Abundance of NKT cells in the salivary glands but absence thereof in the liver and thymus of aly/aly mice with Sjögren syndrome

It is known that ALY/Nsc Jcl-aly/aly (aly/aly) mice that congenitally lack lymph nodes fall victim to Sj?gren syndrome as a function of age. We investigated how TCRint cells of extrathymic origin and TCRhigh cells of thymic origin are distributed in various organs of these mice. Although the distribution of T-cell subsets was not different between control aly/+ and aly/aly mice in youth in any of the tested organs, the proportion of TCRint cells in the liver and spleen of aly/aly mice increased with aging. Usually, TCRint cells in the liver comprise a half-and-half mixture of a NK1. 1(+) subset (i.e., NKT cells) and a NK1.1(-) subset. In constrast, almost all expanding TCRint cells in various immune organs of aly/aly mice were found to be NK1.1(-). A large proportion of lymphocytes, including NK cells and TCRint cells, were also present in the salivary glands of aly/aly mice. Interestingly, these TCRint cells in the salivary glands contained an NK1.1(+) subset (i.e., NKT cells) that used an invariant chain of Valpha14Jalpha281 for TCRalphabeta (>50%). Moreover, gammadeltaT cells that used Vgamma 1, 2, 4/Vdelta 1, 4, 6 mRNAs, different from those of gammadeltaT cells in the liver and intestine, were abundant. Possibly reflecting the in situ generation of these T cells in the salivary glands, the expression of RAG-2 mRNA was evident by the RT-RCR method. These results suggest that (i) inflammatory lymphocytes that evoke Sj?gren syndrome in aly/aly mice are NK cells or TCRint cells (both NK1.1(+) and NK1.1(-) subsets) and (ii) TCRint cells in the salivary glands might be generated in situ.     

Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10-100 ng/mouse) of alpha-galactosyl ceramide (alphaGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following alphaGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to alphaGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-gamma response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG(-/-) or HBs-tg/RAG(-/-)) hosts via the portal vein elicited IFN-gamma responses of liver NK cells in situ. IFN-beta down-regulates the pathogenic IL-12/IFN-gamma cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-beta suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (alphaGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-gamma response of the specifically activated liver NKT cells. In vivo, IFN-beta attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Genetic background influences natural killer cell activation during bacterial peritonitis in mice, and is interleukin 12 and interleukin 18 independent

Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.     

CD1d-restricted NKT cells: an interstrain comparison

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.     

Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice

The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the alpha-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.     

RCAI-8, 9, 18, 19, and 49-52, conformationally restricted analogues of KRN7000 with an azetidine or a pyrrolidine ring: Their synthesis and bioactivity for mouse natural killer T cells to produce cytokines

Conformationally restricted analogues of KRN7000, an alpha-d-galactosyl ceramide, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells to produce cytokines. RCAI-8, 9, 51, and 52 are the analogues with a pyrrolidine ring, and RCAI-18, 19, 49, and 50 are those with an azetidine ring. RCAI-18 was shown to be a potent inducer of cytokine production by mouse NKT cells, while RCAI-51 was a moderately active inducer.     

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.     

RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage: their synthesis and bioactivity for mouse natural killer T cells to produce Th2-biased cytokines

RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage instead of an amide bond, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells. RCAI-17, 22, 24-26, 29, 31, 34-36, and 88 are the aromatic sulfonamide analogs, while RCAI-39 and 40 are the aliphatic ones. RCAI-38 is a C-galactoside analog of RCAI-26, which is the p-toluenesulfonamide analog of KRN7000. According to their bioassay, these sulfonamide analogs were shown to be the stimulants of mouse NKT cells to induce the production of Th2-biased cytokines in vitro, while RCAI-38 did not induce any cytokine production.     

Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex

The NK1.1 cell surface receptor, which belongs to the NKR-P1 gene cluster, has been bred onto nonobese diabetic (NOD) mice for two purposes. The first was to tag NK and NKT cells for easier experimental identification of those subsets and better analysis of their implication in type 1 diabetes. The second was to produce a congenic strain carrying Idd6, a susceptibility locus that has been repeatedly mapped in the vicinity of the NKR-P1 gene cluster and the NK complex, to explore the impact of this locus upon autoimmune diabetes. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice manifest reduced disease incidence and improved NK and NKT cell performance, as compared with wild-type NOD mice. The association of those two features in the same congenic strain constitutes a strong argument in favor of Idd6 being associated to the NK complex. This could explain at the same time the multiple alterations of innate immunity reported in NOD mice and the fact that disease onset can be readily modified by boosting the innate immune system of the mouse.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

