Gamma interferon production by hepatic NK T cells during Escherichia coli infection is resistant to the inhibitory effects of oxidative stress

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-gamma) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-gamma in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-gamma-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-gamma-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1(+) CD3(-)), conventional T cells (NK1.1(-) CD3(+)), and NK T cells (NK1.1(+) CD3(+)). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-gamma production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-gamma expression by a subset of NK T cells, which did not bind alpha-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-gamma-expressing cells were also predominantly CD8(+) and bore gamma delta T-cell antigen receptors. The residual IFN-gamma response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.     

The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes

In vivo administration of NKT cell ligand, alpha-galactosylceramide (alpha-GalCer), caused the activation of NKT cells to induce a strong NK activity and cytokine production by CD1d-restricted mechanisms. Surprisingly, we also found that alpha-GalCer induced the activation of immunoregulatory cells involved in acquired immunity. Specifically, in vivo administration of alpha-GalCer resulted in the induction of the early activation marker CD69 on CD4(+) T cells, CD8(+) T cells and B cells in addition to macrophages and NKT cells. However, no significant induction of CD69 was observed on cells from CD1d- or V(alpha)14 NKT-deficient mice, indicating an essential role for the interaction between NKT cells and CD1d-expressing dendritic cells (DC) in the activation of acquired immunity in response to alpha-GalCer. Indeed, in vivo injection of alpha-GalCer resulted not only in the activation of NKT cells but also in the generation of CD69(+)CD8(+) T cells possessing both cytotoxic T lymphocyte (CTL) activity and IFN-gamma-producing ability. Tumor-specific CTL generation was also accelerated by alpha-GalCer. The critical role of CD40-CD40 ligand (CD40L)-mediated NKT-DC interaction during the development of CD69(+)CD8(+) CTL by alpha-GalCer was demonstrated by blocking experiments using anti-CD40L mAb. These findings provide direct evidence for a critical role of CD1d-restricted NKT cells and DC in bridging innate and acquired immunity.     

Characterization of early gamma interferon (IFN-gamma) expression during murine listeriosis: identification of NK1.1+ CD11c+ cells as the primary IFN-gamma-expressing cells

Though it is well established that gamma interferon (IFN-gamma) is crucial to the early innate defense of murine listeriosis, its sources remain controversial. In this study, intracellular cytokine staining of IFN-gamma-expressing splenocytes early after Listeria monocytogenes infection revealed that NK1.1(+), CD11c(+), CD8(+) T, and CD4(+) T cells expressed IFN-gamma 24 h after infection. Contrary to the previous report, most IFN-gamma(+) dendritic cells (DC) were CD8alpha(-) DC. Unexpectedly, almost all CD11c(+) IFN-gamma-expressing cells also expressed NK1.1. These NK1.1(+) CD11c(+) cells represented primary IFN-gamma-expressing cells after infection. In situ studies showed these NK1.1(+) CD11c(+) cells were recruited to the borders of infectious foci and expressed IFN-gamma. A significant NK1.1(+) CD11c(+) population was found in uninfected spleen, lymph node, blood, and bone marrow cells. And its number increased significantly in spleen, lymph node, and bone marrow after L. monocytogenes infection. Using interleukin-12 (IL-12) p40(-/-) mice, IFN-gamma expression was found to be largely IL-12 p40 dependent, and the number of IFN-gamma-expressing cells was only about one-third of that of wild-type mice. Moreover, the IFN-gamma expression was absolutely dependent on live L. monocytogenes infection, as no IFN-gamma was detected after inoculation of heat-killed L. monocytogenes. Our findings not only provide an insight into IFN-gamma expression after in vivo infection but may also change the current perceptions of DC and natural killer cells.     

