The regulatory CD4+CD25+ T cells have a limited role on pathogenesis of infection with Trypanosoma cruzi

Recent reports have established an important role of CD4+CD25+ T cells in the immune regulation of infectious diseases, autoimmune disorders and cancer. In the present work, we investigated whether these cells had a regulatory role during Trypanosoma cruzi infection, using the Colombian strain. Inactivation of CD4+CD25+ cells in vivo conferred mice slightly more resistant to infection with the Colombian strain of T. cruzi, as evidenced by lower parasitemia and mortality rates. The augmented resistance to infection with Colombian strain did correlate with increased activation of effector CD4 cells. It was antibody-independent, since no difference in levels of IgM, IgG, IgG1 and IgG2a(b) recognizing T. cruzi antigens was observed throughout the infection of CD25-inactivated and control mice. Regarding pathogenesis, inflammatory infiltrate and frequency of CD4 and CD8 T cells or macrophages in the cardiac tissue was similar in both groups. Together, our data indicate that CD4+CD25+ cells have a limited role on host resistance during early T. cruzi infection. Despite exhaustive investigation, we did not observe any role for these regulatory cells in the pathogenesis of experimental chronic Chagas' disease.     

Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucleotide induces protective mucosal and systemic trypanosoma cruzi immunity involving CD8+ CTL and B cell-mediated cross-priming

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.     

Cutting edge: small molecule CD40 ligand mimetics promote control of parasitemia and enhance T cells producing IFN-gamma during experimental Trypanosoma cruzi infection

Host resistance to Trypanosoma cruzi infection depends on a type 1 response characterized by a strong production of IL-12 and IFN-gamma. Amplifying this response through CD40 triggering results in control of parasitemia. Two newly synthesized molecules (<3 kDa) mimicking trimeric CD40L (mini CD40Ls(-1) and (-2)) bind to CD40, activate murine dendritic cells, and elicit IL-12 production. Wild-type but not CD40 knockout mice exhibited a sharp decrease of parasitemia and mortality when inoculated with T. cruzi mixed with miniCD40Ls. Moreover, the immunosuppression induced by T. cruzi infection was impaired in mice treated with miniCD40Ls, as shown by proliferation of splenic lymphocytes, percentage of CD8(+) T cells, and IFN-gamma production. Mice surviving T. cruzi infection in the presence of miniCD40L(-1) were immunized against a challenge infection. Our results indicate that CD40L mimetics are effective in vivo and promote the control of T. cruzi infection by overcoming the immunosuppression usually induced by the parasites.     

CD8+ T-Cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes

CD8+ T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8+ T cells in T. cruzi-infected mice and humans. This analysis demonstrates that in both hosts the CD8+ T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi-infected mice, with more than 30% of the CD8+ T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level.     

Trypanosoma cruzi infection from the view of CD8+ T cell immunity--an infection model for developing T cell vaccine

Chagas' disease is caused by Trypanosoma cruzi (T. cruzi) which was once prevalent in Central and South America. Although the recent success in Triatoma vector control has made the disease being possibly "extinct" in the near future, the development of effective preventive and therapeutic vaccines is still necessary to prevent the resurgence of the neglected infection. In addition to the importance for containing the disease, T. cruzi infection presents unique features for elucidating hosts' immune responses against intracellular infectious agents. Due to its biological capacity for invading into principally any types of cells and for causing systemic infection which damages particularly muscle and neural cells, T cell immunity is critical for resolving its infection. Although T cell-mediated immune responses have been, so far, extensively investigated in viral and bacterial infections, parasitic infection such as malaria has presented epoch-making discovery in T cell immunity. Recent advances in the analyses of T cell-mediated immune responses against T. cruzi infection now make this infectious disease potentially more suitable for detecting subtle immunological changes in hosts' immune defense upon modifying immune system. The current review focuses on the usefulness of T. cruzi infection as a model for developing effective CD8(+) T cell-mediated vaccine against intracellular infectious agents.     

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.     

Why is HIV a pathogen?

The pathogenesis of HIV begins with a profound depletion of CD4+ T cells in the gut followed by a long period of clinically silent but dynamic virus replication and diversification with high host cell turnover before the onset of AIDS. The AIDS-defining opportunistic infections and tumors mark the end-point of a long balancing act between virus and host that occurs when CD4+ T cell numbers fall below a level that can sustain immunity. Comparative studies of lentivirus infections in other species show that AIDS is not an inevitable outcome of infection because simian immunodeficiency virus in natural hosts seldom causes disease. What distinguishes pathogenic from 'passenger' infection is a systemic activation of immune responses followed by destruction of the integrity of lymphoid follicles. Macrophage and dendritic cell infection also contribute to pathogenesis. Maedi-Visna virus infection in sheep, which targets these cells but not T lymphocytes, also leads to progressive disease and death that resembles the wasting and brain diseases of HIV without the T cell immunodeficiency. Thus, lessons from pathogenic and nonpathogenic lentivirus infections provide insight into the complex syndrome called AIDS.     

