Influenza virus-induced dendritic cell maturation is associated with the induction of strong T cell immunity to a coadministered,normally nonimmunogenic protein

We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-gamma, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-gamma. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2-3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently.     

Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.     

Nonreplicating recombinant vaccinia virus expressing CD40 ligand enhances APC capacity to stimulate specific CD4+ and CD8+ T cell responses

Recombinant poxviruses expressing immunomodulatory molecules together with specific antigens represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in vivo, that coexpression of CD80 and CD86 costimulatory molecules enhances the immunogenic capacity of a recombinant vaccinia virus (rVV) encoding different tumor-associated antigens. To further investigate the capacity of these vectors to provide ligands for different costimulatory pathways relevant in the generation of T cell responses, we constructed a recombinant virus (rVV) expressing CD40 ligand or CD154 (CD154rVV). Upon binding the CD40 receptor expressed on antigen presenting cells (APC), this molecule, physiologically expressed on activated CD4+ T cells, increases their antigen presentation and immunostimulatory capacities. Therefore, we evaluated the effects of CD154rVV infection on APC activation and its consequences on T cell stimulation. CD154rVV infection of autologous fibroblasts, monocytes, or iDC promoted the expression of a number of cytokines, including GM-CSF, TNF-alpha, and IL-15 in iDC. Most importantly, IL-12 p40 gene expression and protein secretion were induced by CD154rVV but not by wild-type VV (WT VV) in either CD14+ cells or iDC, and these effects could be blocked by anti-CD40 monoclonal antibodies. Furthermore, phenotypic characterization of CD154rVV infected iDC revealed enhanced expression of CD83 and CD86 surface markers as compared with wild-type vaccinia virus infection. As expected, VV infection triggered cytokines gene expression in cultures including APC and T cells from VV immune donors. However, cytokine genes typically expressed by T cell receptor triggered T cells such as those encoding IL-2 and IFN-gamma, or T cell proliferation, were detectable to a significantly higher extent in CD154rVV infected cultures, as compared with WT VV. Activation of specific CD8+ T cells was then investigated using MART-1/Melan-A(27-35) epitope as the model of tumor-associated antigen (TAA). In the presence of CD154rVV activated APCs, significantly higher numbers of specific cytotoxic CD8+ T cells were detected, as compared with cultures performed in the presence of WT VV or in the absence of virus. Taken together, these data indicate that functional CD154 expression from rVV infected cells promotes APC activation, thereby enhancing antigen-specific T cell generation. Such a recombinant vector might help bypass the requirement for activated helper cells during CTL priming, thus qualifying as a potentially relevant vector in the generation of CD8+ T cell responses in cancer immunotherapy.     

Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection

T lymphocytes play a primary role in recovery from viral infections and in antiviral immunity. Although viral-specific CD8+ and CD4+ T cells have been shown to be able to lyse virally infected targets in vitro and promote recovery from lethal infection in vivo, the role of CD4+ T lymphocytes and their mechanism(s) of action in viral immunity are not well understood. The ability to further dissect the role that CD4+ T cells play in the immune response to a number of pathogens has been greatly enhanced by evidence for more extensive heterogeneity among the CD4+ T lymphocytes. To further examine the role of CD4+ T cells in the immune response to influenza infection, we have generated influenza virus-specific CD4+ T cell clones from influenza-primed BALB/c mice with differential cytokine secretion profiles that are defined as T helper type 1 (Th1) clones by the production of interleukin 2 (IL-2) and interferon gamma (IFN-gamma), or as Th2 clones by the production of IL-4, IL-5, and IL-10. Our studies have revealed that Th1 clones are cytolytic in vitro and protective against lethal challenge with virus in vivo, whereas Th2 clones are noncytolytic and not protective. Upon further evaluation of these clonal populations we have shown that not only are the Th2 clones nonprotective, but that pulmonary pathology is exacerbated as compared with control mice as evidenced by delayed viral clearance and massive pulmonary eosinophilia. These data suggest that virus-specific CD4+ T cells of the Th2 subset may not play a primary role in virus clearance and recovery and may lead to immune mediated potentiation of injury.     

Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells.