CD1d-restricted NKT cells contribute to malarial splenomegaly and enhance parasite-specific antibody responses

CD1d-restricted NKT cells are a novel T cell lineage with unusual features. They co-express some NK cell receptors and recognize glycolipid antigens through an invariant T cell receptor (TCR) in the context of CD1d molecules. Upon activation through the TCR, NKT cells produce large amounts of IFN-gamma and IL-4. It has been proposed that rapid cytokine output by activated NKT cells may induce bystander activation of other lymphoid lineages. The impact of CD1d-restricted NKT cell activation in the induction of B cell-mediated immune responses to infection is still unclear. We show here that CD1-restricted NKT cells contribute to malarial splenomegaly associated with expansion of the splenic B cell pool and enhance parasite-specific antibody formation in response to Plasmodium berghei infection. The increased B cell-mediated response correlates with the ability of NKT cells to promote Th2 immune responses. Additionally, antibody responses against the glycosylphosphatidylinositol (GPI)-anchored protein merozoite surface protein 1 (MSP-1) were found to be significantly lower in CD1(-/-) mice compared to wild-type animals. P. berghei-infected MHC class II (MHCII)(-/-) mice also generated antibodies against MSP-1, suggesting that antibody production against GPI-anchored antigens in response to malaria infection can arise from both MHCII-dependent and independent pathways.     

Natural killer T-cell populations in C57BL/6 and NK1.1 congenic BALB.NK mice-a novel thymic subset defined in BALB.NK mice

Natural killer (NK) T lymphocytes are a subpopulation of T lymphocytes regarded as early regulators of immune responses. The majority of NKT cells are restricted by the CD1d molecule. NKT cells have mostly been studied in one single mouse strain, C57BL/6 (B6), because of the absence of NK1.1 in other common mouse strains, and the lack of other reliable surface markers for CD1d-restricted cells. To investigate NKT cell subsets in a mouse strain of a genetic background different from B6, we have back-crossed the NKT cell marker NK1.1 from the B6 mouse to the BALB/c mouse strain. We show that NKT cells in the congenic BALB.B6-NK1.1(b) mouse share many characteristics with their B6 counterparts, but seem to be deficient in the functional NKT cell subtype characterized by low interleukin-4 and high interferon-gamma production, and surface expression of CD49b but not CD69. Moreover, in the thymus but not the spleen of BALB.B6-NK1.1(b) mice we find a novel Valpha14-Jalpha18 invariant NKT cell subset which is devoid of a set of NK markers, suggesting that these cells represent a less differentiated NKT cell stage, and carries high levels of the T-cell receptor and uses a skewed T-cell receptor Vbeta-repertoire.     

Adoptive transfer of ex vivo immune-programmed NKT lymphocytes alleviates immune-mediated colitis

T lymphocyte-expressing natural killer (NK) cell markers (NKT cells) play a role in immune regulation. Our aim was to evaluate the in vivo effect of adoptive transfer of immune-programmed NKT cells. Colitis was induced in C57/B6 mice by 2,4,6-trinitrobenzenesulfonic acid. NKT, CD4, CD8 lymphocytes, and dendritic cells (DC) were prepared from spleens of naive mice, animals with colitis, and animals with colitis that were orally tolerized. Subsets of splenocytes, NKT, CD4, and CD8 and NKT+CD4, NKT+CD8, and NKT+DC lymphocytes were prepared. Assessment of the T helper cell type 1 (Th1)/Th2 cytokine secretion paradigm in vitro was performed before and following exposure to the antigen. Adoptive transfer of ex vivo immune-programmed lymphocytes from each group was performed into recipient mice, followed by colitis induction. Ex vivo exposure of NKT cells harvested from mice with colitis-to-colitis proteins [colitis-extracted proteins (CEP)] led to a Th2 cytokine shift. The interleukin (IL)-4/interferon-gamma (IFN-gamma) ratio increased for NKT harvested from colitis-harboring mice following exposure to CEP. Adoptive transfer of NKT lymphocytes harvested from colitis-harboring mice, which were ex vivo-educated, significantly alleviated experimental colitis in vivo. Intrahepatic NKT lymphocytes increased significantly in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP, similar to mice transplanted with NKT lymphocytes harvested from tolerized donors. Exposure of NKT cells to the disease-target antigen induced a significant increase in the IL-4/IFN-gamma cytokine ratio. Adoptive transfer of a relatively small number of immune-programmed NKT cells induced a systemic Th1 to Th2-immune shift and alleviated immune-mediated colitis.     

