Challenge of Chronically Infected Mice with Homologous Trypanosoma cruzi Parasites Enhances the Immune Response but Does Not Modify Cardiopathy: Implications for the Design of a Therapeutic Vaccine

Chagas disease is a Trypanosoma cruzi-induced zoonosis that has no natural cure. Local damage induced by the parasite and the immune response causes chronic heart and digestive lesions. Efforts to develop a therapeutic vaccine that boosts the immune response to completely clear the parasite are needed because there is no effective treatment for chronically infected patients. In an attempt to modify the host-parasite equilibrium to increase parasite destruction, we analyzed cardiopathy and the immune response in chronically infected mice that were challenged with live homologous parasites. Challenge with a single dose of parasite increased CD4⁺ and CD8⁺ T cell populations, gamma interferon (IFN-γ) production, and serum-specific IgG levels. However, subpatent parasitemias and cardiac tissue were not affected. Because of the short duration of the immune boost after a single challenge, we next evaluated the impact of four parasite doses, administered 3 weeks apart. At 1 to 2 months after the last dose, the numbers of CD4⁺ T cells and IFN-γ-producing CD4⁺ memory cells and the CD4⁺ T cell proliferative response to T. cruzi antigen were increased in the spleen. The frequency of IFN-γ-producing CD8⁺ memory cells in the blood was also increased. However, the sustained challenge did not favor TH1 development; rather, it induced an increase in serum-specific IgG1 levels and mixed TH1/TH2 cytokine production. Moreover, there were no significant changes in cardiac lesions and subpatent parasitemias. In conclusion, we believe that this study may help in elucidating the necessary elements for a successful therapeutic vaccine which may reduce cardiomyopathy in chronically infected human patients.     

Regulatory T Cells Modulate Staphylococcal Enterotoxin B-Induced Effector T-Cell Activation and Acceleration of Colitis

Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after reconstitution with CD4⁺ CD45RB^high or CD4⁺ CD45RB^high plus CD4⁺ CD45RB^low T cells. Mice were sacrificed at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to produce any clinical effect on SCID mice reconstituted with CD4⁺ CD45RB^high and CD4⁺ CD45RB^low T cells, but feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone. The latter was associated with an increase in the number of CD4⁺ Vβ8⁺ T cells expressing CD69 and a significantly lower number of CD4⁺ CD25⁺ Foxp3⁺ T cells. These changes were not observed in SCID mice reconstituted with both CD45RB^high and CD45RB^low T cells. In addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.     

Generation of Trypanosoma cruzi-Specific CD8+ T-Cell Immunity Is Unaffected by the Absence of Type I Interferon Signaling

