Differential control of IFN-gamma and IL-2 production during Trypanosoma cruzi infection

In murine infection with Trypanosoma cruzi, immune responsiveness to parasite and non-parasite Ag becomes suppressed during the acute phase of infection, and this suppression is known to extend to the production of IL-2. To determine whether suppression of lymphokine production was specific for IL-2, or was a generalized phenomenon involving suppressed production of other lymphokines, we have begun an investigation of the ability of mice to produce of a number of lymphokines during infection, initially addressing this question by studying IFN-gamma production. Supernatants from Con A-stimulated spleen cells from infected resistant (C57B1/6) and susceptible (C3H) mice were assayed for IFN-gamma. Supernatants known to be suppressed with respect to IL-2 production from both mouse strains contained IFN-gamma at or above that of supernatants from normal spleen cells. Samples were assayed in an IFN bioassay to ensure that the IFN-gamma detected by ELISA was biologically active. Thus, suppression during T. cruzi infection does not extend to the production of all lymphokines. The stimulation of IFN-gamma production was confirmed by detection of IFN-gamma mRNA in unstimulated spleen cells from infected animals, and in Con A, Con A + PMA, and in some cases, parasite Ag-stimulated spleen cells from infected animals. IFN-gamma mRNA levels in mitogen-stimulated spleen cells equalled or exceeded those found in similarly stimulated normal cells. In contrast, stimulated spleen cells from infected animals had reduced levels of IL-2 mRNA relative to normal spleen cells. Thus at both the protein and mRNA level, IFN-gamma production is stimulated by T. cruzi infection, whereas IL-2 production is suppressed. Serum IFN-gamma in infected C57B1/6 and C3H mice was detected 8 days after infection, peaked on day 20 of infection, and subsequently fell, but remained detectable at low levels throughout the life of infected mice. Infected animals were depleted of cell populations known to be capable of producing IFN-gamma, and Thy-1+, CD4-, CD8-, NK- cells, and to a lesser degree, CD4+ and CD8+ cells were found to be responsible for the production of IFN-gamma during infection. We also report that IL-2 can induce IFN-gamma production in vitro and in vivo by spleen cells from infected animals, and that IL-2 can synergize with epimastigote or trypomastigote antigen to produce high levels of IFN-gamma comparable to those found in supernatants from mitogen-stimulated cells.     

The role of B cells in the establishment of T cell response in mice infected with an intracellular bacteria, Listeria monocytogenes.

To clarify the role of B cells in the establishment of T cell response against intracellular bacteria, B-cell-deficient (muMT-/-) mice were infected with an intracellular bacteria, Listeria monocytogenes, and T cell response against the bacteria was analyzed. On day 6 of primary Listeria infection, spleen T cells of the muMT-/- mice showed significantly lower levels of proliferative response and IFN-gamma production than those of normal infected mice after in vitro stimulation with listerial antigen. Even in the secondary Listeria infection after immunization with viable bacteria, spleen T cells of the muMT-/- mice proliferated and produced IFN-gamma against listerial antigen at significantly lower levels than those of normal immunized mice. These results demonstrate participation of B cells in priming of Listeria-specific T cells in vivo. However, B cells failed to present Listeria antigen to Listeria-specific T cells in vitro unless Listeria antigen was solubilized. Furthermore, transfer of immune serum from Listeria-infected normal mice failed to enhance the Listeria-specific T cell response of muMT-/- mice. The results indicate that B cells support the T cell response against intracellular bacteria through a mechanism other than their Ig production or antigen presentation function.     