We investigated the role of antigen-presenting cells in early interferon (IFN)-gamma production in normal and recombinase activating gene 2-deficient (Rag-2(-/-)) mice in response to Listeria monocytogenes (LM) infection and interleukin (IL)-12 administration. Levels of serum IFN-gamma in Rag-2(-/-) mice were comparable to those of normal mice upon either LM infection or IL-12 injection. Depletion of natural killer (NK) cells by administration of anti-asialoGM1 antibodies had little effect on IFN-gamma levels in the sera of Rag-2(-/-) mice after LM infection or IL-12 injection. Incubation of splenocytes from NK cell-depleted Rag-2(-/-) mice with LM resulted in the production of IFN-gamma that was completely blocked by addition of anti-IL-12 antibodies. Both dendritic cells (DCs) and monocytes purified from splenocytes were capable of producing IFN-gamma when cultured in the presence of IL-12. Intracellular immunofluorescence analysis confirmed the IFN-gamma production from DCs. It was further shown that IFN-gamma was produced predominantly by CD8alpha+ lymphoid DCs rather than CD8alpha- myeloid DCs. Collectively, our data indicated that DCs are potent in producing IFN-gamma in response to IL-12 produced by bacterial infection and play an important role in innate immunity and subsequent T helper cell type 1 development in vivo.     

Cytokine-inducing activity and antitumor effect of a liposome-incorporated interferon-gamma-inducing molecule derived from OK-432, a streptococcal preparation

An interferon-gamma (IFN-gamma)-inducing molecule (OK-PSA) has been purified from OK-432 by an affinity chromatographic technique performed on cyanogen bromide-activated Sepharose 4B-bound TS-2 monoclonal antibody, which neutralizes IFN-gamma-inducing activity of OK-432. OK-PSA has striking anti-tumor activity in vivo and in vitro. In the current study, the liposomes were used to improve the delivery of the agent (OK-PSA) to effector cells and to increase the therapeutic effect. Significantly less OK-PSA encapsulated into liposomes (Lipo-OK-PSA) than OK-PSA alone (1/100 or less of OK-PSA alone) was required to induce IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, interleukin-1 beta (IL-1 beta), natural killer, and lymphokine-activated killer activities by human peripheral blood mononuclear cells and mouse spleen cells. Furthermore, higher levels of these activities were detected in peripheral blood mononuclear cells and mouse spleen cells treated with Lipo-OK-PSA than in those treated with OK-PSA. All of these activities induced by Lipo-OK-PSA were almost completely neutralized by anti-asialo-GM1 antibody and complement (p < 0.001). In in vivo experiments, Lipo-OK-PSA elicited striking anti-tumor activity on syngeneic Meth-A tumor-bearing and colon 26-bearing BALB/c mice and on salivary gland tumor-bearing nude mice far better than did OK-PSA. Furthermore, high levels of natural killer and lymphokine-activated killer activities and a significant increase in the number of cells positive for asialo-GM1, IFN-gamma, TNF-alpha, or IL-1 beta were detected in the spleen cells derived from the animals given Lipo-OK-PSA compared with those given saline. These findings clearly indicate that OK-PSA plays an important role in the anti-tumor efficiency of OK-432, and that, for the most part, liposome encapsulation of this molecule markedly accelerates its effect mediated by asialo-GM1-positive cells (mainly natural killer cells).     

Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H.

Antiviral roles of natural killer (NK) cell subsets were examined in C57BL/6 mice infected with murine cytomegalovirus (MCMV) and other viruses, including lymphocytic choriomeningitis virus (LCMV), vaccinia virus (VV), and mouse hepatitis virus (MHV). Each virus vigorously induced an NK cell infiltrate into the peritoneal cavity and liver, causing some redistributions of NK cell subsets defined by monoclonal antibody (mAb) directed against Ly49A, C/I, D, and G2. Striking results were seen with a mAb (1F8) reactive with the positively signaling molecule Ly49H, present in MCMV-resistant C57BL/6 mice. mAb 1F8 also stains Ly49 C and I, but exclusion of those reactivities with mAb 5E6, which recognizes Ly49 C and I, indicated that Ly49H(+) cells infiltrated the peritoneal cavity and liver and were particularly effective at synthesizing interferon gamma. Depletion of 1F8(+) but not 5E6(+) cells in vivo by mAb injections enhanced MCMV titers by 20-1,000-fold in the spleen and approximately fivefold in the liver. Titers of LCMV or VV were not enhanced. These anti-MCMV effects were attributed to prototypical NK1.1(+)CD3(-) NK cells and not to NK1.1(+)CD3(+) "NK/T" cells. This is the first evidence that control of a virus infection in vivo is mediated by a distinct NK cell subset.     