Trypanosoma cruzi is a protozoan parasite that causes human Chagas’ disease, a leading source of congestive heart failure in Central and South America. CD8⁺ T cells are critical for control of T. cruzi infection, and CD8⁺ T cells recognizing the immunodominant trans-sialidase gene-encoded peptide TSKB20 (ANYKFTLV) account for approximately 30% of the total CD8⁺ T-cell population at the peak of infection in C57BL/6 mice. Type I interferons (IFN-I) are pleiotropic cytokines that play a critical role in both innate and adaptive immunity against a variety of infections, but their induction and their role in infection are dictated by the infectious agent. Because type I IFNs and IFN-responsive genes are evident early after T. cruzi infection of host cells, we examined the influence of IFN-I on the development of CD8⁺ T-cell responses during this infection. Mice lacking the receptor for IFN-I (IFNARKO) and their wild-type counterparts both developed chronic infections and generated similar frequencies of immunodominant TSKB20- and subdominant TSKB18-specific CD8⁺ T cells following T. cruzi infection. In contrast, peak TSKB20-specific CD8⁺ T-cell responses generated during infection with vaccinia virus engineered to express TSKB20 were approximately 2.5-fold lower in IFNARKO mice than B6 mice, although after viral clearance, the frequencies of TSKB20-specific CD8⁺ T cells stabilized at similar levels. Together, these data suggest that IFN-I induction and biology are dependent upon the microbial context and emphasize the need to investigate various infection models for a full understanding of CD8⁺ T-cell development.CD8⁺ T cells are an essential contributor to the control of the intracellular protozoan pathogen Trypanosoma cruzi, the causative agent of human Chagas'' disease. T. cruzi trypomastigotes can infect virtually any nucleated cell, and once in the host cell cytoplasm, transform into and replicate as amastigotes. The cytoplasmic localization of T. cruzi allows for parasite-derived proteins to be available for processing and presentation through the major histocompatibility complex class I (MHC-I) pathway, and obliteration of this pathway leads to early death for T. cruzi-infected mice (21, 22). T. cruzi-infected mice depleted of CD8⁺ T cells also succumb to infection during the acute phase (within 35 days after infection) (20). The annotated genome of T. cruzi contains more than 12,000 genes (5), yet despite this genetic complexity and the resultant enormous number of potential epitopes, the CD8⁺ T-cell response to T. cruzi is focused on only a few peptides. In B6 mice infected with the Brazil strain of T. cruzi, CD8⁺ T cells recognizing TSKB20 (ANYKFTLV) and TSKB18 (ANYDFTLV) expand significantly and represent over 40% of the total CD8⁺ T-cell population at the peak of the immune response (12).Type I interferons (IFN-I) are a complex set of innate cytokines induced early after infection. IFN-β and the multiple IFN-α species all act through a heterodimeric receptor (IFNAR) and have pleiotropic effects on both innate and adaptive immune responses, including MHC upregulation, B-cell isotype switching, and NK cell activation, as well as providing direct survival signals to CD8⁺ and CD4⁺ T cells (16). Although type I IFNs appear to be essential for antiviral innate response (19), their necessity in other infections is less well documented. Recently, type I interferons have also been shown to govern the expansion of antiviral CD8⁺ T cells (1, 7, 8, 10, 18).Global cytokine analysis of T. cruzi-infected cells has demonstrated that infection of host cells in vitro by T. cruzi is relatively “quiet,” with the most notable response being the production of IFN-I and IFN-I-responsive genes (ISGs) (24). In vivo studies have also shown early induction of IFN-I and ISGs (3, 17). Given the evidence for early production of IFN-I in T. cruzi infection and the role this cytokine has been shown to play in the generation of protective immunity in other infection models, in this study we have examined the contribution of IFN-I to the immunodominant CD8⁺ T-cell response to T. cruzi.     

Immunodepression Induced by Trypanosoma cruzi and Mouse Hepatitis Virus Type 3 Is Associated with Thymus Apoptosis

Trypanosoma cruzi-infected mice show disturbance in the peripheral immune system such as polyclonal lymphocyte activation, autoantibody production, and immunosuppression of T lymphocytes. Previous observations in our laboratory showed that some stocks of T. cruzi can be contaminated with mouse hepatitis virus type 3 (MHV-3). Literature has shown that MHV-3 infection induces immunologic disorders characterized by thymic involution with marked cell depletion. However, the effects of interactions between MHV-3 and the parasite on the immune system are not well understood. In the present study specific-pathogen-free CBA mice were inoculated with MHV-3, alone or associated with different stocks of T. cruzi. Concurrent murine virus infection resulted in increased pathogenicity of T. cruzi infection shown by profound thymic atrophy; loss of cortical thymocytes; depletion of Thy1.2⁺, CD4⁺, and CD8⁺ cells; enhancement of in situ labeling of nuclear DNA fragmentation; and eventually, death of the animals. Such lines of evidence show that the mechanism underlying this thymic atrophy is associated with apoptosis. These results also suggest that MHV-3 can account for the increased immunosuppression observed during experimental infection with the parasite.     

