Susceptibility of interleukin-2-deficient mice to Toxoplasma gondii is associated with a defect in the production of gamma interferon

Costimulation through the B7-CD28 interaction is an important second signal for T-cell activation, and previous studies have shown that CD28(-/-) mice infected with Toxoplasma gondii generate suboptimal CD4(+) T-cell responses, associated with a defect in production of the T-cell growth factor interleukin-2 (IL-2). To address the role of IL-2 in the expansion of T cells during toxoplasmosis, IL-2(-/-) mice were infected with T. gondii and their ability to generate a protective T-cell response was assessed. Although IL-2(-/-) mice produced normal levels of IL-12p40, they had reduced levels of gamma interferon (IFN-gamma) in serum, had an increased parasite burden, and succumbed to infection with T. gondii within 20 days. Fluorescence-activated cell sorter analysis revealed that, although uninfected IL-2(-/-) mice had an increased number of activated T cells compared with uninfected IL-2(+/+) mice, following infection they were unable to further upregulate this population. Examination of the ability of splenocytes from uninfected and infected mice to produce IFN-gamma revealed that IL-2(-/-) mice were hyporesponsive to stimulation with anti-CD3 or parasite antigen compared with wild-type mice, and the addition of IL-2 alone or in combination with IL-12 or stimulation with phorbol myristate acetate and ionomycin did not restore the production of IFN-gamma. Together, these studies reveal that IL-2(-/-) mice are unable to generate a protective IFN-gamma response following infection with T. gondii and suggest that IL-2(-/-) mice have an intrinsic defect in their ability to activate and expand IFN-gamma-producing T cells required for resistance to T. gondii.     

Contribution of interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 ligand pathways to the development of a pathological T-cell response in IL-10-deficient mice

The ability of interleukin-10 (IL-10) to suppress accessory cell functions required for optimal T-cell activation makes it an important inhibitor of cell-mediated immunity. Thus, after infection with the protozoan parasite Toxoplasma gondii, IL-10 knockout (KO) mice develop a CD4(+)-T-cell-dependent shock-like reaction with high levels of IL-12 and gamma interferon (IFN-gamma) in serum, leading to death of mice during the acute phase of infection. Previous studies from this laboratory have shown that simultaneous blockade of CD28 and CD40 can prevent this lethal reaction by inhibiting the production of IFN-gamma. However, the blockade of costimulation did not affect systemic levels of IL-12. To better understand the relationship between IL-12 and the CD28 and CD40 pathways in mediating immune hyperactivity, antagonists of these factors were used to determine their effects on the development of a pathological T-cell response in IL-10 KO mice. Blockade of IL-12 or the CD28/B7 interaction alone did not affect survival; however, the combined blockade of both pathways resulted in decreased production of IFN-gamma and the survival of IL-10 KO mice. To assess the role of the two ligands for CD28, B7.1 and B7.2, IL-10 KO mice were treated with alphaIL-12 plus alphaB7.1 or alphaB7.2 or the combination of all three antibodies. These studies revealed that blockade of both B7 molecules is required for decreased production of IFN-gamma and survival of infected IL-10 KO mice, suggesting that B7.1 and B7.2 can contribute to the lethal shock-like reaction in IL-10 KO mice. In contrast, neutralization of IL-12 and blockade of the CD40/CD40 ligand (CD40L) interaction in vivo did not alter the production of IFN-gamma and only resulted in a small delay in time to death of mice. Together, these data suggest that the CD28/B7 interaction has a central role in the development of a pathological T-cell response in IL-10 KO mice, which is distinct from the role of the CD40/CD40L and IL-12 pathways.     

CD40 restrains in vivo growth of Toxoplasma gondii independently of gamma interferon

CD40-CD154 interaction is pivotal for resistance against numerous pathogens. However, it is not known if this pathway can also enhance in vivo resistance in gamma interferon (IFN-gamma)-deficient hosts. This is an important question because patients and mice with defects in type 1 cytokine response can control a variety of pathogens. While blockade of endogenous CD154 resulted in a remarkable increase in parasite load in IFN-gamma-/- mice infected with Toxoplasma gondii, in vivo administration of a stimulatory anti-CD40 monoclonal antibody markedly reduced parasite load. This latter effect took place even in T-cell-depleted mice and was accompanied by induction of macrophage toxoplasmacidal activity. CD40 stimulation restricted T. gondii replication independently of STAT1, p47 GTPases, and nitric oxide. In vivo CD40 ligation enhanced tumor necrosis factor alpha (TNF-alpha) production by T. gondii-infected macrophages. In addition, CD40 stimulation required the presence of TNF receptor 2 to reduce parasite load in vivo. These results suggest that CD40-CD154 interaction regulates IFN-gamma-independent mechanisms of host protection through induction of macrophage antimicrobial activity and modulation of TNF-alpha signaling.     

