A heritable defect in IL-12 signaling in B10.Q/J mice. II. Effect on acute resistance to Toxoplasma gondii and rescue by IL-18 treatment

This study documents a defect in IL-12-dependent IFN-gamma responses in a substrain (B10.Q-H2-(q)/SgJ) of B10.Q mice that manifests as an acute susceptibility to infection by the intracellular protozoan pathogen, Toxoplasma gondii. Despite robust systemic production of IL-12, infected B10.Q/J animals fail to mount an early IFN-gamma response after parasite inoculation. Genetic experiments revealed that the host resistance and IFN-gamma production defects are determined by a single autosomal recessive locus distinct from the Stat4 gene. Nonetheless, a delayed IL-12-mediated IFN-gamma response emerges in later stages of acute infection but is unable to prevent host mortality. IL-18 administration restores, in an IL-12-dependent manner, the early IFN-gamma response and host resistance of B10.Q/J animals. These in vivo studies indicate that the partially impaired IL-12 responsiveness in B10.Q/J mice can result in defective host resistance and demonstrate a therapeutic function for IL-18 in reversing a genetically based immunodeficiency in IL-12-dependent IFN-gamma production.     

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.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.     

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.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to Toxoplasma gondii

To define the role of NF-kappa B in the development of T cell responses required for resistance to Toxoplasma gondii, mice in which T cells are transgenic for a degradation-resistant (Delta N) form of I kappa B alpha, an inhibitor of NF-kappa B, were challenged with T. gondii and their response to infection compared with control mice. I kappa B alpha(Delta N)-transgenic (Tg) mice succumbed to T. gondii infection between days 12 and 35, and death was associated with an increased parasite burden compared with wild-type (Wt) controls. Analysis of the responses of infected mice revealed that IL-12 responses were comparable between strains, but Tg mice had a marked reduction in systemic levels of IFN-gamma, the major mediator of resistance to T. gondii. In addition, the infection-induced increase in NK cell activity observed in Wt mice was absent from Tg mice and this correlated with NK cell expression of the transgene. Infection-induced activation of CD4(+) T cells was similar in Wt and Tg mice, but expansion of activated CD4(+)T cells was markedly reduced in the Tg mice. This difference in T cell numbers correlated with a reduced capacity of these cells to proliferate after stimulation and was associated with a major defect in the ability of CD4(+) T cells from infected mice to produce IFN-gamma. Together, these studies reveal that inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to T. gondii.     

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.     

Tyrphostin B42 inhibits IL-12-induced tyrosine phosphorylation and activation of Janus kinase-2 and prevents experimental allergic encephalomyelitis

IL-12 is a macrophage-derived cytokine that induces proliferation, cytokine production, and cytotoxic activity of T and NK cells. Signaling through its receptor, IL-12 induces these cellular responses by tyrosine phosphorylation and activation of Janus kinase-2 (Jak-2), Tyk-2, Stat3, and Stat4. We have used tyrphostin B42 (AG490), a Jak-2 inhibitor, to determine the role of Jak-2 kinase in IL-12 signaling and IL-12-induced T cell functions. Treatment of activated T cells with tyrphostin B42 inhibited the IL-12-induced tyrosine phosphorylation and activation of Jak-2 without affecting Tyk-2 kinase. In contrast, treatment with tyrphostin A1 inhibited the tyrosine phosphorylation of Tyk-2 but not that of Jak-2 kinase. Inhibition of either Jak-2 or Tyk-2 leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat3, but not of Stat4, protein. While inhibition of Jak-2 lead to programmed cell death, the inhibition of Jak-2 or Tyk-2 resulted a decrease in IFN-gamma production. We have further tested the in vivo effects of tyrphostin B42 in experimental allergic encephalomyelitis, a Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin B42 decreased the proliferation and IFN-gamma production of neural Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced the incidence and severity of active and passive EAE. These results suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling and IL-12-mediated Th1 differentiation in vivo.     

Regulatory role of peritoneal NK1.1+ alpha beta T cells in IL-12 production during Salmonella infection.