Coinfection with Heligmosomoides polygyrus fails to establish CD8+ T-cell immunity against Toxoplasma gondii

CD8⁺ T-cell immunity is important for long-term protection against Toxoplasma gondii infection. However, a Th1 cytokine environment, especially the presence of gamma interferon (IFN-γ), is essential for the development of primary CD8⁺ T-cell immunity against this obligate intracellular pathogen. Earlier studies from our laboratory have demonstrated that mice lacking optimal IFN-γ levels fail to develop robust CD8⁺ T-cell immunity against T. gondii. In the present study, induction of primary CD8⁺ T-cell immune response against T. gondii infection was evaluated in mice infected earlier with Heligmosomoides polygyrus, a gastrointestinal worm known to evoke a polarized Th2 response in the host. In the early stage of T. gondii infection, both CD4 and CD8⁺ T-cell responses against the parasite were suppressed in the dually infected mice. At the later stages, however, T. gondii-specific CD4⁺ T-cell immunity recovered, while CD8⁺ T-cell responses remained low. Unlike in mice infected with T. gondii alone, depletion of CD4⁺ T cells in the dually infected mice led to reactivation of chronic infection, leading to Toxoplasma-related encephalitis. Our observations strongly suggest that prior infection with a Th2 cytokine-polarizing pathogen can inhibit the development of CD8⁺ T-cell immune response against T. gondii, thus compromising long-term protection against a protozoan parasite. This is the first study to examine the generation of CD8⁺ T-cell immune response in a parasitic nematode and protozoan coinfection model that has important implications for infections where a CD8⁺ T-cell response is critical for host protection and reduced infection pathology.     

Effector CD8+ T lymphocytes against liver stages of Plasmodium yoelii do not require gamma interferon for antiparasite activity

The protective immune response against liver stages of the malaria parasite critically requires CD8⁺ T cells. Although the nature of the effector mechanism utilized by these cells to repress parasite development remains unclear, a critical role for gamma interferon (IFN-γ) has been widely assumed based on circumstantial evidence. However, the requirement for CD8⁺ T-cell-mediated IFN-γ production in protective immunity to this pathogen has not been directly tested. In this report, we use an adoptive transfer strategy with circumsporozoite (CS) protein-specific transgenic T cells to examine the role of CD8⁺ T-cell-derived IFN-γ production in Plasmodium yoelii-infected mice. We show that despite a marginal reduction in the expansion of naive IFN-γ-deficient CS-specific transgenic T cells, their antiparasite activity remains intact. Further, adoptively transferred IFN-γ-deficient CD8⁺ T cells were as efficient as their wild-type counterparts in limiting parasite growth in naive mice. Taken together, these studies demonstrate that IFN-γ secretion by CS-specific CD8⁺ T cells is not essential to protect mice against live sporozoite challenge.     

Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection

DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8⁺ cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2^b and H-2^d mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2^b mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2K^b-restricted TSA-1_515–522 epitope. In H-2^d mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8⁺-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2^b and 16 of 18 (89%) H-2^d mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas’ disease.