Blockade of costimulation prevents infection-induced immunopathology in interleukin-10-deficient mice

Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-gamma) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-gamma or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-gamma and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-gamma but not IL-12. Further reduction of IFN-gamma production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.     

Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1-type immunopathology mediated by local production of IFN-gamma, TNF-alpha, and NO. To analyze whether the proinflammatory cytokines IL-12 and IL-18 play a role in the induction of immunopathology, IL-12p35/p40(-/-) and IL-18(-/-) mice were orally infected with T. gondii. Wild-type mice developed massive necrosis in their small intestines and died 7-10 days post infection. Even though IL-12p35/40(-/-) mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL-18(-/-) mice died during the acute phase of infection. Compared to wild-type mice, IL-12p35/p40(-/-) but not IL-18(-/-) mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite-associated inflammatory foci in their livers. IFN-gamma production was similar in infected wild-type and IL-18(-/-) mice but significantly decreased in IL-12p35/p40(-/-) mice. Treatment of mice with anti-IL-12- or anti-IL-18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL-12 and IL-18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL-12 is dominant over IL-18 in the host defense against parasite replication. Therefore, neutralization of IL-18 (rather than TNF-alpha, IL-12, and IFN-gamma) may be a safe strategy for the treatment of Th1-associated diseases.     

Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii.

Interleukin-10 (IL-10) is a cytokine which can inhibit T-cell and natural killer (NK) cell functions associated with cell-mediated immunity to intracellular infections. The production of IL-10 by mice infected with Toxoplasma gondii has been implicated in the suppression of lymphocyte proliferation observed during acute toxoplasmosis, as well as susceptibility to infection with this parasite. We have used C57BL/6 mice which lack a functional IL-10 gene (IL-10(-/-) mice) to investigate the role of IL-10 in acute toxoplasmosis. Intraperitoneal infection of IL-10(-/-) mice with T. gondii resulted in 100% mortality by day 13, whereas wild-type C57BL/6 (WT) mice survived acute infection. IL-10(-/-) mice infected with T. gondii had significantly higher serum levels of IL-12 and gamma interferon (IFN-gamma) than WT mice. Early mortality of infected IL-10(-/-) mice was prevented by treatment with IL-10 and significantly delayed by neutralizing antibodies to IL-12 and IFN-gamma. Further studies revealed that SCID/IL-10(-/-) mice infected with T. gondii had delayed time to death compared to IL-10(-/-) mice, indicating that lymphocytes contributed to death of IL-10(-/-) mice. In addition, infected SCID/IL-10(-/-) mice survived longer than infected SCID mice. These latter data indicate that in mice lacking lymphocytes, endogenous IL-10 is associated with increased susceptibility to T. gondii. However, the lack of IL-10 does not alter the infection-induced suppression of T cell and NK cell functions. Our experiments reveal that IL-10 is associated with protection or increased susceptibility to infection with T. gondii, depending on whether mice possess lymphocytes, and demonstrate the important roles of IL-12 and IFN-gamma in the early infection-induced mortality observed in the IL-10(-/-) mice.     

Role of interleukin-1beta in activating the CD11c(high) CD45RB- dendritic cell subset and priming Leishmania amazonensis-specific CD4+ T cells in vitro and in vivo

Cutaneous leishmaniasis associated with Leishmania amazonensis infection is characterized by uncontrolled parasite replication and profound immunosuppression; however, the underlying mechanisms remain largely unclear. One possibility is that the L. amazonensis parasite modulates antigen-presenting cells, favoring the generation of pathogenic Th cells that are capable of recruiting leukocytes but insufficient to fully activate their microbicidal activities. To test this possibility, we infected bone marrow-derived dendritic cells (DCs) of C57BL/6 mice with L. amazonensis or Leishmania major promastigotes and assessed the activation of DC subsets and their capacity in priming CD4(+) T cells in vitro. In comparison to L. major controls, L. amazonensis-infected DCs secreted lower levels of interleukin-1alpha (IL-1alpha) and IL-1beta, were less potent in activating the IL-12p40-producing CD11c(high) CD45RB(-) CD83(+) CD40(+) DC subset, and preferentially activated CD4(+) T cells with a IFN-gamma(low) IL-10(high) IL-17(high) phenotype. Although the addition of IL-1beta at the time of infection markedly enhanced DC activation and T-cell priming, it did not skew the cytokine profile of DCs and pathogenic Th cells, as local injection of IL-1beta following L. amazonensis infection accelerated Th cell activation and disease progression. This study suggests that intrinsic defects at the level of DC activation are responsible for the susceptible phenotype in L. amazonensis-infected hosts and that this parasite may have evolved unique mechanisms to interfere with innate and adaptive immunity.     