NK1.1+ alpha beta T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1+ alpha beta T cells during murine salmonellosis, mice lacking NK1.1+ alpha beta T cells by disruption of TCR beta (TCR beta-/-), beta 2m (beta 2m-/-), or J alpha 281 (J alpha 281-/-) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCR alpha beta stimulation, whereas those in TCR beta-/-, beta 2m-/-, or J alpha 281-/- mice showed no IL-4 production upon the stimulation, indicating that NK1.1+ alpha beta T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1+ alpha beta T cells after Salmonella infection accompanied by increased serum IFN-gamma level. The aberrantly increased IL-12 production in infected TCR beta-/- or J alpha 281-/- mice was suppressed by adoptive transfer of T cells containing NK1.1+ alpha beta T cells but not by the transfer of T cells depleted of NK1.1+ alpha beta T cells or T cells from J alpha 281-/- mice. Taken together, it is suggested that NK1. 1+ alpha beta T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Characterization of lymphocyte subsets in the bronchiolar lymph nodes of BALB/c mice infected with cilia-associated respiratory bacillus

Cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, extracellular bacterium that causes chronic respiratory tract disease in rodents. Infected mice develop microscopic lesions characterized by a primary lymphocytic response followed by macrophage and neutrophilic infiltration. To characterize the lymphocytic subsets that respond to CAR bacillus infection, BALB/c mice were inoculated with 10(5) CAR bacillus bacteria. At seven weeks after inoculation, mice were euthanized and the tracheobronchiolar and hilar lymph nodes were collected and stained for cell surface markers to T cells (CD3, CD4, and CD8), B cells (B220, CD5), natural killer (NK) cells (pan-NK) and intracellular interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). Flow cytometric analysis of lymph nodes from CAR bacillus-infected mice revealed 11% increase in frequency of B cells (R220+), 12% increase in the frequency of double-negative (CD4-CD8-CD3+) T cells, and slight increase in the B-1 subset of B cells (B220+CD5+). There was no change in the frequency of NK cells. The CAR bacillus-infected mice had an overall decrease in the frequency of T cells. Intracellular cytokine staining revealed distinct populations of T cells producing IL-10 and IFN-gamma, and IL-10 production from B cells; NK cells were not a substantial source of IFN-gamma. To our knowledge, this is the first characterization of lymphocytic responses and suggestion that B cells and double-negative T cells may be principally responsible for the lesions associated with CAR bacillus infection.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4(+) T cells (CD4(Stat3)(-/-)) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4(Stat3)(-/-) mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-gamma-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4(+) T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-gamma while these double-expressors are absent in CD4(Stat3)(-/-) and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4(Stat3)(-/-) mouse because of the reduction in the expression of activated alpha4/beta1 integrins on CD4(Stat3)(-/-) T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4(Stat3)(-/-) mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4(+) T cells results in an intrinsic developmental defect that renders CD4(Stat3)(-/-) resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-gamma, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.     

IL-12 receptor beta 2 (IL-12R beta 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites

Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.     

NK cell-like behavior of Valpha14i NK T cells during MCMV infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Valpha14 invariant natural killer T cell response to MCMV has not been examined. We found that Valpha14i NK T cells become activated and produce significant levels of IFN-gamma, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Valpha14i NK T cells into MCMV-infected CD1d(-/-) mice demonstrate that CD1d is dispensable for Valpha14i NK T cell activation. In contrast, both IFN-alpha/beta and IL-12 are required for optimal activation. Valpha14i NK T cell-derived IFN-gamma is partially dependent on IFN-alpha/beta but highly dependent on IL-12. Valpha14i NK T cells contribute to the immune response to MCMV and amplify NK cell-derived IFN-gamma. Importantly, mortality is increased in CD1d(-/-) mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Valpha14i NK T cells that act as effector T cells during bacterial infection, but have NK cell-like behavior during the innate immune response to MCMV infection.     

Murine plasmacytoid dendritic cells produce IFN-gamma upon IL-4 stimulation

IL-4 plays a key role in inducing IL-4 production in CD4+ T cells, functioning as an important determinant for Th2 cell differentiation. We show here that IL-4 induces IFN-gamma production in B220+ plasmacytoid dendritic cells (PDCs). By searching for cell populations that produce IFN-gamma upon IL-4 stimulation, we found that PDCs were a major IFN-gamma-producing cell upon IL-4 stimulation in wild-type and Rag-2-/- splenocytes. Isolated PDCs, but not CD11b+ DCs or CD8+ DCs, produced IFN-gamma upon IL-4 stimulation. In vivo, the depletion of PDCs by anti-Ly6G/C Ab prevented IFN-gamma production induced by IL-4 administration. We also found that IL-4 induced IFN-gamma production, but not IL-12 or IFN-alpha production, in PDCs and also strongly enhanced CpG oligodeoxynucleotide-induced IFN-gamma production, but not CpG oligodeoxynucleotide-induced IL-12 or IFN-alpha production. However, IL-4 did not induce IFN-gamma production in Stat6-/- PDCs. Moreover, IL-4 induced Stat4 expression in PDCs through a Stat6-dependent mechanism, and only the Stat4-expressing PDCs produced IFN-gamma. Furthermore, IL-4 did not induce IFN-gamma production in Stat4-/- PDCs. These results indicate that PDCs preferentially produce IFN-gamma upon IL-4 stimulation by Stat6- and Stat4-dependent mechanisms.