Chagas’ disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a lifelong health problem in Central and South America, where an estimated 18 million people are infected with this parasite and 90 million are at risk of infection (35, 65). Following a short-lived acute-phase illness characterized by fever and a patent parasitemia, infected individuals enter a nearly aparasitemic asymptomatic chronic phase, where most remain for the remainder of their lifetime. However, at 10 to 20 years postinfection nearly 30% of infected individuals develop severe cardiomyopathy, which is responsible for most of the 50,000 deaths caused by Chagas’ disease each year (45). Although reduviid vector control and blood bank screening measures have had a major impact in reducing transmission of T. cruzi (65), the operational costs to maintain such control programs, behavioral differences among vector species, existence of animal reservoirs, persistence of parasites in chronically infected patients, and lack of adequate chemotherapies to treat the infection will likely prevent these control measures alone from completely eradicating T. cruzi. An additional approach that could contribute significantly to control the transmission of Chagas’ disease is the development of anti-T. cruzi vaccines. To date, however, vaccine production for T. cruzi has been a low priority despite the current knowledge about the protective roles that antibodies, type 1 cytokines, and CD8⁺ T cells play in resistance to experimental T. cruzi infections (53).During T. cruzi infection, both chagasic patients and experimental animals produce strong immune responses to molecules from the infective nonreplicative trypomastigote stage and the replicative amastigote forms (3, 4, 14, 29). Among these, trypomastigote surface antigen 1 (TSA-1) (15, 38), a major trypomastigote surface antigen and the first identified member of the trans-sialidase gene superfamily (48), is a target of protective immune responses in mice (61, 66). Immunization with an amino-proximal fragment of TSA-1 induces a strong antibody response and protects mice against an otherwise lethal challenge with T. cruzi (66). Our studies have recently identified TSA-1 as the first bona fide target of CD8⁺ cytotoxic T lymphocytes (CTL) in T. cruzi-infected mice and demonstrated that the adoptive transfer of TSA-1-specific gamma interferon (IFN-γ)- and tumor necrosis factor alpha-producing CTL lines protects naive animals against lethal T. cruzi infection (61). Moreover, we have recently determined that TSA-1 and amastigote surface protein-1 and -2 (33, 44), which are also recognized by murine CTL (32), represent three target molecules of T. cruzi-specific human CD8⁺ CTL (62). These studies demonstrated the validity of the mouse model to identify target antigens of protective anti-T. cruzi immune responses and provide a strong incentive for the development of vaccines as a potential control measure against Chagas’ disease. For this purpose, and given the success of plasmid DNA vaccination in specifically stimulating a broad spectrum of immune responses to the vector-encoded target antigen (12), we have chosen to investigate DNA-based immunization as a system to generate vaccine-induced resistance against T. cruzi and have used TSA-1 as a model antigen for its initial evaluation. In this report we document that intramuscular injection of BALB/c and C57BL/6J mice with TSA-1-encoding plasmid DNA induces antibodies, CTL, and significant protection against lethal challenge with T. cruzi.     

Trypanosoma cruzi Infection in Tumor Necrosis Factor Receptor p55-Deficient Mice

Tumor necrosis factor receptor p55 (TNFRp55) mediates host resistance to several pathogens by allowing microbicidal activities of phagocytes. In the studies reported here, TNFRp55^−/− mice infected with the intracellular parasite Trypanosoma cruzi showed clearly higher parasitemia and cumulative mortality than wild-type (WT) controls did. However, gamma interferon (IFN-γ)-activated macrophages from TNFRp55^−/− mice produced control levels of nitric oxide and killed the parasite efficiently in vitro. Trypanocidal mechanisms of nonphagocytic cells (myocardial fibroblasts) from both TNFRp55^−/− and WT mice were also activated by IFN-γ in a dose-dependent way. However, IFN-γ-activated TNFRp55^−/− nonphagocytes showed less effective killing of T. cruzi than WT control nonphagocytes, even when interleukin 1β (IL-1β) was added as a costimulator. In vivo, T. cruzi-infected TNFRp55^−/− mice and WT mice released similar levels of NO and showed similar levels of IFN-γ mRNA and inducible nitric oxide synthase mRNA in their tissues. Instead, increased susceptibility to T. cruzi of TNFRp55^−/− mice was associated with reduced levels of parasite-specific immunoglobulin G (IgG) (but not IgM) antibodies during infection, which is probably linked to abnormal B-cell differentiation in secondary lymphoid tissues of the mutant mice. Surprisingly, T. cruzi-infected TNFRp55^−/− mice showed increased inflammatory and necrotic lesions in several tissues, especially in skeletal muscles, indicating that TNFRp55 plays an important role in controlling the inflammatory process. Accordingly, levels of Mn²⁺ superoxide dismutase mRNA, a TNF-induced enzyme which protects the cell from the toxic effects of superoxide, were lower in mutant than in WT infected mice.     

An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible to Mycobacterium tuberculosis

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10³ or 10⁵ CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44⁻ CD45RB⁺ cells disappeared within 2 weeks postinfection, while the number of CD44⁺ CD45RB^−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3⁺ CD4⁺ CD8⁻ T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ⁺ IL-4⁻) clone and one Th0-like (IFN-γ⁺ IL-4⁺ IL-10⁺) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.     