体外阻断CD137-CD137L途径控制小鼠移植物抗宿主病的研究

目的探讨体外阻断活化的供鼠T淋巴细胞CD137-CD137L共刺激途径控制小鼠异基因骨髓移植（allo-BMT）后急性移植物抗宿主病（aGVHD）及其机制。方法供、受鼠的淋巴细胞体外混合培养，分别加入抗CD137L单抗或不加抗CD137L单抗培养后与供鼠骨髓细胞混合移植给受鼠。采用流式细胞术检测移植后受鼠T细胞亚群的变化，RT-PCR法检测细胞因子mRNA表达水平的变化，观察移植后受鼠GVHD的临床及病理改变。结果与未用抗CD137L单抗组相比，应用抗CD137L单抗组CD3^＋CD8^＋T细胞明显降低（P＜0．01）；IFN-γ表达水平明显减低（P＜0．01），IL-10的表达水平明显升高（P＜0．01）。移植后未预防GVHD组（A组）小鼠移植后15d内均死于aGVHD。采用甲氨蝶呤＋环孢素预防GVHD组（B组）小鼠100％发生aGVHD，但临床及病理改变程度较A组轻，平均存活时间[（9．5±2．5）d]较A组[（7．5±1．5）d]略有延长。采用抗CD137L单抗预防GVHD组（C组）受鼠aGVHD的发生率为70％，程度比A、B两组轻，与A、B两组相比生存率明显提高（P＜0．01），平均存活时间[（16．0±2．5）d]明显延长（P＜0．01），30％的小鼠生存时间大于30d。结论抗CD137L单抗体外阻断CD137-CD137L共刺激途径能有效控制小鼠GVHD，可能与影响Ⅰ类和Ⅱ类T细胞因子平衡有关。     

Combined interleukin 1/interleukin 2 therapy of mice injected with highly metastatic Friend leukemia cells: host antitumor mechanisms and marked effects on established metastases

Peritumoral injection of recombinant human interleukin 1 beta (IL-1 beta) in mice transplanted subcutaneously with Friend erythroleukemia cells (FLC) resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. In contract, IL-2 treatment alone did not significantly inhibit the development of FLC metastases. A synergistic antitumor effect was observed after combined IL-1/IL-2 therapy of these mice. The antitumor action of IL-1/IL-2 treatment was abolished or markedly reduced in mice treated with antibodies to CD4 or CD8 antigens, whereas antibodies to asialo-GM1 were ineffective. A clear-cut increase in the percentage of CD4+ cells was observed in the spleens of cytokine-treated mice on days 17 and 23. On day 23 of cytokine therapy, CD8+ cells were increased in both spleens and lymph nodes. On day 17, infiltrates of host-reactive cells (i.e., lymphocytes, granulocytes, and monocytes) were observed in both spleen and liver from FLC-injected mice treated with IL-1/IL-2, in association with tumor cells. On days 17 and 23, spleen cells and cells recovered from mesenteric lymph nodes of IL-1/IL-2-treated mice exerted a potent antitumor effect as determined by Winn assay experiments. This antitumor activity was abolished by preincubation of spleen cells with anti-CD8 antibody, but not by treatment with antibodies to asialo-GM1; antibodies to CD4 exerted only a slight effect. Combined IL-1/IL-2 therapy was more effective on established (i.e., 6-7-d) FLC tumors than on early (i.e., 1-d) tumor-transplanted mice. IL-1/IL-2 treatments were also highly effective in increasing survival time of mice from which the subcutaneous primary tumors were excised 7 d after FLC injection. These data indicate that in mice injected with FLC, the antitumor effects of IL-1/IL-2 are mediated by CD4+ and CD8+ cells (but not NK cells), and suggest that this combined cytokine treatment may be effective against established metastatic tumors.     

Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection

Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN-gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN-gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease.     

Effect of anti-interleukin 12 treatment on murine lyme borreliosis.