Differential innate immune cell activation and proinflammatory response in Anaplasma phagocytophilum infection

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The critical role of gamma interferon (IFN-gamma) for induction of severe inflammatory histopathology, even in the absence of a significant bacterial load, was previously demonstrated in a murine model of HGA. We hypothesized that NK, NKT, and possibly CD8(+) cytotoxic T cells participate in the development of histopathologic lesions with A. phagocytophilum infection. Mice were mock infected or infected with low- or high-passage A. phagocytophilum and assayed for hepatic histopathology and splenocyte immunophenotype during the first 21 days after infection. Compared to high-passage A. phagocytophilum-infected mice, low-passage A. phagocytophilum-infected mice had more severe hepatic lesions and increased apoptosis. The hepatic histopathology severity in low-passage A. phagocytophilum-infected mice peaked on day 2 at the time of peak plasma IFN-gamma levels and gradually decreased through day 21. Low-passage A. phagocytophilum-infected mice also showed significantly increased levels of lymphocyte NK1.1/FasL expression on days 4 to 7 corresponding to early, severe hepatic inflammation, whereas the levels of NKT cells were substantially lower on day 4, suggesting that there was NKT cell involvement. This result supports the concept that NK1.1(+) cells, including NK and NKT cells, are major components in the early pathogenesis of A. phagocytophilum infection.     

Characterization of the phenotype and function of CD8(+), alpha / beta(+) NKT cells from tumor-bearing mice that show a natural killer cell activity and lyse multiple tumor targets

Natural Killer (NK) T cells are a specialized T cell population that co-expresses receptors of the NK lineage with the alpha / beta TCR receptor and other T cell surface markers. Their functions, regulation and relationship to other cells in the immune system are not fully understood. This report demonstrates that tumor-bearing C57BL / 6 mice have a population of NKT cells that co-express CD8 and CD161 (NK1.1) surface markers. These cells are maintained in long-term culture with T helper 2 (Th2) cytokine interleukin-4 (IL-4), but produce large amounts of Th1 cytokine interferon-gamma (IFN-gamma) following activation. NK1.1(+)CD8(+) T cells show a potent NK-like cytotoxic activity against multiple tumor targets, and lysis is independent of major histocompatibility complex (MHC)-class I or non-classical MHC-class I molecules (Qa, TL). The NK1.1(+)CD8(+) T cells express Vbeta14 chain of the TCR. These NKT cells are not CD1d restricted, and their cytotoxic activity is CD1d independent. Therefore, they represent a unique subset of T cells with an unknown restriction element which produce large quantities of IFN-gamma following expansion with IL-4. Furthermore, their cytotoxic activity is enhanced by B7 co-stimulatory molecules present on tumor cells. CD161(+) T cells that are expanded in tumor-bearing hosts may function as a part of the innate immune system with potential role(s) in tumor surveillance.     

Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen

To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.     

Differential contribution of dendritic cell CD1d to NKT cell-enhanced humoral immunity and CD8+ T cell activation

CD1d-restricted type I NKT cells provide help for specific antibody production. B cells, which have captured and presented a T-dependent, antigen-derived peptide on MHC class II and CD1d-binding glycolipid α-GC on CD1d, respectively, activate Th and NKT cells to elicit B cell help. However, the role of the DC CD1d in humoral immunity remains unknown. We therefore constructed mixed bone marrow chimeras containing CD1d-expressing, DTR-transgenic DCs and CD1d(+) or CD1d(-) nontransgenic DCs. Following DT-mediated DC ablation and immunization, we observed that the primary and secondary antibody responses were equivalent in the presence of CD1d(+) and CD1d(-) DCs. In contrast, a total ablation of DCs delayed the primary antibody response. Further experiments revealed that depletion of CD1d(+) DCs blocked in vivo expansion of antigen-specific cytotoxic (CD8(+)) T lymphocytes. These results provide a clear demonstration that although CD1d expression on DCs is essential for NKT-enhanced CD8(+) T cell expansion, it is dispensable for specific antibody production.     