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

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

CD8+-T-Cell Immunity against Toxoplasma gondii Can Be Induced but Not Maintained in Mice Lacking Conventional CD4+ T Cells

T-cell immunity is critical for survival of hosts infected with Toxoplasma gondii. Among the cells in the T-cell population, CD8(+) T cells are considered the major effector cells against this parasite. It is believed that CD4(+) T cells may be crucial for induction of the CD8(+)-T-cell response against T. gondii. In the present study, CD4(-/-) mice were used to evaluate the role of conventional CD4(+) T cells in the immune response against T. gondii infection. CD4(-/-) mice infected with T. gondii exhibited lower gamma interferon (IFN-gamma) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly, T. gondii infection induced a normal antigen-specific CD8(+)-T-cell immune response in CD4(-/-) mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-gamma production by the CD8(+)-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8(+)-T-cell immunity. At 180 days after infection, the CD8(+)-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-gamma assays. Loss of CD8(+)-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8(+) T cells from CD4(-/-) donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8(+)-T-cell immunity can be induced in the absence of conventional CD4(+) T cells, it cannot be maintained without such cells.     

CD40-CD40 ligand costimulation is not required for initiation and maintenance of a Th1-type response to Leishmania major infection

Although previous studies demonstrated a requirement for CD40-CD40 ligand (CD40L) interaction in the development of resistance to Leishmania infection, we recently showed that mice lacking the gene for CD40L (CD40L(-/-) mice) can control Leishmania major infection when they are infected with reduced numbers of parasites. In this study, we examine the cytokine pattern in healing versus nonhealing CD40L(-/-) mice and investigated whether CD40 activation is required for resistance to reinfection. We observed that CD4(+) cells in healed CD40L(-/-) mice produce high levels of gamma interferon compared to cells from nonhealing, high-dose-inoculated mice. In addition, we observed a higher frequency of interleukin-12 (IL-12)- producing cells and a reduced number of IL-4-producing cells in mice infected with reduced numbers of parasites. Importantly, we found that healed CD40L(-/-) mice are highly resistant to reinfection with a large parasite inoculum. In addition, by comparing the cytokine patterns at an early and late stage of infection in nonhealing CD40L(-/-) mice, we demonstrated that nonhealing CD40L(-/-) mice produce a weak Th1-type response during the early stage of infection, but this response wanes as a Th2-type response emerges during late stages of infection. Anti-IL-4 antibody treatment, starting either at the beginning of infection or at week 4 postinfection enabled CD40L(-/-) mice to control a high-dose infection. Together, these results show that CD40-CD40L interaction, although important for IL-12 production in high-dose infections, is not required for either the development or maintenance of resistance in mice infected with reduced numbers of parasites.     

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.     

Organ- and disease-stage-specific regulation of Toxoplasma gondii-specific CD8-T-cell responses by CD4 T cells

Toxoplasma gondii induces a persistent central nervous system infection, which may be lethally reactivated in AIDS patients with low CD4 T-cell numbers. To analyze the role of CD4 T cells for the regulation of parasite-specific CD8 T cells, mice were infected with transgenic T. gondii expressing the CD8 T-cell antigen beta-galactosidase (beta-Gal). Depletion of CD4 T cells prior to infection did not affect frequencies of beta-Gal(876-884)-specific (consisting of residues 876 to 884 of beta-Gal) CD8 T cells but resulted in a pronounced reduction of intracerebral beta-Gal-specific gamma interferon (IFN-gamma)-producing and cytolytic CD8 T cells. After cessation of anti-CD4 treatment a normal T. gondii-specific CD4 T-cell response developed, but IFN-gamma production of intracerebral beta-Gal-specific CD8 T cells remained impaired. The important supportive role of CD4 T cells for the optimal functional activity of intracerebral CD8 T cells was also observed in mice that had been depleted of CD4 T cells during chronic toxoplasmosis. Reinfection of chronically infected mice that had been depleted of CD4 T cells during either the acute or chronic stage of infection resulted in an enhanced proliferation of beta-Gal-specific IFN-gamma-producing splenic CD8 T cells. However, reinfection of chronically infected mice that had been depleted of CD4 T cells in the acute stage of infection did not reverse the impaired IFN-gamma production of intracerebral CD8 T cells. Collectively, these findings illustrate that CD4 T cells are not required for the induction and maintenance of parasite-specific CD8 T cells but, depending on the stage of infection, the infected organ and parasite challenge infection regulate the functional activity of intracerebral CD8 T cells.     

