Pretreatment with recombinant Flt3 ligand partially protects against progressive cutaneous leishmaniasis in susceptible BALB/c mice

Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-gamma)-producing Th1-type CD4(+) T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 microg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-gamma and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.     

Substance P regulates natural killer cell interferon-gamma production and resistance to Pseudomonas aeruginosa infection

Studies have shown that after Pseudomonas aeruginosa (P. aeruginosa) corneal infection, BALB/c mice that are capable of resolving the disease, locally produce IFN-gamma. As T cells are not detected in the infected cornea of these mice, antibody depletion was used to test whether NK cells produce the cytokine. After depletion, decreased corneal IFN-gamma mRNA and increased disease severity, bacterial load, and PMN infiltrate resulted. Further work determined if substance P (SP), a pro-inflammatory neuropeptide, participated in regulation of this response. To this end, mice were treated with the SP antagonist, spantide I that blocks SP interaction with neurokinin-1, its major receptor. The treatment significantly decreased corneal IFN-gamma and IL-18 protein levels and corneal perforation resulted. In vitro experiments using isolated splenic NK cells confirmed their ability to respond to IL-18 and SP and to secrete IFN-gamma protein. We conclude: that for development of the BALB/c resistance response, NK cells are required to produce IFN-gamma; that the cells express the neurokinin-1 receptor; and that SP directly regulates IFN-gamma production through this receptor. The data suggest a unique link between the nervous system and development of innate immunity in the cornea.     

Multiple signals are required for maturation of human dendritic cells mobilized in vivo with Flt3 ligand

The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (Flt3L) is a growth factor for hematopoietic progenitors and induces expansion of the two distinct lineages of dendritic cells (DC) that have been described in humans. These two lineages, DC1 and DC2, have been described according to their ability to induce naive T cell differentiation to T helper cell type 1 (Th1) and Th2 effector cells, respectively. The immunoregulatory potential of DC1 and DC2 depends on their state of maturation and activation, which can be mediated by several molecules. Because monocyte-derived DC1 produce interleukin-12 (IL-12) when stimulated with CD40 ligand (CD40L), we hypothesized that similar results would be obtained with DC1 mobilized by Flt3L. Unexpectedly, we found that immature DC expanded in vivo by Flt3L treatment could not be stimulated to produce IL-12 in vitro using CD40L and/or interferon-gamma (IFN-gamma) alone. Instead, we found that Flt3L-mobilized DC from cancer patients require a sequence of specific signals for maturation, which included initial treatment with granulocyte macrophage-colony stimulating factor followed by a combination of maturation signals such as CD40L and IFN-gamma. Flt3L-mobilized DC matured in this manner possessed greater T cell-stimulatory function than nonmatured DC. The ability to generate phenotypically mature, IL-12-producing DC1 from peripheral blood mononuclear cells mobilized by Flt3L will have important implications for the development of effective cancer immunotherapy strategies.     

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.     

A role for plasmacytoid dendritic cells in the rapid IL-18-dependent activation of NK cells following HSV-1 infection

Natural killer (NK) cells play a crucial role in the initial response to viral infections but the mechanisms controlling their activation are unclear. We show a rapid and transient activation of NK cells that results in the production of IFN-gamma immediately following infection with herpes simplex virus type 1 (HSV-1). Activation of NK cells leading to synthesis of IFN-gamma was not mediated by a direct interaction with virus but required the presence of additional cell types and was largely dependent on the cytokine IL-18, but not IL-12. HSV-1-induced IFN-gamma expression by NK cells in vitro was impaired in spleen cultures depleted of CD11c(+) cells. Conversely, coculture of NK cells with virus-exposed conventional DC or plasmacytoid (p)DC restored the production of IFN-gamma, indicating that multiple DC subsets could mediate NK cell activation. While conventional DC populations stimulated NK cells independently of IL-18, they were less effective than pDC in promoting NK cell IFN-gamma expression. In contrast, the potent stimulation of NK cells by pDC was dependent on IL-18 as pDC from IL-18-deficient mice only activated a similar proportion of NK cells as conventional DC. These data identify IL-18 as a crucial factor for pDC-mediated NK cell regulation.     