In vivo administration of recombinant IFN-gamma induces macrophage activation, and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections

Recombinant murine IFN-gamma (rMu-IFN-gamma) was demonstrated to be a potent in vivo activator of mouse peritoneal macrophages to kill Trypanosoma cruzi in vitro and to be capable of conferring protection against death from acute T. cruzi infection. Following i.p. injections of rMu-IFN-gamma, resident peritoneal macrophages were cultured and infected with T. cruzi in vitro. Numbers of intracellular parasites were determined at different times thereafter. Ten or 100 micrograms (1 microgram = 6.5 X 10(5) U) of Mu-IFN-gamma, injected both 24 and 4 h before macrophage harvest, induced up to 99% inhibition of T. cruzi. One microgram of rMu-IFN-gamma was not effective under these conditions. In vitro inhibition of T. cruzi by peritoneal macrophages occurred by 24 h after infection and continued until at least 120 h after infection. There were no significant differences in initial parasite uptake by macrophages from IFN-gamma-treated or control mice, indicating that the rMu-IFN-gamma induced parasite killing. One i.p. dose of 10 micrograms was as effective as two doses if the single injection was given 24 h before macrophage harvest. In subsequent experiments, mice were given multiple injections of 10 micrograms rMu-IFN-gamma beginning 24 h before or 2 h after infection with virulent T. cruzi. Mice treated with rMu-IFN-gamma had significantly lower parasitemias and decreased morbidity compared with control mice. Proliferative responses to Con A and antibody responses to SRBC were not significantly lowered in IFN-gamma-treated mice, in contrast to untreated infected controls. All of the IFN-gamma-treated mice survived acute T. cruzi infection, whereas 100% of saline-treated infected mice died. It was demonstrated in this study that rMu-IFN-gamma activated mouse macrophages in vivo to kill T. cruzi and that rMu-IFN-gamma significantly reduced morbidity and immune suppression, and eliminated mortality resulting from acute infection with this parasite.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.     

Induction of CTLA-4-mediated anergy contributes to persistent colonization in the murine model of gastric Helicobacter pylori infection

Helicobacter pylori infection induces gastric inflammation but the host fails to generate protective immunity. Therefore, we evaluated the immunologic mechanisms that contribute to the failure of the T cells to promote active immunity to H. pylori in the mouse model of H. pylori infection. Spleen cells from infected C57BL/6 mice underwent significantly less proliferation and cytokine production than cells from immune mice upon in vitro stimulation with H. pylori lysate. Similar results were observed when stimulating with Ag-pulsed macrophages demonstrating that hyporesponsiveness was not due to a direct effect of H. pylori virulence factors on the T cells. Ag-specific hyporesponsiveness could be reversed by the addition of high-dose IL-2 but not by removal of CD4(+)CD25(+) T cells, indicating that hyporesponsiveness was due to anergy and not due to active suppression. Cells from infected mice lacked significant suppressor activity as shown by the failure to reduce the recall response of cells from immune mice in coculture at physiologic ratios. Direct blockade of CTLA-4 using anti-CTLA-4 Fabs or indirect blockade using CTLA-4 Ig plus anti-CD28 Ab resulted in significantly increased T cell activation in vitro. The importance of CTLA-4 in establishing anergy was confirmed in an in vivo model of H. pylori infection in which mice that received anti-CTLA-4 Fabs responded to H. pylori challenge with significantly greater inflammation and significantly reduced bacterial load. These results suggest that CTLA-4 engagement induces and maintains functional inactivation of H. pylori-specific T cells during H. pylori infection resulting in a reduced immune response.     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.     

V gamma 1 gammadelta T cells regulate type-1/type-2 immune responses and participate in the resistance to infection and development of heart inflammation in Trypanosoma cruzi-infected BALB/c mice