Different parasite inocula determine the modulation of the immune response and outcome of experimental Trypanosoma cruzi infection

During infection, the host response develops effector mechanisms to combat the parasite. However, this response can become uncontrolled or regulated by mechanisms that modulate the inflammatory reaction. The number of parasites that infects the host, such as trypomastigotes in Chagas disease, may also influence immune activation and disease pathology. We evaluated the inflammation and immune regulation that follows Trypanosoma cruzi infection with low (300), intermediate (3000) or high (30 000) parasite loads. Our results showed that the load of parasite inoculum influenced disease outcome: the higher the number of parasites in the inoculum, the lower were the survival rates. There was a strong association between parasitism and inflammatory infiltrate in the heart and the parasite inoculum determined cytokine interplay in this tissue, as shown by increased interferon‐γ, tumour necrosis factor‐α, interleukin‐17 (IL‐17) and IL‐23 in the 300 and 30 000 inoculum groups, higher IL‐4 and IL‐10 in the intermediate‐inoculum mice, and elevated IL‐6 production in the heart of mice in the 3000 and 30 000 groups. The number of T cells and antigen‐presenting cells was augmented in the infected groups, especially for the splenic CD4⁺ CD25⁺ regulatory T cells expressing CD45RB^low, GITR, PD‐1 and FoxP3 in the group with the highest inoculum. Interestingly, these mice also presented an apparent decrease in CD4⁺ CD25⁺ FoxP3⁺ cells in the cardiac infiltrate, in contrast to the intermediate inoculum group, which showed elevated numbers of these regulatory leucocytes in the heart. Finally, our results demonstrated that parasite load during T. cruzi infection is linked to the response pattern that will result in parasite/inflammation control or tissue damage.     

Cytotoxic-T-lymphocyte-associated antigen 4 blockade abrogates protection by regulatory T cells in a mouse model of microbially induced innate immune-driven colitis

Cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) expressed at high levels by CD4⁺ CD25⁺ CD45RB^low regulatory T cells (Treg) is essential to their homeostatic and immunoregulatory functions. However, its relevance to anti-inflammatory roles of Treg in the context of colitogenic innate immune response during pathogenic bacterial infections has not been examined. We showed earlier in Rag2-deficient 129/SvEv mice that Treg cells are capable of suppressing colitis and colon cancer triggered by Helicobacter hepaticus, a widespread murine enterohepatic pathogen. Using this model, we now examined the effects of antibody blockade of CTLA-4 on Treg function during innate immune inflammatory response. Consistent with our previous findings, we found that a single adoptive transfer of Treg cells prior to infection prevented colitis development despite persistent H. hepaticus infection in recipient mice. However, when infected mice were injected with anti-CTLA-4 antibody along with Treg cell transfer, they developed a severe acute colitis with poor body condition that was not observed in Rag2^−/− mice without Treg cell transfer. Despite high numbers of Foxp3⁺ Treg cells, evident by immunohistochemical analyses in situ, the CTLA-4 antibody-treated mice had severely inflamed colonic mucosa and increased rather than decreased expression levels of cytokines gamma interferon and interleukin-2. These findings indicate that antibody blockade of CTLA-4 clearly abrogates Treg cell ability to suppress innate immune-driven colitis and suggest that Treg cell CTLA-4 cognate interactions may be necessary to maintain homeostasis among cells of innate immunity.     

Fas-FasL Interaction Involved in Pathogenesis of Ocular Toxoplasmosis in Mice

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8⁺ and CD4⁺ T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8⁺ than CD4⁺ T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.     

Relative importance of T-cell subsets in monocytotropic ehrlichiosis: a novel effector mechanism involved in Ehrlichia-induced immunopathology in murine ehrlichiosis

Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4⁺ Th1 cells, the expansion of tumor necrosis factor alpha (TNF-α)-producing CD8⁺ T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-α. Here, we investigated the role of CD4⁺ and CD8⁺ T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (10³ and 10⁵ bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4⁺ T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8⁺ T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8⁺ T cells abrogated TNF-α and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4⁺ T-cell numbers, and increased the frequency of Ehrlichia-specific CD4⁺ Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4⁺ T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4⁺ and CD8⁺ T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8⁺ T cells mediate Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4⁺ T-cell apoptosis.     