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.     

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.     

T cell response and persistence of the microsporidia

The microsporidia are a diverse phylum of obligate intracellular parasites related to the fungi that cause significant and sometimes life-threatening disease in immune-compromised hosts, such as AIDS and organ transplant patients. More recently, their role in causing pathology in immune-competent populations has also been appreciated. Interestingly, in several instances, the microsporidia have been shown to persist in their hosts long term, causing at opposite ends of the spectrum either an intractable chronic diarrhea and wasting in patients with advanced-stage AIDS or asymptomatic shedding of spores in healthy populations. Much remains to be studied regarding the immune response to these pathogens, but it seems clear that CD8+ T cells are essential in clearing infection. However, in the infection models examined thus far, the role for CD4+ T cells is unclear at best. Here, we discuss the possible reasons and ramifications of what may be a weak primary CD4+ T cell response against Encephalitozoon cuniculi. Given the central role of the CD4+ T cell in other models of adaptive immunity, a better appreciation of its role in responding to microsporidia may provide insight into the survival strategies of these pathogens, which allow them to persist in hosts of varied immune status.     

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-alpha blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-alpha) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-alpha levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-alpha, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-alpha+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-alpha treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-alpha-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-alpha treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.     

Virally induced CD4+ T cell depletion is not sufficient to induce AIDS in a natural host

Peripheral blood CD4+ T cell counts are a key measure for assessing disease progression and need for antiretroviral therapy in HIV-infected patients. More recently, studies have demonstrated a dramatic depletion of mucosal CD4+ T cells during acute infection that is maintained during chronic pathogenic HIV as well as SIV infection. A different clinical disease course is observed during the infection of natural hosts of SIV infection, such as sooty mangabeys (Cercocebus atys), which typically do not progress to AIDS. Previous studies have determined that SIV+ mangabeys generally maintain healthy levels of CD4+ T cells despite having viral replication comparable to HIV-infected patients. In this study, we identify the emergence of a multitropic (R5/X4/R8-using) SIV infection after 43 or 71 wk postinfection in two mangabeys that is associated with an extreme, persistent (>5.5 years), and generalized loss of CD4+ T cells (5-80 cells/microl of blood) in the absence of clinical signs of AIDS. This study demonstrates that generalized CD4+ T cell depletion from the blood and mucosal tissues is not sufficient to induce AIDS in this natural host species. Rather, AIDS pathogenesis appears to be the cumulative result of multiple aberrant immunologic parameters that include CD4+ T cell depletion, generalized immune activation, and depletion/dysfunction of non-CD4+ T cells. Therefore, these data provide a rationale for investigating multifaceted therapeutic strategies to prevent progression to AIDS, even following dramatic CD4 depletion, such that HIV+ humans can survive normal life spans analogous to what occurs naturally in SIV+ mangabeys.     

CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to filarial parasites in vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.     

Persistence and function of central and effector memory CD4+ T cells following infection with a gastrointestinal helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.     

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.     

Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease

In this study, we document the development of stable, antigen-independent CD8+ T cell memory after drug-induced cure of a chronic infection. By establishing a system for drug cure of chronic Trypanosoma cruzi infection, we present the first extensively documented case of total parasite clearance after drug treatment of this infection. Cure resulted in the emergence of a stable, parasite-specific CD8+ T cell population with the characteristics of central memory cells, based upon expression of CD62L, CCR7, CD127, CD122, Bcl-2 and a reduced immediate in vivo CTL function. CD8+ T cells from treated and cured mice also expanded more rapidly and provided greater protection following challenge than those from chronically infected mice. These results show that complete pathogen clearance results in stable, antigen-independent and protective T cell memory, despite the potentially exhausting effects of prior long-term exposure to antigen in this chronic infection.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.     

Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8+ T cells

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-gamma-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-gamma-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.     

Modulation of the CD8+-T-cell response by CD4+ CD25+ regulatory T cells in patients with hepatitis B virus infection

CD4+ CD25+ regulatory T cells have been shown to maintain peripheral tolerance against self and foreign antigens. In this study we analyzed the effect of circulating CD4+ CD25+ T cells on CD8+-T-cell responses of patients with chronic and resolved hepatitis B virus (HBV) infection. We demonstrated that circulating CD4+ CD25+ T cells modulate the function and expansion of HBV-specific CD8+ cells ex vivo in all patients, regardless of whether they have chronic or resolved HBV infection. The possible role of CD4+ CD25+ T cells in the pathogenesis of chronic HBV infection is not supported by these data. However, these results might have implications for optimizing future immunotherapeutic approaches to HBV treatment.