The effect of anti-interleukin (IL-12 treatment on Lyme borreliosis in C3H/HeN (C3H) mice was assessed because other studies have implicated CD4+ T cell helper (Th) type 1 responses in the genesis of disease caused by Borrelia burgdorferi. Infection of inbred mice with B. burgdorferi results in varying degrees of arthritis: BALB/c mice develop mild disease and C3H mice develop severe arthritis that is most pronounced 2-4 wk after infection. Since IL-12 is a major inducer of Th1 responses, we blocked this cytokine in vivo in B. burgdorferi infected C3H mice, and evaluated the effects of treatment on the development of arthritis at the peak of acute joint inflammation (14 d) and in the resolution phase (60 d) of disease. As expected, intraperitoneal administration of an anti-IL-12 monoclonal antibody (mAb) to C3H mice resulted in a decrease in both IFN-gamma and B. burgdorferi-specific IgG2a in serum, indicative of diminished Th1 responses. No IL-4 production was detected in serum of anti-IL-12 mAb treated or control mice. IgG1 and IgG2b levels did not increase in B. burgdorferi infected mice treated with anti-IL-12 mAb compared with controls suggesting that Th2 responses were not affected. Nevertheless, CD4+ T cells from both control and anti-IL-12 mAb treated mice had similar in vitro responses to B. burgdorferi antigens. Treatment with anti-IL-12 mAb produced a significant reduction in peak arthritis severity, and an increase in the number of spirochetes in ear tissue. These data show that treatment of B. burgdorferi infected mice with anti-IL-12 mAb results in a reduction of the Th1 and/or innate immune responses in vivo and a reduction in the severity of acute murine Lyme arthritis.     

Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell-dependent, interferon gamma-independent mechanism

BALB/c mice infected with Leishmania major develop fatal, progressive disease, despite an immune response characterized by expansion of CD4+ T cells in the draining lymph nodes. The immune response has been further characterized by a lack of IFN-gamma mRNA, but increased IL-4 mRNA in lymphoid tissues, and striking elevation of serum IgE. Treatment of infected BALB/c mice with rIFN-gamma at doses shown to be beneficial in other protozoan infections was insufficient to ameliorate L. major infection. In contrast, neutralization of IL-4 by six weekly injections of mAb 11B11 led to attenuation of disease in 100% of animals, and complete cure in 85%. Resolution of disease required the presence of T cells, and recovered mice remained resistant to reinfection at 12 wk. This immunity was adoptively transferable and was dependent on both CD4+ and CD8+ cells. Although administration of anti-IL-4 was associated with fourfold increase in IFN-gamma mRNA in lymph node cells draining the lesion, the coadministration of neutralizing R4 6A2 anti-IFN-gamma mAb had no effect on resistance to disease. This was in marked contrast to resolution of disease in both resistant C57BL/6- and GK1.5-pretreated BALB/c mice that was abrogated by in vivo treatment with anti-IFN-gamma. These data suggest a novel mechanism of cellular immunity established by interference with the development of Th2 cells during infection.     

Low-dose vaccinia virus-mediated cytokine gene therapy of glioma

Recombinant viruses can produce cytokines in tumors mobilizing an immune response to tumor cells. In this study, the authors investigated gene expression, in vivo antitumor efficacy, and safety of attenuated recombinant vaccinia virus (rVV) carrying murine cytokine genes interleukin (IL)-2 (rVV-mIL-2), IL-12 (rVV-mIL-12), and both IL-2 and IL-12 (rVV-2-12) in an athymic nude mice model. Significant tumor inhibition (p < 0.05) was observed in a preestablished subcutaneously implanted C6 glioma model using rVVs at doses ranging from 10(2) to 10(7) plaque forming units (PFU). An antitumor effect did not depend on the dose of the rVV-mIL-2 and rVV-mIL-12 viruses. All constructed rVVs induced a high level of cytokine expression in vitro and in vivo. Most groups injected with high doses of recombinant viruses encoding cytokine genes (10(5) to 10(7) PFU) showed signs of cytokine toxicity, whereas in the low-dose treatment groups (10(2) to 10(3) PFU) toxicity was greatly reduced. The antitumor activity of rVV-mIL-12 was associated with increases in both the percentage and number of natural killer T cells in the spleen. Local detection of interferon-y and tumor necrosis factor-alpha was also correlated with tumor growth arrest induced by the treatment. High-dose VV control vector per se induced tumor inhibition by activating Mac-1+ cells in blood, but the antitumor effect was less pronounced compared with rVV-carrying cytokine genes (p < 0.05). These results suggest that attenuated recombinant strains of VV at low doses may potentially be efficient vectors for cancer immunotherapy.     