Interleukin-4-promoted T helper 2 responses enhance Nippostrongylus brasiliensis-induced pulmonary pathology

The role of CD4⁺ T-cell interleukin-4 (IL-4) receptor alpha (IL-4Rα) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4⁺ T-cell IL-4Rα knockout (KO) mice with the parasitic nematode Nippostrongylus brasiliensis, which induces strong host TH2 responses. Although N. brasiliensis expulsion was not affected in CD4⁺ T-cell IL-4Rα KO mice, the associated lung pathology was reduced. Infected CD4⁺ T-cell IL-4Rα KO mice showed abrogation of airway mucus production. Furthermore, CD4⁺ T-cell IL-4Rα KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4⁺ T cells was reduced in infected CD4⁺ T-cell IL-4Rα KO mice. Restimulation of tissue-derived CD4⁺ T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4⁺ T-cell IL-4Rα KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4⁺ T-cell IL-4Rα KO mice. Restimulation with N. brasiliensis antigen of total lung cell populations and populations with CD4⁺ T cells depleted showed that CD4⁺ T cells were a key TH2 cytokine source. These data demonstrated that CD4⁺ T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated with N. brasiliensis infections.     

CD4+ T-cell dependence of primary CD8+ T-cell response against vaccinia virus depends upon route of infection and viral dose

CD4⁺ T-cell help (CD4 help) plays a pivotal role in CD8⁺ T-cell responses against viral infections. However, the role in primary CD8⁺ T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8⁺ T-cell responses to vaccinia virus (VACV) in MHC class II^−/− mice. CD4 help deficiency diminished the generation of VACV-specific CD8⁺ T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8⁺ T cells in i.p. infected MHC II^−/− mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II^−/− mice when a small viral dose was used. These data suggested that primary CD8⁺ T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8⁺ T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8⁺ T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8⁺ T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8⁺ T cells than i.p. infection.     

2-Gy whole-body irradiation significantly alters the balance of CD4+CD25? T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice

CD4⁺CD25⁺ T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4⁺CD25⁺Foxp3⁺ Treg cells has not been determined. The proportion, phenotypes and function of CD4⁺CD25⁺ Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁺Foxp3⁺ Treg cells to CD4⁺ T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4⁺CD25⁺ Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4⁺CD25⁻ T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4⁺CD25⁺Foxp3⁺ Treg cells than in CD4⁺CD25⁻ T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.     

CD8+ T cells are associated with severe gastritis in Helicobacter pylori-infected mice in the absence of CD4+ T cells

Helicobacter pylori infection results in the development of chronic gastritis, and CD4⁺ T cells are a major component of the gastric cellular infiltrate. To examine whether CD4⁺ T cells are important in initiating and maintaining H. pylori-induced gastritis, mice deficient in CD4⁺ T cells (B6.BM1.GK 1.5 mice [GK 1.5 mice]) were infected with H. pylori. We found that as in normal mice, H. pylori-specific antibodies, mostly of the immunoglobulin M isotype, developed in GK 1.5 mice but were unable to cure H. pylori infection. Further, while the stomachs of H. pylori-infected GK 1.5 mice were more heavily infiltrated with CD8⁺ T cells and B cells, mice deficient in both CD4⁺ and CD8⁺ T cells developed mild inflammation comparable to the level observed for C57BL/6 mice. These observations suggest that CD4⁺ T cells may play an important role in regulating or suppressing gastric CD8⁺ T cells which, in the absence of CD4⁺ T cells, may mediate more-severe disease. These studies have revealed a potentially important role for CD8⁺ T cells in the gastric disease resulting from H. pylori infection.     

