated with strong Th2 responses, still remain elusive. In this study, we show that during the course of murine schistosomiasis, iNKT cells exhibit an activated phenotype and that following schistosome egg encounter in the liver, hepatic iNKT cells produce both IFN-gamma and IL-4 in vivo. We also report that schistosome egg-sensitized dendritic cells (DCs) activate, in a CD1d-dependent manner, iNKT cells to secrete IFN-gamma and IL-4 in vitro. Interestingly, transfer of egg-sensitized DCs promotes a strong Th2 response in recipient wild-type mice, but not in mice that lack iNKT cells. Engagement of TLRs in DCs is not necessary for iNKT cell stimulation in response to egg-sensitized DCs, suggesting an alternative pathway of activation. Finally, we propose that self, rather than parasite-derived, CD1d-restricted ligands are implicated in iNKT cell stimulation. Taken together, our data show for the first time that helminths can activate iNKT cells to produce immunoregulatory cytokines in vivo, enabling them to influence the adaptive immune response.     

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Dendritic cells present lytic antigens and maintain function throughout persistent gamma-herpesvirus infection

The activation and maintenance of Ag-specific CD8(+) T cells is central to the long-term control of persistent infections. These killer T cells act to continuously scan and remove reservoirs of pathogen that have eluded the acute immune response. Acutely cleared viral infections depend almost exclusively on dendritic cells (DC) to present Ags to, and to activate, the CD8(+) T cell response. Paradoxically, persistent pathogens often infect professional APCs such as DC, in addition to infecting a broad range of nonprofessional APC, raising the possibility that many cell types could present viral Ags and activate T cells. We addressed whether in persistent viral infection with murine gammaherpesviruses, DC or non-DC, such as B cells and macrophages, were required to maintain the continued activation of Ag-specific CD8(+) T cells. We found that presentation of the surrogate Ag, OVA, expressed under a lytic promoter to CD8(+) T cells during persistent infection was largely restricted to DC, with little contribution from other lymphoid resident cells, such as B cells. This is despite the fact that B cells harbor a very large reservoir of latent virus. Our results support that, during persistent viral infection, continual presentation of lytic Ags by DC leads to T cell activation critical for maintaining CD8(+) T cells capable of limiting persistent viral infection.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).     

Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids

NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.     

Role of interleukin-12 in determining differential kinetics of invariant natural killer T cells in response to differential burden of Listeria monocytogenes

Invariant (i) natural killer (NK) T cells are unique T lymphocytes expressing NKR-P1B/C (NK1.1), which recognize glycolipids, notably alpha-galactosylceramide (alpha-GalCer) presented by CD1d. The characteristic phenotype of these iNKT cells undergoes dramatic changes following Listeria monocytogenes infection, and interleukin (IL)-12 is involved in these alterations. Here we show that liver iNKT cells in mice are differentially influenced by the load of infection. Liver alpha-GalCer/CD1d tetramer-reactive (alpha-GalCer/CD1d(+)) T cells expressing NK1.1 became undetectable by day 2 following L. monocytogenes infection and concomitantly cells lacking NK1.1 increased regardless of the severity of infection. Whereas alpha-GalCer/CD1d(+)NK1.1(+) T cells remained virtually undetectable on day 4 following low-dose infection, considerable numbers of these cells were detected in high-dose-infected mice. Whereas numbers of IL-12 producers in the liver on day 4 post infection were comparable in low- and high-dose-infected mice without in vitro restimulation with heat-killed Listeria, those were more prominent in low-dose-infected mice than in high-dose-infected mice after restimulation despite the fact that higher numbers of macrophages and granulocytes infiltrated the liver in high-dose-infected mice than in low-dose-infected mice. Our results indicate that NK1.1 surface expression on iNKT cells is differentially modulated by the burden of infection, and suggest that a high bacterial load probably causes loss of IL-12 production.     

Invariant natural killer T cells from rhesus macaque spleen and peripheral blood are phenotypically and functionally distinct populations

BACKGROUND: Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo. METHODS: The frequency, phenotype and intracellular cytokine production of V alpha 24+ 6B11+ invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR. RESULTS: The frequencies of peripheral blood (PB) and spleen V alpha 24+ 6B11+ iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4+ CD8+ and CD3+ CD56+ co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB. CONCLUSION: Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.