IL-4 induces in vivo production of IFN-gamma by NK and NKT cells

Although IL-4 and IFN-gamma often have opposite effects and suppress each other's production by T cells, IL-4 can stimulate IFN-gamma production. To characterize this, we injected mice with IL-4 and quantified IFN-gamma production with the in vivo cytokine capture assay. IL-4 induced Stat6-dependent IFN-gamma production by NK and, to a lesser extent, NKT cells, but not conventional T cells, in 2-4 h. Increased IFN-gamma production persisted at a constant rate for >24 h, but eventually declined, even with continuing IL-4 stimulation. This eventual decline in IFN-gamma production was accompanied by a decrease in NK and T cell numbers. Consistent with a dominant role for NK cells in IL-4-stimulated IFN-gamma secretion, IL-4 induction of IFN-gamma was B and T cell-independent; suppressed by an anti-IL-2Rbeta mAb that eliminates most NK and NKT cells; reduced in Stat4-deficient mice, which have decreased numbers of NK cells; and absent in Rag2/gamma(c)-double-deficient mice, which lack T, B, and NK cells. IL-4-induced IFN-gamma production was not affected by neutralizing IL-12p40 and was increased by neutralizing IL-2. IL-13, which signals through the type 2 IL-4R and mimics many IL-4 effects, failed to stimulate IFN-gamma production and, in most experiments, suppressed basal IFN-gamma production. Thus, IL-4, acting through the type 1 IL-4R, induces Stat6-dependent IFN-gamma secretion by NK and NKT cells. This explains how IL-4 can contribute to Th1 cytokine-associated immune effector functions and suggests how IL-13 can have stronger proallergic effects than IL-4.     

Role of SLAM in NKT cell development revealed by transgenic complementation in NOD mice

Allelic variation of SLAM expression on CD4(+)CD8(+) thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4(+)CD8(+) thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4(+)CD8(+) thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4(+)CD8(+) thymocytes and produce larger numbers of NKT cells. Transgenic enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradoxically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells.     

Identification of IFN-gamma-producing cells in IL-12/IL-18-treated mice

Both IL-12 and IL-18 have been characterized as effective IFN-gamma-inducing cytokines. Concomitant treatment with IL-12 and IL-18 has been shown to synergistically induce IFN-gamma and may be an effective therapy for treating cancer, allergy, and infectious diseases. To understand the mechanisms underlying the strong induction of IFN-gamma by IL-12/IL-18 in mice, we focused our studies on the IFN-gamma-producing cells in various lymphoid organs and tissues and utilized the intracellular cytokine staining method to detect such cells in situ. After combined treatment with IL-12 and IL-18, IFN-gamma-positive cells in C57BL/6 mice were detected in the liver (12.18%), spleen (0.68%), bone marrow (1.80%), and peritoneum (2.12%), but not in the thymus or lymph nodes (<0.05 and <0.08%, respectively). A two-color staining method revealed that the majority of IFN-gamma-producing cells in the liver were NK1.1(+) cells, while those in the spleen were mostly CD3(+) cells, and to a lesser degree NK1.1(+) cells. Both CD4(+) and CD8(+) cells in the liver and in the spleen produced IFN-gamma. The CD19(+) B cell population was not definitely shown to produce IFN-gamma in our induction experiments. NKT cells, which are a subpopulation of NK1. 1(+) CD3(+) cells, were diminished in the liver and did not seem to contribute to IFN-gamma production arising from IL-12/IL-18 treatment. Further in vitro experiments confirmed the responsiveness of hepatic mononuclear cells to IL-12/IL-18 stimulation. This study is the first to show the IFN-gamma-producing mechanisms of IL-12/IL-18 treatment at the phenotypic level.     

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.     

The Th1/Th2 balance does not account for the difference of susceptibility of mouse strains to Theiler's virus persistent infection.

Theiler's virus causes a persistent infection with demyelination that is studied as a model for multiple sclerosis. Inbred strains of mice differ in their susceptibility to viral persistence due to both H-2 and non-H-2 genes. A locus with a major effect on persistence has been mapped on chromosome 10, close to the Ifng locus, using a cross between susceptible SJL/J and resistant B10.S mice. We now confirm the existence of this locus using two lines of congenic mice bearing the B10.S Ifng locus on an SJL/J background, and we describe a deletion in the promoter of the Ifng gene of the SJL/J mouse. We studied the expression of IFN-gamma, IL-2, IL-10, and IL-12 in the brains of SJL/J mice, B10.S mice, and the two lines of congenic mice during the first 2 wk following inoculation. We found a greater expression of IFN-gamma and IL-2 mRNA in the brains of B10.S mice compared with those of SJL/J mice. Also, the ratio of IL-12 to IL-10 mRNA levels was higher in B10.S mice. However, the cytokine profiles were the same for the two lines of resistant congenic mice and for susceptible SJL/J mice. Therefore, the difference of Th1/Th2 balance between the B10.S and SJL/J mice is not due to the Ifng locus and does not account for the difference of susceptibility of these mice to persistent infection.     

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

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