Many different cell populations or lineages participate in the resistance to Trypanosoma cruzi infection. gammadelta T cells may also take part in a network of interactions that lead to control of T. cruzi infection with minimal tissue damage by controlling alphabeta T cell activation, as was previously suggested. However, the gammadelta T cell population is not homogeneous and its functions might vary, depending on T cell receptor usage or distinct stimulatory conditions. In this study, we show that the in vivo depletion of V gamma 1-bearing gammadelta T cells, prior to the infection of BALB/c mice with the Y strain of T. cruzi, induces an increased susceptibility to the infection with lower amounts of IFN-gamma being produced by conventional CD4+ or CD8+ T cells. In addition, the production of IL-4 by spleen T cells in V gamma 1-depleted mice was increased and the production of IL-10 remained unchanged. Since V gamma 1(+) gammadelta T cell depletion diminished the conversion of naive to memory/activated CD4 T cells and the production of IFN-gamma during the acute infection, these cells appear to function as helper cells for conventional CD4+ Th1 cells. Depletion of V gamma 1(+) cells also reduced the infection-induced inflammatory infiltrate in the heart and skeletal muscle. More importantly, V gamma 1(+) cells were required for up-regulation of CD40L in CD4+ and CD8+ T cells during infection. These results show that a subset of gammadelta T cells (V gamma 1(+)), which is an important component of the innate immune response, up-regulates the type 1 arm of the adaptative immune response, during T. cruzi infection.     

A role for CD44 in the production of IFN-gamma and immunopathology during infection with Toxoplasma gondii

The interaction of activated CD44 with its ligand, low m.w. hyaluronan, is involved in inflammation, but no role has been identified for this interaction in the regulation of an immune response to infection. In these studies, infection of C57BL/6 mice with Toxoplasma gondii resulted in increased expression of CD44 on T cells, B cells, NK cells, and macrophages, and a small percentage of CD4(+) T cells express an activated form of CD44. Administration of anti-CD44 to infected mice prevented the development of a CD4(+) T cell-dependent, infection-induced inflammatory response in the small intestine characterized by the overproduction of IFN-gamma. The protective effect of anti-CD44 treatment was associated with reduced production of IFN-gamma, but not IL-12, in vivo and in vitro. Furthermore, the addition of low m.w. hyaluronan to cultures of splenocytes or purified CD4(+) T cells from infected mice resulted in the production of high levels of IFN-gamma, which was dependent on IL-12 and TCR stimulation. Together, these results identify a novel role for CD44 in the regulation of IFN-gamma production by CD4(+) T cells during infection and demonstrate a role for CD44 in the regulation of infection-induced immune pathology.     

Immunisation with a major Trypanosoma cruzi antigen promotes pro-inflammatory cytokines, nitric oxide production and increases TLR2 expression

Innate and adaptive immunity collaborate in the protection of intracellular pathogens including Trypanosoma cruzi infection. However, the parasite molecules that regulate the host immune response have not been fully identified. We previously demonstrated that the immunisation of C57BL/6 mice with cruzipain, an immunogenic T. cruzi glycoprotein, induced a strong specific T-cell response. In this study, we demonstrated that active immunisation with cruzipain was able to stimulate nitric oxide (NO) production by splenocytes. Immune cells also showed increased inducible nitric oxide synthase protein and mRNA expression. Spleen adherent cells secreted high levels of IFN-gamma and IL-12. Microbicidal activity in vitro was mainly mediated by reactive nitrogen intermediaries and IFN-gamma, as demonstrated by the inhibitory effects of NO synthase inhibitor or by IFN-gamma neutralisation. Specific T-cells were essential for NO, IFN-gamma and TNF-alpha production. Furthermore, we reported that cruzipain enhanced CD80 and major histocompatibility complex-II molecule surface expression on F4/80+ spleen cells. Interestingly, we also showed that cruzipain up-regulated toll like receptor-2 expression, not only in F4/80+ but also in total spleen cells which may be involved in the effector immune response. Our findings suggest that a single parasite antigen such as cruzipain, through adaptive immune cells and cytokines, can modulate the macrophage response not only as antigen presenting cells, but also as effector cells displaying enhanced microbicidal activity with reactive nitrogen intermediary participation. This may represent a mechanism that contributes to the immunoregulatory process during Chagas disease.     