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.     

Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon.

Gamma interferon (IFN-gamma) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock. To address the cellular source of IFN-gamma production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-gamma RNA was abundant in splenic CD4+ and CD8+ T cells and in a T- and B-cell-depleted population of splenocytes containing 34% NK1.1+ natural killer (NK) cells. Because interleukin 12 (IL-12) is a known inducer of IFN-gamma synthesis by cultured T cells and NK cells, we examined whether IL-12 might be involved in IFN-gamma release during endotoxemia. mRNA encoding the p40 subunit of IL-12 increased markedly in the spleens of C57BL/6 mice at 2 h after LPS injection, whereas p35 IL-12 mRNA was constitutively expressed at all times. Bioactive IL-12 (p70 heterodimer) was detected in mouse serum at 2 to 4 h after LPS injection. Similar results were obtained using a p40 subunit-specific enzyme-linked immunosorbent assay. Endotoxin-insensitive C3H/HeJ mice generated threefold less IL-12 p70 and IFN-gamma at these times than endotoxin-sensitive C3H/HeOuJ mice. Pretreatment of mice with polyclonal anti-mouse IL-12 antibody reduced IFN-gamma levels present at 6 h post-LPS nearly sixfold in three separate experiments. These studies support a role for IL-12 as a proximal stimulator of IFN-gamma release during endotoxemia.     

Modulation of CD11C+ splenic dendritic cell functions in murine visceral leishmaniasis: correlation with parasite replication in the spleen

BALB/c mice resolve Leishmania donovani infection in the liver over an 8-12-week period. However, after an initial phase of 2-4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post-infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-specific antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was purified from 30-day post-infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-gamma (IFN-gamma) production by Leishmania antigen-primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-gamma by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.     

Endogenous interleukin-12 is involved in resistance to Brucella abortus infection.

Protective immunity against Brucella abortus is mediated by acquired cellular resistance, with gamma interferon (IFN-gamma)-producing T cells playing a key role. Interleukin-12 (IL-12) is a cytokine that has a profound effect on the induction of IFN-gamma-producing Th1 and NK cells. Here we report that depletion of endogenous IL-12 before infection of mice significantly exacerbated brucella infection. IL-12-depleted mice also had reduced splenomegaly resulting from infection and showed a decrease in percentage and absolute numbers of macrophages compared with those in control infected mice. Furthermore, spleen cells from IL-12-depleted mice had a reduced ability to produce nitrite, a product of activated macrophages. This could be the result of the low production of IFN-gamma by splenic T cells observed in the IL-12-depleted mice. The mechanism whereby IL-12 controls antibacterial resistance is discussed.     

Impaired Th1 responses in mice deficient in Epstein-Barr virus-induced gene 3 and challenged with physiological doses of Leishmania major

Protection against Leishmania major is dependent on IL-12 release from L. major-infected dendritic cells (DC) that induce IFN-gamma-producing Th1/Tc1 cells. IL-27, a novel member of the IL-12 family, is a heterodimer composed of p28 and IL-12p40-related Epstein-Barr virus-induced gene 3 (EBI3), and was shown to be produced by DC. In this study, we utilized EBI3-deficient mice to investigate the role of IL-27 in leishmaniasis using physiological low-dose infections that mimic natural transmissions. Lesions in EBI3(-/-) mice were significantly larger between weeks 3 and 10 post infection, reaching up to approximately threefold increased lesion volumes compared to wild types. In parallel, dermal lesions of EBI3(-/-) mice contained greater parasite numbers, reaching a peak load that was 2-log higher than in C57BL/6 mice. However, lesions in EBI3(-/-) and wild-type mice resolved after 12 weeks. At early time points, the antigen-specific cytokine response in EBI3(-/-) lymph nodes showed increased levels of IL-4, IL-10 and IL-13 and decreased IFN-gamma production. IL-27 production was restricted to the DC population, since the majority of EBI3 expression in lymph nodes of infected mice was found in CD11c(+) cells. In conclusion, our data show that DC-derived IL-27 is critical for the timely initiation of efficient anti-parasite Th1 immunity early in infections.     