Activation of CD4+ T cells in the presence of a nondepleting monoclonal antibody to CD4 induces a Th2-type response in vitro

In vitro experiments using purified rat CD4+ T cells in primary and secondary mixed leukocyte cultures (MLC) have been carried out to explore the mechanism of inhibition of cell-mediated autoimmune disease in the rat by a nondepleting monoclonal antibody (mAb) to CD4. Previous work has shown that W3/25, a mouse anti-rat CD4 mAb of immunoglobulin G1 isotype, completely prevents the development of the paralysis associated with experimental allergic encephalomyelitis (EAE) in Lewis rats, but does so without eliminating the encephalitogenic T cells. The in vitro experiments described in this study have shown that when CD4+ T cells were activated in the presence of the anti-CD4 mAb in a primary MLC, the synthesis of interferon (IFN) gamma, but not interleukin (IL) 2, was completely inhibited. After secondary stimulation, now in the absence of the mAb, the synthesis of IL-4 and IL-13 mRNA was greatly enhanced compared with that observed from CD4+ T cells derived from primary cultures in which the mAb was omitted. As IL-4 and IL-13 are known to antagonize cell-mediated immune reactions, and as EAE is cell- mediated disease, the data suggest that the W3/25 mAb controls EAE by modifying the cytokine repertoire of T cells that respond to the encephalitogen. The capacity for the mAb to suppress IFN-gamma synthesis provides, in part, an explanation for this change in cytokine production. These findings are discussed in terms of what is known of the factors that determine which cytokine genes are expressed on T cell activation. Possible implications for the evolution of T cell responses in human immunodeficiency virus infection are also discussed.     

IL-4-deficient Balb/c mice resist infection with Leishmania major

Mice with a genetically engineered deficiency for either IL-4 or IFN- gamma R1 (single mutants), and IL-4/IFN-gamma R1 (double mutants) on the Balb/c and 129Sv background were used to study the course of infection with Leishmania major. In contrast to genetically resistant 129Sv wildtype mice, IL-4/IFN-gamma R1 double mutant mice developed fetal disease with parasite dissemination to visceral organs similar to mice lacking IFN-gamma R1 only. Balb/c mice, which are exquisitely susceptible to L. major, were rendered resistant to infection by disruption of the IL-4 gene. As compared to homozygous IL-4+/- mice, heterozygous IL-4+/- mice, heterozygous IL-4+/- animals consistently developed smaller lesions with less ulceration and necrosis, indicating the likelihood of gene-dosage effects. This implicates that the magnitude of the IL-4 response determines the severity of disease. CD4+ T cells of IL-4-deficient mice showed impaired Th2 cell development, as assessed by quantitative RT-PCR of characteristic cytokines. Development of resistance is not explained by default Th1 development, because this was observed only at very late stages of infection. Moreover, the induction of inflammatory cytokines (e.g., IL-1 alpha, IL- 1 beta, TNF-alpha, IL-12) together with iNOS in the lesion and draining lymph nodes was not altered in the absence of IL-4.     

Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor.