Protection against lethal toxoplasmosis in mice by an avirulent strain of Toxoplasma gondii: stimulation of IFN-gamma and TNF-alpha response

In this study, we examined whether the PTN strain (isolated from an AIDS patient) of Toxoplasma gondii could induce cross-protection in mice against infection with a lethal dose of the PLK strain. Mice were first infected with tachyzoites (5 x 10(5)) of PTN and 5 days later challenged with PLK (1 x 10(5), LD(90)) parasites. None of these mice succumbed to infection until day 21 after infection, whereas 100% of the mice given the same dose of PLK infection alone died between 5 and 11 days after infection. The protection was accompanied by an increased expansion of NK cells and CD4 + T cells. This condition was associated by increased production of IFN-gamma and an augmented number of IFN-gamma-producing cells in the spleen. Further, PTN + PLK-infected mice showed higher production of TNF-alpha and nitrite compared to PLK-infected mice. Mice infected with the PTN strain had an enhanced capacity to activate the immune system early in infection since they produced higher levels of IFN-gamma, TNF-alpha, and NO than PLK-infected mice. Administration of anti-IFN-gamma mAb or anti-asialo GM1 antibody resulted in 100 and 20% mortality, respectively, in PTN-infected mice but no death in PTN + PLK-infected mice. Together, these results suggest that early production of IFN-gamma and NK-cell activity is important in protection against PTN infection, whereas in PTN + PLK infection components of adaptive immunity rapidly developed following elaboration of an effective early innate immune response.     

Prostaglandins mediate suppression of lymphocyte proliferation and cytokine synthesis in acute Trypanosoma cruzi infection

Suppression of host lymphoproliferative responses to mitogens and Ag is characteristically seen during acute infection with the protozoan parasite Trypanosoma cruzi. We investigated the reciprocal regulation of prostaglandins (PG), TNF-alpha, and nitric oxide (NO) production and their effects on cytokine production and lymphoproliferative responses to parasite Ag and to Con A by spleen cells (SC) from T.-cruzi-infected mice. Large amounts of PGE2, TNF-alpha, and NO were produced during infection. TNF-alpha stimulated PG and NO synthesis, while both mediators inhibited TNF-alpha synthesis. Blocking PG also reduced NO synthesis indicating that PG stimulate NO production. Treatment with indomethacin or NMLA stimulated lymphoproliferation on days 6 and 22 of infection; on day 14, when suppression of proliferation and NO production was maximal, combined inhibition of NO and PG production restored parasite Ag specific and Con A proliferative responses. Blocking PG or NO production increased IL-2, IFN-gamma, and TNF-alpha, but not IL-12 production by SC; IL-10 levels were not reduced. Indomethacin-treated infected mice had higher mortality compared to untreated infected animals. The data indicate that PG, together with NO and TNF-alpha, participate in a complex circuit that controls lymphoproliferative and cytokine responses in T. cruzi infection.     

Circulating levels of cyclooxygenase metabolites in experimental Trypanosoma cruzi infections

Trypanosoma cruzi induces inflammatory reactions in several tissues. The production of prostaglandin F2alpha, 6-keto-prostaglandin F1alpha and thromboxane B2, known to regulate the immune response and to participate in inflammatory reactions, was studied in mice experimentally infected with T. cruzi. The generation of nitric oxide (NO), which could be regulated by cyclooxygenase metabolites, was also evaluated. In the acute infection the extension of inflammatory infiltrates in skeletal muscle as well as the circulating levels of cyclooxygenase metabolites and NO were higher in resistant C3H mice than in susceptible BALB/c mice. In addition, the spontaneous release of NO by spleen cells increased earlier in the C3H mouse strain. In the chronic infections, the tissue inflammatory reaction was still prominent in both groups of mice, but a moderate increase of thromboxane B2 concentration and in NO released by spleen cells was observed only in C3H mice. This comparative study shows that these mediators could be mainly related to protective mechanisms in the acute phase, but seem not to be involved in its maintenance in the chronic T. cruzi infections.     