Critical role of type 1 cytokines in controlling initial infection with Burkholderia mallei

Burkholderia mallei is a gram-negative bacterium which causes the potentially fatal disease glanders in humans; however, there is little information concerning cell-mediated immunity to this pathogen. The role of gamma interferon (IFN-gamma) during B. mallei infection was investigated using a disease model in which infected BALB/c mice normally die between 40 and 60 days postinfection. IFN-gamma knockout mice infected with B. mallei died within 2 to 3 days after infection, and there was uncontrolled bacterial replication in several organs, demonstrating the essential role of IFN-gamma in the innate immune response to this pathogen. Increased levels of IFN-gamma, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 were detected in the sera of immunocompetent mice in response to infection, and splenic mRNA expression of IFN-gamma, IL-6, IL-12p35, and IL-27 was elevated 24 h postinfection. The effects of IL-18, IL-27, and IL-12 on stimulation of the rapid IFN-gamma production were investigated in vitro by analyzing IFN-gamma production in the presence of heat-killed B. mallei. IL-12 was essential for IFN-gamma production in vitro; IL-18 was also involved in induction of IFN-gamma, but IL-27 was not required for IFN-gamma production in response to heat-killed B. mallei. The main cellular sources of IFN-gamma were identified in vitro as NK cells, CD8+ T cells, and TCRgammadelta T cells. Our data show that B. mallei is susceptible to cell-mediated immune responses which promote expression of type 1 cytokines. This suggests that development of effective vaccines against glanders should target the production of IFN-gamma.     

Human monocyte-derived and CD83(+) blood dendritic cells enhance NK cell-mediated cytotoxicity

Dendritic cells (DC) are known to be the most potent APC and to stimulate antigen-specific T cell responses. Recently it was reported that murine DC were also capable of modulating the innate immunity by stimulating NK cells through cell-to-cell contact. In the present study, we examined whether human DC could affect NK activity. Both monocyte-derived and CD83(+) blood DC were tested. The addition of DC to cultures of CD56(+) cells resulted in the significant dose-dependent enhancement of the killing activity against various NK-sensitive targets. The resultant activity was comparable to that induced by optimal concentrations of various cytokines, including IL-2, IL-12, IL-15 and IFN-gamma. Interestingly, DC enhanced the cytotoxicity of CD3(-)CD56(+) NK cells, but not that of CD3(+)CD56(+) T cells. Experiments using transwells clearly demonstrated that the enhancement of NK activity by DC was mediated by soluble factors produced by DC. The culture supernatants of DC also stimulated NK activity. The treatment of both DC and their supernatants with anti-human IL-12 or IL-18 antibodies did not block the enhancement of NK cell-mediated cytolysis by DC, indicating that other factor(s) produced by DC were responsible for the enhancement of NK activity. These results suggest that human myeloid DC can modulate innate immunity by enhancing NK activity.     

Endotoxin-induced gamma interferon production: contributing cell types and key regulatory factors

Gamma interferon (IFN-gamma) is an important mediator of endotoxin (lipopolysaccharide [LPS])-induced immune responses. However, the specific cell types that produce IFN-gamma in response to LPS and the cellular factors that regulate LPS-induced IFN-gamma production have not been fully determined. The present studies were undertaken to characterize the cell populations that produce IFN-gamma after LPS challenge in the spleens of mice and to determine the regulatory factors that modulate LPS-induced production of IFN-gamma. Our studies show that the levels of splenic IFN-gamma mRNA and protein production peak at 6 and 8 h, respectively, after systemic LPS challenge. Approximately 60% of IFN-gamma-producing cells are natural killer (NK) cells (CD3(-)DX5(+)) and 25% are NKT cells (CD3(+)DX5(+)). Most of the remaining IFN-gamma-producing cells are T cells (CD3(+)DX5(-)), macrophages, and dendritic cells. Functionally, interleukin-12 (IL-12) is the major IFN-gamma-stimulating factor after LPS challenge, with costimulation provided by IL-15, IL-18, and B7 proteins. IL-10 is a major inhibitor of LPS-induced IFN-gamma production. Unlike intact heat-killed gram-negative and gram-positive bacteria, the class II major histocompatibility complex did not play a functional role in LPS-induced IFN-gamma production. LPS is a potent stimulus for splenic IL-10, IL-12 p40, and IL-15 mRNA expression, whereas IL-12 p35 and IL-18 mRNAs, as well as B7 proteins, are constitutively expressed in the mouse spleen. Of the factors studied, IL-18 serves as the most potent costimulus with IL-12 for IFN-gamma production, followed by IL-15 and B7 proteins. These data demonstrate that NK cells and NKT cells are the most abundant IFN-gamma-producing cells in the mouse spleen after LPS challenge and that IL-10 and IL-12 are key functional regulators of LPS-induced IFN-gamma production.     