Most biologic responses to IL-2 have been attributed to interaction of IL-2 with a high affinity receptor which consists of a heterodimer composed of two distinct IL-2-binding proteins (IL-2R alpha/IL-2R beta). However, both low affinity IL-2R alpha (55 kD) and intermediate affinity IL-2R beta (70-75 kD) also appear to be expressed independently on the cell surface. We investigated the receptor-specific regulatory effects of IL-2 on cytokine production in unstimulated and activated T cells. T cells were activated by stimulation of the antigen receptor complex with anti-CD3 mAb. IL-2 (10(2) U/ml, 1 nM) stimulation of resting cells resulted in a fivefold increase in GM-CSF release but in only minimal IFN-gamma release. IL-2 markedly augmented mRNA expression of GM-CSF but not IFN-gamma in unstimulated T cells. IL-2R beta mAb but not IL-2R alpha mAb decreased IL-2-induced GM-CSF release and mRNA expression from unstimulated T cells. IL-2 concentrations required for GM-CSF release from resting cells suggested ligand binding to an intermediate affinity receptor. GM-CSF and IFN-gamma release from activated T cells increased four- to fivefold in response to 1 nM IL-2 and IL-2 augmented both GM-CSF and IFN-gamma mRNA. IL-2R beta mAb but not IL-2R alpha mAb reduced GM-CSF release and mRNA expression in activated T cells stimulated with 1 nM IL-2. IL-2R alpha blockade markedly decreased IL-2-induced IFN-gamma release and mRNA expression from activated cells, while IL-2R beta blockade had little effect on IFN-gamma production in activated cells. IL-2R alpha blockade altered the affinity of the receptor mediating activated cell GM-CSF release from a high affinity to an intermediate affinity state. These studies indicate an independent role for IL-2R beta in mediating GM-CSF production from T cells. They also suggest that unstimulated and activated T cells, which express distinct IL-2 receptor moieties, mediate release of separate lymphokines and that different subunits of the IL-2 receptor may play an important role in the regulation of cytokine production.     

Natural killer cells are a source of interferon gamma that drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice

Infection of mice with the protozoan Leishmania major provides an excellent model to define the factors involved in T helper (Th) subset development, since Th1 cells confer protection in resistant strains of mice, whereas Th2 cells are associated with the fatal outcome of susceptible mice. We previously found that interferon gamma (IFN-gamma) was required for Th1 cell development after infection of mice with L. major. In this report, we evaluate the contribution of natural killer (NK) cells to IFN-gamma levels early in L. major infection. NK cell activity was higher in resistant C3H/HeN mice than in susceptible BALB/c mice during the first week of infection, and removal of NK cells significantly decreased IFN-gamma levels and promoted interleukin 4 (IL- 4) production in both the draining lymph nodes and spleen. IFN-gamma production by NK cells required the presence of CD4+ T cells or IL-2, but not CD8+ T cells. Enhanced disease, as measured by parasite numbers and lesion development, was observed in NK cell-depleted mice. Furthermore, a comparison of the NK cell response and the subsequent parasite burden in several inbred strains of mice demonstrated that NK cells mediate early resistance to L. major. Together, these data indicate that the stimulation of NK cells, through the production of IFN-gamma, plays an important role in initiating Th1 cell differentiation in leishmaniasis and in controlling early resistance to L. major.     

Macrophages are a significant source of type 1 cytokines during mycobacterial infection

T-helper 1 (Th1) cells are believed to be the major producer of the type 1 cytokine interferon-gamma (IFN-gamma) in cell-mediated immunity against intracellular infection. We have investigated the ability of macrophages to release type 1 cytokines and their regulatory mechanisms using both in vivo and in vitro models of pulmonary mycobacterial infection. During pulmonary infection by live Mycobacterium bovis bacilli Calmette-Guérin (BCG) in wild-type mice, lung macrophages released interleukin-12 (IL-12), IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), and expressed surface activation markers. However, macrophages in infected IL-12(-/-) mice released TNF-alpha but not IFN-gamma and lacked surface activation makers. In freshly isolated lung macrophages from naive IL-2(-/-) mice, mycobacteria alone released TNF-alpha but not IFN-gamma, whereas exogenously added IL-12 alone released a minimum of IFN-gamma. However, these macrophages released large quantities of IFN-gamma upon stimulation with both mycobacteria and IL-12. In contrast, mycobacteria and exogenous IFN-gamma released only a minimum of endogenous IFN-gamma. Endogenous IL-18 (IFN-gamma-inducing factor) played little role in IFN-gamma responses by macrophages stimulated by mycobacteria and IL-12. Our data reveal that macrophages are a significant source of type 1 cytokines during mycobacterial infection and that both IL-12 and intracellular pathogens are required for the release of IFN-gamma but not TNF-alpha. These findings suggest that macrophages regulate cell-mediated immunity by releasing not only IL-12 and TNF-alpha but also IFN-gamma and that full activation of IFN-gamma response in macrophages is tightly regulated.