Innate immune response to encephalomyocarditis virus infection mediated by CD1d

CD1d-reactive natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and/or Th2 cytokines, can activate antigen-presenting cell (APC) interleukin-12 (IL-12) production, and are implicated in the regulation of adaptive immune responses. The role of the CD1d system was assessed during infection with encephalomyocarditis virus (EMCV-D), a picornavirus that causes acute diabetes, paralysis and myocarditis. EMCV-D resistance depends on IL-12-mediated interferon-gamma (IFN-gamma) production. CD1d-deficient mice, which also lack CD1d-reactive NKT cells, were substantially more sensitive to infection with EMCV-D. Infected CD1d knockout mice had decreased IL-12 levels in vitro and in vivo, and indeed were protected by treatment with exogenous IL-12. IFN-gamma production in CD1d knockout mice was decreased compared with that in wild-type (WT) mice in response to EMCV-D in vitro, although differences were not detected in vivo. Treatment with anti-asialo-GM1 antibody, to deplete NK cells, caused a marked increase in susceptibility of WT mice to EMCV-D infection, whereas CD1d knockout mice were little affected, suggesting that NK-cell-mediated protection is CD1d-dependent. Therefore, these data indicate that CD1d is essential for optimal responses to acute picornaviral infection. We propose that CD1d-reactive T cells respond to early immune signals and function in the innate immune response to a physiological viral infection by rapidly augmenting APC IL-12 production and activating NK cells.     

Study of the NKT cell subsets with superantigen SEB-activated tolerance

AIM: To study the NKT cell subsets and their differentiation. METHODS: Splenic lymphocytes from C57BL/J mice that had received SEB treatment were collected as effector cells on the 10(th) day. The cells were cultured in medium containing ConA, LPS and IL-2 for 3 days and measured their response to mitogens and cytokine. The inhibitory action of the effector cells was examined. The effector cells were cultured with normal lymphocytes and above mitogens or cytokine for 3 day. The cells proliferation was assessed with MTT method.The NKT cell subsets among these effector cells with the tolerance function were analyzed and their differentiation sources and correlation of functions were detected by flow cytometry. RESULTS: The response of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.80+/-0.04, 0.60+/-0.03 and 0.55+/-0.07 in control groups to 0.60+/-0.05, 0.30+/-0.05 and 0.27+/-0.04 in effector groups, respectively (P<0.01, n=3).The inhibitory ability of effectors cells against the response of normal lymphocytes to ConA, LPS and IL-2 were clearly observed. They inhibited the response of normal lymphocytes to several mitogens and cytokine. And the A values of cell proliferation were decreased to 0.26+/-0.02, 0.48+/-0.04 and 0.34+/-0.02, respectively (P<0.01, n=3). The CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRV8(+)NK1.1(+) NKT cell subsets among SEB-activated effector cells with tolerance function were significantly increased and shown that they come from T cell population. And the CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells by ConA or SEB-activated were shown coming from NK cell population. CONCLUSION: The effector cells with tolerance function activated by superantigen SEB relate to CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRVbeta8(+)NK1.1(+) NKT cell subsets. The NKT cell subsets come from T cells. The CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells differentiating from NK cells are not involved in the regulation of tolerance.     

Homeostasis of V alpha 14i NKT cells

CD1d-reactive natural killer T (NKT) cells with an invariant V alpha 14 rearrangement (V alpha 14i) are a distinct subset of T lymphocytes that likely have important immune-regulatory functions. Little is known regarding the factors responsible for their peripheral survival. Using alpha-galactosylceramide-containing CD1d tetramers to detect V alpha 14i NKT cells, we show here that the expansion of V alpha 14i NKT cells in lymphopenic mice was not dependent on CD1d expression and was unaffected by the presence of host NKT cells. Additionally, we found that IL-15 was important in the expansion and/or survival of V alpha 14i NKT cells, with IL-7 playing a lesser role. These results demonstrate that the homeostatic requirements for CD1d-restricted NKT cells, which are CD4(+) or CD4(-)CD8(-), resemble those of CD8(+) memory T cells. We propose that this expansion and/or survival in the periphery of V alpha 14i NKT cells is affected by competition for IL-15, and that IL-15-requiring cells-such as NK cells and CD8(+) memory cells-may define the V alpha 14i NKT cell niche.     