The increased potential for the production of inflammatory cytokines by Kupffer cells and splenic macrophages eight days after thermal injury

Burn patients often experience a devastating inflammatory response to infection within the first two weeks after thermal injury. The inflammatory cytokines IL-6, TNF and IL-1 have been implicated in this condition but most studies have focused on the abnormal levels of cytokines in the plasma. In this study the production of cytokines was compared for Kupffer cells versus splenic macrophages; endotoxin (LPS) stimulation versus no stimulation; and burn (post burn days 1, 3 and 8) versus no burn (control). Corresponding serum levels of IL-6 were also determined. Kupffer cells from normal or burned animals were shown to produce much higher amounts of the inflammatory cytokines than that produced by splenic macrophages. An exception to this was the equal production of TNF by LPS-stimulated hepatic and splenic cells. Both LPS-stimulated Kupffer cells and splenic macrophages produced larger amounts of the cytokines than that produced by the unstimulated cells. There was a significant effect of thermal injury on cytokine production by LPS-stimulated Kupffer cells at post burn day 8 and on TNF production by stimulated splenic macrophages also at post burn day eight. Although there was a statistically significant effect of thermal injury at post burn day 8 on IL-1 production by unstimulated splenic macrophages, the absolute amount of cytokine produced was very small. The results suggest that by post burn day 8 the cells may have become primed to respond to a stimulus such as endotoxin (LPS), a condition that could arise in a burn patient from sepsis. Strangely, the large spike in serum IL-6 level occurred at post burn day one and the level of the cytokine returned nearly to the control value on post burn days 3 and 8.     

Interleukin-18 in combination with IL-2 enhances natural killer cell activity without inducing large amounts of IFN-gamma in vivo.

Interleukin-18 (IL-18) is known to synergistically enhance murine natural killer (NK) cell activity in vitro when combined with either IL-12 or IL-2. However, it has also been demonstrated that simultaneous administration of IL-18 and IL-12 to mice induces extraordinarily large amounts of interferon-gamma (IFN-gamma) in the serum. In this study, we examined the effects of a combination of IL-18 and IL-2 on in vivo NK cell activation in parallel with the induction of toxicity. In contrast to the IL-18 and IL-12 combination, the combined administration of IL-18 and IL-2 to BALB/c mice for 3 days induced neither high levels of IFN-gamma production nor other visible side effects. When compared with treatment with IL-18 or IL-2 alone, the combined treatment resulted in a significant increase in the number of DX-5 (pan-NK cells marker)-positive cells in spleens and a marked enhancement of splenic NK activity, as determined by standard cytotoxicity assays. Enhanced splenic cytotoxicity generated in the mice treated with both IL-18 and IL-2 was also observed against syngeneic Colon 26 adenocarcinoma cells. Consistent with this in vitro observation, combined treatment produced a significantly stronger inhibitory effect on the pulmonary metastases following i.v. injection of Colon 26 tumor cells than treatment with either cytokine alone. These results suggest that IL-18 combined with IL-2 potentiates in vivo NK cell activity and contributes to inhibition of tumor metastasis without inducing significant toxicity.     