IL-18, but not IL-12, is required for optimal cytokine production by influenza virus-specific CD8+ T cells

The potent innate cytokines IL-12 and IL-18 are considered to be important antigen-independent mediators of IFN-gamma production by NK cells and T lymphocytes. The present analysis addresses the physiological role of IL-12 and IL-18 in the generation of virus-specific CD8+ T cells. Both wt C57BL/6J (B6) mice and mice with disrupted IL-12p40 (IL-12p40(-/-)) or IL-18 (IL-18(-/-)) genes were infected with an influenza A virus and the characteristics of the resultant epitope-specific CD8+ T cell responses were compared. While IL-12 appeared to have no notable effect on either virus growth or on CD8+ T cell response profiles, the absence of IL-18 was associated with delayed virus clearance from the lung and, despite normal numbers, a significantly reduced production of IFN-gamma, TNF-alpha, and IL-2 by epitope-specific CD8+ T cells. While this cytokine phenotype was broadly maintained in IL-12p40/IL-18 double-knockout mice, no evidence was seen for any additive effect. Together, our results suggest that IL-18, but not IL-12, induces optimal, antigen-specific production of key cytokines by CD8+ T cells for the efficient clearance of influenza virus from the lungs of infected mice.     

Role of CD40 Ligand in Mycobacterium avium Infection

Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4(+) cell counts of <50 cells/microliters, suggesting a possible involvement of a T cell-macrophage interaction for the elimination of M. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. avium infection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth of M. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% +/- 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 micrograms/ml) inhibited intracellular growth of M. avium by 76.9% +/- 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium-infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment of M. avium-infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection.     

Vaccine-induced protection against Leishmania amazonensis is obtained in the absence of IL-12/23p40

Protozoa of the genus Leishmania are intracellular parasites of macrophages and may cause diverse clinical forms of leishmaniasis, including cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Infection with L. major in mice indicates that a protective immune response is achieved when Th1 cells are developed. Thus, adoptive or vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated immunity and IFN-gamma production. Induction of a Th1 response is dependent on the presence of IL-12 whilst lymphocytes are activated. This study was aimed at evaluating the role of IL-12 during infection with L. amazonensis and after vaccination with Leishvacin (killed Leishmania amazonensis promastigotes), since the role of this cytokine in vaccine-induced immunity with this preparation in experimental models or in humans is not yet elucidated. Hence, C57BL/6 interleukin-12-deficient mice (IL-12p40(-/-)) and wild-type controls (wt) were infected with L. amazonensis and the course of infection, parasite burden and cytokine production were compared. IL-12p40(-/-) mice were more susceptible to L. amazonensis than wt: lesions and parasite burden were larger in IL-12p40(-/-) when compared to wt. Interestingly, IL-4 was not produced in the absence of IL-12 in response to infection with L. amazonensis. To evaluate the role of IL-12 in the vaccine-induced immunity against L. amazonensis infection, IL-12p40(-/-) wt mice were vaccinated in the base of the tail and subsequently challenged with L. amazonensis in the footpads. Surprisingly, vaccinated IL-12p40(-/-) mice developed smaller lesions and had fewer parasites in footpads than non-vaccinated controls. Lymph node and spleen cells from vaccinated IL-12p40(-/-) mice did not produce high levels of IFN-gamma in response do in vitro stimulus with antigen. Hence, partial protection against infection with L. amazonensis could be obtained in the absence of functional IL-12 and a typical Th1 response.     

Opportunistic infections and retrovirus-induced immunodeficiency: studies of acute and chronic infections with Toxoplasma gondii in mice infected with LP-BM5 murine leukemia viruses.