Cytokines production of U5A2-13-positive T cells by stimulation with glycolipid alpha-galactosylceramide

We have previously established and reported a novel monoclonal antibody (mAb), U5A2-13, which recognizes a phenotypically similar population of natural killer (NK)-like T cells. Using U5A2-13 mAb, we now describe the functional properties of U5A2-13(+) T cells in both NK1.1-positive or -negative mouse strains. Similar to NK1.1(+) T cells, hepatic U5A2-13(+) T cells of C57BL/6 (NK1.1(+) strain) mice, but not U5A2-13(-) T cells, could be induced to produce large amounts of IL-4 and IFN-gamma by stimulation with glycolipid alpha-galactosylceramide (alpha-GalCer) present on dendritic cells (DC) in a dose-dependent manner. The abundant production of these cytokines from U5A2-13(+) T cells of BALB/c (NK1.1(-) strain) mice is similar to that noted in C57BL/6 mice. Cytokine production by cultures stimulated with DC of beta2-microglobulin-deficient mice was significantly less than that of cultures stimulated with DC of intact mice. Overall, U5A2-13(+) T cells recognize alpha-GalCer presented by CD1d, indicating that U5A2-13(+) T cells can be used to analyze NK-like T cell function in various strains of mice.     

Modulatory effect of killed Propionibacterium acnes and its purified soluble polysaccharide on peritoneal exudate cells from C57Bl/6 mice: major NKT cell recruitment and increased cytotoxicity

Propionibacterium acnes has been described as a potent adjuvant to immune responses in vitro and in vivo. Presently, we analysed the modulation of peritoneal exudate cells (PEC) by heat-killed P. acnes or its purified soluble polysaccharide (PS), both injected intraperitoneally in C57Bl/6 mice, aiming at their recruitment and cytotoxicity. Both treatments induced an increase in macrophages, immature dendritic cells, B1a lymphocytes and NK1.1(+) CD3(+) cells. The bacterium caused a remarkable increase in a NK1.1(+) CD3(+) CD4(-) CD8(-) cells subpopulation, whereas the PS component seemed responsible for the recruitment of mainly macrophage cells. To assess P. acnes and PS adjuvant effect on PEC cytotoxicity we evaluated their in vitro effect on murine B16F10 melanoma cells. The effector cells from the heat-killed bacteria and PS-treated groups lysed melanoma cells in co-cultures with PEC. Mice genetically deficient in IFN-gamma, when stimulated with P. acnes or PS, had reduced PEC cytotoxicity, and the cytotoxic effect was completely abrogated in PEC from iNOS(-/-) mice. The tumoricidal activity of PEC from P. acnes-treated mice was mediated by macrophages and NKT cells stimulated with IL-12. In PS-treated mice the cytotoxicity was mediated mainly by macrophages. Moreover, both treatments increased IL-4 and IFN-gamma production by NKT cells. In conclusion, we show that P. acnes act mainly by recruiting and activating NKT double-negative cells in PEC, which were shown to be tumoricidal in vitro when induced by IL-12. Macrophages induced by both P. acnes and PS have their antitumour effect dependent on NO production.     

Invariant NKT cells regulate experimental autoimmune uveitis through inhibition of Th17 differentiation

Although NKT cells have been implicated in diverse immunomodulatory responses, the effector mechanisms underlying the NKT cell-mediated regulation of pathogenic T helper cells are not well understood. Here, we show that invariant NKT cells inhibited the differentiation of CD4(+) T cells into Th17 cells both in vitro and in vivo. The number of IL-17-producing CD4(+) T cells was reduced following co-culture with purified NK1.1(+) TCR(+) cells from WT, but not from CD1d(-/-) or Jα18(-/-) , mice. Co-cultured NKT cells from either cytokine-deficient (IL-4(-/-) , IL-10(-/-) , or IFN-γ(-/-) ) or WT mice efficiently inhibited Th17 differentiation. The contact-dependent mechanisms of NKT cell-mediated regulation of Th17 differentiation were confirmed using transwell co-culture experiments. On the contrary, the suppression of Th1 differentiation was dependent on IL-4 derived from the NKT cells. The in vivo regulatory capacity of NKT cells on Th17 cells was confirmed using an experimental autoimmune uveitis model induced with human IRBP(1-20) (IRBP, interphotoreceptor retinoid-binding protein) peptide. NKT cell-deficient mice (CD1d(-/-) or Jα18(-/-) ) demonstrated an increased disease severity, which was reversed by the transfer of WT or cytokine-deficient (IL-4(-/-) , IL-10(-/-) , or IFN-γ(-/-) ) NKT cells. Our results indicate that invariant NKT cells inhibited autoimmune uveitis predominantly through the cytokine-independent inhibition of Th17 differentiation.     