Induction of M2-like macrophages in recipient NOD-scid mice by allogeneic donor CD4+CD25+ regulatory T cells

CD4⁺CD25⁺ regulatory T cells (Tregs) play an important role in maintaining host immune tolerance via regulation of the phenotype and function of the innate and adaptive immune cells. Whether allogeneic CD4⁺CD25⁺ Tregs can regulate recipient mouse macrophages is unknown. The effect of allogeneic donor CD4⁺CD25⁺ Tregs on recipient mouse resident F4/80⁺macrophages was investigated using a mouse model in which allogeneic donor CD4⁺CD25⁺ Tregs were adoptively transferred into the peritoneal cavity of host NOD-scid mice. The phenotype and function of the recipient macrophages were then assayed. The peritoneal F4/80⁺ macrophages in the recipient mice that received the allogeneic CD4⁺CD25⁺ Tregs expressed significantly higher levels of CD23 and programmed cell death-ligand 1(PD-L1) and lower levels of CD80, CD86, CD40 and MHC II molecules compared to the mice that received either allogeneic CD4⁺CD25⁻ T cells (Teffs) or no cells. The resident F4/80⁺ macrophages of the recipient mice injected with the allogeneic donor CD4⁺CD25⁺ Tregs displayed significantly increased phagocytosis of chicken red blood cells (cRBCs) and arginase activity together with increased IL-10 production, whereas these macrophages also showed decreased immunogenicity and nitric oxide (NO) production. Blocking arginase partially but significantly reversed the effects of CD4⁺CD25⁺ Tregs with regard to the induction of the M2 macrophages in vivo. Therefore, the allogeneic donor CD4⁺CD25⁺ Tregs can induce the M2 macrophages in recipient mice at least in part via an arginase pathway. We have provided in vivo evidence to support the unknown pathways by which allogeneic donor CD4⁺CD25⁺ Tregs regulate innate immunity in recipient mice by promoting the differentiation of M2 macrophages.     

Intranasal Vaccinations with the trans-Sialidase Antigen plus CpG Adjuvant Induce Mucosal Immunity Protective against Conjunctival Trypanosoma cruzi Challenges

Trypanosoma cruzi is an intracellular protozoan parasite capable of infecting through mucosal surfaces. Our laboratory has previously elucidated the anatomical routes of infection after both conjunctival and gastric challenge in mice. We have shown that chronically infected mice develop strong immune responses capable of protecting against subsequent rechallenge with virulent parasites through gastric, conjunctival, and systemic routes of infection. We have also shown that intranasal immunizations with the unique T. cruzi trans-sialidase (TS) antigen protect against gastric and systemic T. cruzi challenge. In the current work we have investigated the ability of purified TS adjuvanted with CpG-containing oligonucleotides to induce immunity against conjunctival T. cruzi challenge. We confirm that intranasal vaccinations with TS plus CpG induce TS-specific T-cell and secretory IgA responses. TS-specific secretory IgA was detectable in the tears of vaccinated mice, the initial body fluid that contacts the parasite during infectious conjunctival exposures. We further show that intranasal vaccinations with TS plus CpG protect against conjunctival T. cruzi challenge, limiting local parasite replication at the site of mucosal invasion and systemic parasite dissemination. We also provide the first direct evidence that mucosal antibodies induced by intranasal TS vaccination can inhibit parasite invasion.Trypanosoma cruzi is an intracellular parasite and the causative agent of Chagas'' disease. An estimated 16 to 18 million people in Latin America are infected, and up to 40% of those will develop the manifestations of chronic Chagas'' disease, including cardiac arrhythmias, cardiomyopathy, megaespophagus, and/or megacolon. To date there is no highly efficacious treatment for chronic infection, nor is a vaccine to prevent infection currently available. The parasite is not capable of infecting the mammalian host through intact skin but can infect through breaks in the skin or mucosal surfaces, such as the gastric mucosa after oral challenge or nasal-associated mucosa after conjunctival contamination (8, 12). The conjunctival route of T. cruzi infection is a common mode of parasite transmission. In fact, unilateral palpebral edema known as Romaña''s sign, which occurs after conjunctival parasite contamination, has been recognized as a marker for acute Chagas'' disease since the 1930s (15).We are currently investigating whether a vaccination protocol using the T. cruzi trans-sialidase (TS) antigen can induce mucosal immunity protective against conjunctival parasite challenge in susceptible BALB/c mice. The TS gene is a member of the largest gene family of T. cruzi and an important virulence factor for parasite infection. T. cruzi is not capable of synthesizing its own sialic acid; however, this molecule is required for host cell invasion by the parasite (5). TS both cleaves sialic acid residues from the surface of host cells and transfers them to the parasite surface. TS is an immunodominant protein inducing strong antibody and cell-mediated responses during human infection (13). The catalytic domain contains an H-2k^d-restricted CD8⁺ T-cell epitope (IYNVGQVSI) and at least one unidentified CD4⁺ T-cell epitope (6). DNA vaccines encoding the catalytic domain of TS have been shown to induce immunity protective against systemic T. cruzi challenge (3, 4, 6, 16). It has also been shown that both CD4⁺ and CD8⁺ T-cell epitopes are required for this TS-specific protective immunity (6). We have recently shown that intranasal vaccination with TS protein adjuvanted with CpG can induce immune responses protective against systemic and oral T. cruzi challenge (11).In the current work we demonstrate that intranasal vaccination with the catalytic domain of TS combined with an oligonucleotide containing Toll-like receptor 9 (TLR-9)-triggering CpG motifs induces strong type 1 cellular immune responses as well as production of mucosal secretory IgA (sIgA) present in fecal extracts (FE) and in tears. We also demonstrate that these immune responses are protective against both systemic and conjunctival T. cruzi challenge and that opsonization with vaccine-induced TS-specific mucosal antibodies can inhibit parasite infection after conjunctival challenge. This is the first direct evidence that a vaccine-induced mucosal antibody response can inhibit T. cruzi infection after conjunctival challenge. These data provide further evidence that the development of vaccines protective against T. cruzi infection is a reasonable research goal and that these efforts should be directed toward generating type 1 immune responses and production of secretory IgA.     