Mice infected with LP-BM5 murine leukemia viruses develop a syndrome, termed mouse AIDS (MAIDS), characterized by increasingly severe immunodeficiency and progressive lymphoproliferation. Virus-infected mice were examined for the ability to resist acute infection and to control chronic infection with the protozoan Toxoplasma gondii, a major opportunistic pathogen of individuals infected with human immunodeficiency virus. Mice infected with the retroviruses for 2 or 4 weeks responded normally to challenge with the parasite, but mice inoculated with the protozoan 8 or 12 weeks after viral infection died with acute disease due to T. gondii. Increased sensitivity to acute infection was associated with a reduced ability to produce gamma interferon (IFN-gamma) and with established changes in CD4+ T-cell function. Mice latently infected with T. gondii and then inoculated with the retrovirus mixture were found to reactivate the parasite infection, with 30 to 40% of dually infected animals dying between 5 and 16 weeks after viral infection. Reactivation was associated with reduced proliferation and impaired production of IFN-gamma in response to stimulation with soluble T. gondii antigens or to concanavalin A. Continuing resistance to lethal reactivation in the remaining mice was shown to require CD8+ T cells and expression of IFN-gamma. In addition, it was found that chronic infection with T. gondii altered the course of MAIDS by inhibiting the progression of splenomegaly and immunodeficiency and reducing the expression of both the helper and etiologic defective viruses. These results support previous studies which indicate that infection with T. gondii is controlled by synergistic interactions between CD4+ and CD8+ T cells, the functions of which are progressively impaired during the course of MAIDS.     

Rapid elimination of Toxoplasma gondii by gamma interferon-primed mouse macrophages is independent of CD40 signaling

Autophagy has been implicated in the intracellular destruction of Toxoplasma gondii by primed macrophages following gamma interferon (IFN-gamma) activation of p47 GTPases. CD40 ligation has also been shown to trigger autophagic elimination of T. gondii independent of IFN-gamma and p47 GTPases. Here we demonstrate that IFN-gamma/p47 GTPase-dependent elimination of T. gondii by strain CPS vaccine-primed macrophages is independent of CD40/tumor necrosis factor signaling. Similar to wild-type controls, both CD40-deficient and tumor necrosis factor receptor 1/2 (TNFR1/2)-deficient macrophages can efficiently eliminate invaded strain GFP-PTG and restrain its replication following priming. In contrast, macrophages from mice lacking the IFN-gamma receptor gene neither clear the parasites nor repress their proliferation. Thus, CD40 and IFN-gamma-induced pathogen elimination might represent two independent resistance pathways, the latter of which plays a primary role in anti-Toxoplasma immunity in mice.     

CD40, but not CD40L, is required for the optimal priming of T cells and control of aerosol M. tuberculosis infection

CD40(-/-) mice succumbed to low-dose aerosol infection with M. tuberculosis due to deficient IL-12 production leading to impaired priming of IFN-gamma T cell responses. In contrast, CD40L(-/-) mice were resistant to M. tuberculosis. This asymmetry in outcome of infection between the two knockout strains is likely due to the existence of an alternative ligand for CD40. Both in vitro M. tuberculosis infection and recombinant M. tuberculosis Hsp70 elicited IL-12 production from WT dendritic cells. This response was absent in both CD40(-/-) dendritic cells and CD40(-/-) mice, suggesting that M. tuberculosis Hsp70 serves as an alternative ligand for CD40 in vivo.     

Modulation of T-cell costimulation as immunotherapy or immunochemotherapy in experimental visceral leishmaniasis

CD40 ligand (CD40L)-deficient C57BL/6 mice failed to control intracellular Leishmania donovani visceral infection, indicating that acquired resistance involves CD40-CD40L signaling and costimulation. Conversely, in wild-type C57BL/6 and BALB/c mice with established visceral infection, injection of agonist anti-CD40 monoclonal antibody (MAb) induced killing of approximately 60% of parasites within liver macrophages, stimulated gamma interferon (IFN-gamma) secretion, and enhanced mononuclear cell recruitment and tissue granuloma formation. Comparable parasite killing was also induced by MAb blockade (inhibition) of cytotoxic T lymphocyte antigen-4 (CTLA-4) which downregulates separate CD28-B7 T-cell costimulation. Optimal killing triggered by both anti-CD40 and anti-CTLA-4 required endogenous IFN-gamma and involved interleukin 12. CD40L(-/-) mice also failed to respond to antileishmanial chemotherapy (antimony), while in normal animals, anti-CD40 and anti-CTLA-4 synergistically enhanced antimony-associated killing. CD40L-CD40 signaling regulates outcome and response to treatment of experimental visceral leishmaniasis, and MAb targeting of T-cell costimulatory pathways (CD40L-CD40 and CD28-B7) yields macrophage activation and immunotherapeutic and immunochemotherapeutic activity.