A role for CD1d-restricted NKT cells in injury-associated T cell suppression

Natural killer T (NKT) cells are known to modulate T cell responses during autoimmunity, tolerance, and antitumor immunity; however, their potential role in regulating the immune response to injury has not been reported. Using a murine model of burn injury, we investigated whether CD1d-restricted NKT cells played a role in the T cell suppression that occurs early after injury. A functional role for CD1d stimulation of NKT cells in the injury-related immune suppression was demonstrated by experiments in which the suppression of antigen (Ag)-specific delayed-type hypersensitivity and in vitro T cell-proliferative responses were prevented if mice were given anti-CD1d monoclonal antibody (mAb) systemically just before injury. The CD1d-NKT cell-dependent suppression of the T cell response after injury occurred in the absence of quantitative changes in NKT cells themselves or CD1d(+) Ag-presenting cells. We observed that elevated production of the immunosuppressive cytokine interleukin (IL)-4 correlated with burn-induced immune dysfunction, and we found that NKT cells but not conventional T cells were the source of IL-4 early after injury. Lastly, we observed that the injury-induced production of NKT cell-derived IL-4 could be blocked by systemic treatment of burn-injured mice with anti-CD1d mAb. Together, our results reveal a novel mechanism involving CD1d stimulation of NKT cells in the onset of T cell suppression that occurs subsequent to injury.     

Thymocyte expression of cathepsin L is essential for NKT cell development

CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor V(alpha)14J(alpha)18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of V(alpha)14(+)NK1.1(+) T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of V(alpha)14(+)NK1.1(+) T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating V(alpha)14(+)NK1.1(+) T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation.     

Expression of functional IL-2 receptors on mature splenic dendritic cells

We report here the expression of functional IL-2 receptor (IL-2R) on mature splenic dendritic cells (DC) and synergistic effect of IL-2 on IFN-gamma production by DC. IL-2 augmented IL-12-dependent IFN-gamma production by DC purified from both splenocytes of wild-type and anti-asialoGM1 Ab-treated Rag-2(-/-) splenocytes devoid of T, B, NK and NKT cells. A neutralizing mAb against IL-2Ralpha blocked such enhancing effect of IL-2 on IFN-gamma production, indicating the presence of functional IL-2R on DC. Synergistic effects of IL-2 were also observed on IFN-gamma production by DC stimulated through CD40 or MHC class II, suggesting that T cell-derived IL-2 can act on DC during antigen presentation. Furthermore, we provide evidence that DC produce IFN-gamma during interaction with allogeneic CD4(+) T cells from IFN-gamma(-/-) mice. These results suggest that IL-2 produced by naive T cells upon antigen stimulation is an important factor during Th0 to Th1 differentiation by inducing IFN-gamma from DC.     

Host resistance and immune deviation in pigeon cytochrome c T-cell receptor transgenic mice infected with Toxoplasma gondii

Resistance to Toxoplasma gondii has been shown to be mediated by gamma interferon (IFN-gamma) produced by NK, CD4(+), and CD8(+) T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4(+) T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance to T. gondii infection of pigeon cytochrome c transgenic (PCC-Tg) Rag-2(-/-) mice in which all CD4(+) T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4(+) T cells. Moreover, the CD4(+) lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, while T. gondii-induced IL-12 cannot trigger IFN-gamma production by CD4(+) T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties of T. gondii infection.