Serum-Mediated Activation of Macrophages Reflects TcVac2 Vaccine Efficacy against Chagas Disease

Chagas disease is endemic in Latin America and an emerging infectious disease in the United States. No effective treatments are available. The TcG1, TcG2, and TcG4 antigens are highly conserved in clinically relevant Trypanosoma cruzi isolates and are recognized by B and T cells in infected hosts. Delivery of these antigens as a DNA prime/protein boost vaccine (TcVac2) elicited lytic antibodies and type 1 CD8⁺ T cells that expanded upon challenge infection and provided >90% control of parasite burden and myocarditis in chagasic mice. Here we determined if peripheral blood can be utilized to capture the TcVac2-induced protection from Chagas disease. We evaluated the serum levels of T. cruzi kinetoplast DNA (TckDNA), T. cruzi 18S ribosomal DNA (Tc18SrDNA), and murine mitochondrial DNA (mtDNA) as indicators of parasite persistence and tissue damage and monitored the effect of sera on macrophage phenotype. Circulating TckDNA/Tc18SrDNA and mtDNA were decreased by >3- to 5-fold and 2-fold, respectively, in vaccinated infected mice compared to nonvaccinated infected mice. Macrophages incubated with sera from vaccinated infected mice exhibited M2 surface markers (CD16, CD32, CD200, and CD206), moderate proliferation, a low oxidative/nitrosative burst, and a regulatory/anti-inflammatory cytokine response (interleukin-4 [IL-4] plus IL-10 > tumor necrosis factor alpha [TNF-α]). In comparison, macrophages incubated with sera from nonvaccinated infected mice exhibited M1 surface markers, vigorous proliferation, a substantial oxidative/nitrosative burst, and a proinflammatory cytokine response (TNF-α ≫ IL-4 plus IL-10). Cardiac infiltration of macrophages and TNF-α and oxidant levels were significantly reduced in TcVac2-immunized chagasic mice. We conclude that circulating TcDNA and mtDNA levels and macrophage phenotype mediated by serum constituents reflect in vivo levels of parasite persistence, tissue damage, and inflammatory/anti-inflammatory state and have potential utility in evaluating disease severity and efficacy of vaccines and drug therapies.