Effects of DNA- and Mycobacterium bovis BCG-based delivery of the Flt3 ligand on protective immunity to Mycobacterium tuberculosis

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo. A DNA fusion of the genes encoding murine Flt3L and M. tuberculosis antigen 85B stimulated enhanced gamma interferon (IFN-gamma) release by T cells and provided better protection against virulent M. tuberculosis than DNA encoding the single components. Vaccination of mice with a recombinant Mycobacterium bovis BCG strain secreting Flt3L (BCG:Flt3L) led to early expansion of DCs compared to immunization with BCG alone, and this effect was associated with increased stimulation of BCG-reactive IFN-gamma-secreting T cells. BCG and BCG:Flt3L provided similar protective efficacies against low-dose aerosol M. tuberculosis; however, immunization of immunodeficient mice revealed that BCG:Flt3L was markedly less virulent than conventional BCG. These results demonstrate the potential of in vivo targeting of DCs to improve antimycobacterial vaccine efficacy.     

IL-1 beta induces dendritic cells to produce IL-12

The cytokine IL-12, a product of dendritic cells (DC), plays a major role in cellular immunity, notably by inducing lymphocytes to produce IFN-gamma. Microbial products, T cell signals and cytokines induce the production of IL-12. Here, IL-1 beta is identified as a new IL-12-inducing agent, acting conjointly with CD40 ligand (CD40L) on human monocyte-derived DC in vitro. The effects of IL-1 beta were dose dependent, specifically blocked by neutralizing antibodies, and were observed both in immature and mature DC. Immature DC secreted more IL-12 than mature DC, but the effects of IL-1 beta were not due to a block of DC maturation as determined by analysis of DC surface markers. The mechanisms of action of IL-1 beta could be contrasted to that of other inducers of IL-12 such as IFN-gamma and lipopolysaccharide (LPS). Either IL-1 beta or IFN-gamma co-induced IL-12 with CD40L but conjointly, IL-1 beta, CD40L and IFN-gamma synergized, inducing very high levels of IL-12. The effects of IL-1 beta differed from those of LPS in that IL-1 beta, unlike LPS, could not induce IL-12 solely after IFN-gamma priming; and when combined with CD40L, IL-1 beta, unlike LPS, induced little IL-10. The mechanism of action of IL-1 beta involves IL-12 alpha mRNA up-regulation, and we show that the combination of CD40L and IL-1 beta induces high levels of IL-12 alpha and IL-12 beta mRNA in DC. Altogether, these results delineate a new mechanism linking adaptive and innate immune responses for the regulation of IL-12 production in DC and for the role of IL-1 beta in the development of cellular immunity.     

Bacillus Calmette Guerin triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.     

An important role of Tyk2 in APC function of dendritic cells for priming CD8+ T cells producing IFN-gamma

Tyrosine kinase 2 (Tyk2) contributes to the signals triggered by IL-12 for IFN-gamma production by NK cells and T cells. We found in this study that Tyk2-deficient (-/-) mice showed increased susceptibility at the early stage after an i.p. infection with Listeria monocytogenes, accompanied by impaired IFN-gamma production. The numbers of both MHC class Ib (H2-M3)- or MHC class Ia (Kb)-restricted CD8+ T cells producing IFN-gamma and exhibiting cytotoxicity were significantly decreased in Tyk2-/- mice after infection with L. monocytogenes. Using an adoptive transfer system of OT-I cells expressing OVA(257-264)/Kb-specific TCR into Tyk2-/- mice followed by challenge with recombinant L. monocytogenes expressing OVA, we found that the defective Tyk2 signaling in the host environment was at least partially responsible for the impaired CD8+ T cytotoxic-1 (Tc1) cell responses in Tyk2-/- mice following the infection. Adoptive transfer with MHC class Ib- or MHC class Ia-binding peptide-pulsed BM-derived DC from Tyk2-/- mice induced lower levels of the Ag-specific CD8+ Tc1 cells producing IFN-gamma. These results suggest that Tyk2 signaling is also important for DC function in the induction of MHC class Ia- and class Ib-restricted CD8+ Tc1 cells following L. monocytogenes infection.