Trypanosoma cruzi: roles for perforin-dependent and perforin-independent immune mechanisms in acute resistance

CD8+ T cells have been shown to be required for acute resistance to infection with the protozoan parasite, Trypanosoma cruzi, the causative agent of Chagas' disease. However, to date, the mechanism by which CD8+ T cells mediate protection in vivo has not been determined. While CD8+ T cells can exhibit cytolytic function, they also secrete cytokines such as IFN-gamma, which is known to mediate protection against T. cruzi infections. To determine whether cytolysis is an important effector function in vivo, we have compared outcomes of T. cruzi infection in normal and perforin-deficient mice. Our results indicate that while perforin-dependent cytolytic mechanisms clearly make a major contribution to acute resistance to T. cruzi infection, this contribution may be strain and challenge dose-dependent, since perforin-deficient mice challenged with lower doses of a less virulent strain survived and were subsequently resistant to challenge with virulent organisms. In vivo depletion studies demonstrated that survival of perforin-deficient mice challenged with low doses of T. cruzi requires both CD4+ and CD8+ T cells and is dependent on IFN-gamma secretion. These studies document the participation of both perforin-dependent cytotoxic and perforin-independent, IFN-gamma-dependent immune mechanisms in acute resistance to T. cruzi infection.     

Trypanosoma cruzi: maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.     

Immune deficiency in chronic Trypanosoma cruzi infection. Recombinant IL-1 restores Th function for antibody production

Mice with chronic Trypanosoma cruzi infections are unable to mount primary responses to T-dependent Ag, such as SRBC. Responses to SRBC were restored in vitro and in vivo with rIL-1. The cellular basis of the immunodeficiency and the mechanism of IL-1 action were investigated. B cells from infected mice were capable of normal levels of PFC production when provided with the appropriate signals, IL-2 plus IL-1. T cells from infected mice were unable to provide Th function to normal B cells. However, Th activity was provided by these cells if IL-1 was added to the cultures. Furthermore, T-depleted spleen cells from infected mice did not make antibody in the presence of normal T cells unless IL-1 was added to the cultures. Neutralizing antibody against IL-2 greatly reduced the augmentation by IL-1 of the antibody response of cells from infected mice. Together these results indicate that splenic B cells from infected mice are capable of antibody production, but that Th function is lacking in the spleens of infected mice. These results suggest that the inability of mice with T. cruzi infection to mount primary antibody responses to T-dependent Ag may be due to a macrophage defect lending to impairment of Th function. These results document the potential of IL-1 in restoring immune competence in an infectious disease model.     

Inhibitory receptors are expressed by Trypanosoma cruzi-specific effector T cells and in hearts of subjects with chronic Chagas disease

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4(+) T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4(+)CTAL-4(+) T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4(+)LIR-1(+) among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3(+) T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease.     

Leishmania sp: comparative study with Toxoplasma gondii and Trypanosoma cruzi in their ability to initialize IL-12 and IFN-gamma synthesis

We compared in vitro and in vivo induction of IL-12 (p40) and IFN-gamma by mouse cells stimulated with Toxoplasma gondii, Trypanosoma cruzi, and different species of Leishmania. Spleen cells cultured in vitro with T. cruzi or T. gondii, but not with Leishmania, produced IL-12 (p40) and IFN-gamma. Accordingly, IL-12 (p40) was produced by macrophages stimulated in vitro with live T. cruzi or T. gondii or membrane glycoconjugates obtained from trypomastigotes or tachyzoites. No IL-12 production was detected when macrophages were stimulated with live parasites or glycoconjugates from Leishmania, regardless of priming with IFN-gamma. In vivo, only T. cruzi and T. gondii induced the synthesis of IL-12 and IFN-gamma by mouse spleen cells after intraperitoneal injection of parasites. When injected subcutaneously, live Leishmania sp. induced IL-12 (p40) and IFN-gamma production by draining lymph node cells, albeit the levels were slightly lower than those induced by infection with T. gondii or T. cruzi using the same route. Together our results indicate that under different conditions, the intracellular protozoa T. gondii and T. cruzi are more potent stimulators of IL-12 and IFN-gamma synthesis by host immune cells than parasites of the genus Leishmania.