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

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

Activation mediated CD4+ T cell unresponsiveness during acute Toxoplasma gondii infection in mice

infection of mice with Toxoplasma gondii has been shown to inducea transient state of immune down-regulation. Earlier reportshave demonstrated the role of cytokines, in particular IL-10,in this host response. Here evidence is presented that T.gondll,a major opportunistic pathogen of the newborn and those withAIDS, is able to induce CD4⁺ T cell population Increased involume by day 7 post-infection and expressed T cell maturationmarkers (CD44^hl, Il-2r^hl,Mel-14^fo). Further noted was a clonalactivation of several CD4+ T cells subsets expressing the V_ßchain of the TCR. At day 7 post-infection, partial reductionof all CD4⁺ T cells to mltogen or parasite antigen stimulationwas observed, In particular V_ß5 T cells. Additionof rlL-2 partially restored the CD4⁺ T cell proliferative responsein Vitro. The T cell activation marker CTLA-4 could not be detectedand te co-stimulatory molecule, CD28, was down-regulated. Elctrophoneticand morphologic analysis of these cells post0culture demostrateda DNA fragmentation pattern and cell death consistent with apoptosis.These studies demonstrate for the first time in a protozoanparasite that activation-induced CD4⁺ T cell unresponslvenessoccurs during actue T.gondll infection in mice, and may be importantin immune down-regulation and parasite persistence in the infectedhost.0     

Removal of Toxoplasma gondii Cysts from the Brain by Perforin-Mediated Activity of CD8+ T Cells

Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8⁺ immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8⁺ T cells does not require their production of interferon-γ, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8⁺ T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting many warm-blooded mammals including humans. Acute infection is characterized by proliferation of tachyzoites and is known to cause various diseases including lymphadenitis and congenital infection of fetuses.¹ Interferon (IFN)-γ-mediated immune responses limit proliferation of tachyzoites, but the parasite establishes a chronic infection by forming cysts, which can contain hundreds to thousands of bradyzoites, primarily in the brain. Chronic infection with T. gondii is one of the most common parasitic infections in humans. It is estimated that 5 × 10⁸ people worldwide are chronically infected with this parasite.² The tissue cysts remain largely quiescent for the life of the host, but can reactivate and cause life-threatening toxoplasmic encephalitis in immunocompromised patients, such as those with AIDS, neoplastic diseases and organ transplants.^3,4 In immunocompetent individuals, recent studies suggested that T. gondii is an important cause of cryptogenic epilepsy,^5,6 and is likely involved in the etiology of schizophrenia.^7,8 The tissue cyst is not affected by any of the current drug treatments and it has been generally regarded as untouchable. However, the immune responses against T. gondii cysts remain largely unexplored.Resistance to T. gondii is under genetic control in humans^9,10 and mice.^11,12 BALB/c mice are genetically resistant and have only small numbers of cysts in their brains at 2 to 3 months after infection.^11,12 These mice may be able to prevent formation of cysts by efficiently controlling proliferation of tachyzoites during the acute stage of infection. However, it is also possible that the immune system of these animals has the capability to recognize T. gondii cysts and eliminate them from their brains. To examine whether immune cells have an activity to remove cysts that have already been formed in the brain, we adoptively transferred immune cells obtained from chronically infected BALB/c mice into infected, sulfadiazine-treated athymic nude or severe combined immunodeficient (SCID) mice, both of which lack T cells and developed large numbers of cysts in their brains. We present evidence for a potent capability of CD8⁺ immune T cells to eliminate T. gondii cysts from the brains through their perforin-mediated activity.     

Granule-dependent killing of Toxoplasma gondii by CD8+ T cells

Immunization of mice with live bradyzoites of a low-virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T-cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T-cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH-infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H-uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon-gamma (IFN-gamma) than did naive splenic cells; however, the production of nitric oxide (NO) by RH-infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH-infected macrophages, but it did not affect the production of IFN-gamma. Treatment of CD8+ T-enriched splenic cells from the immunized mice with concanamycin A, but not an anti-Fas ligand monoclonal antibody, significantly reduced their anti-proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule-dependent cytotoxic activities.     

Donor CD8+ T Cells Prevent Toxoplasma gondii De-Encystation but Fail To Rescue the Exhausted Endogenous CD8+ T Cell Population

Functional exhaustion of CD8⁺ T cells due to increased expression of inhibitory molecule PD-1 (Programmed Death-1) causes reactivation of latent disease during later phases of chronic toxoplasmosis. Onset of disease recrudescence results in decreased parasite cyst burden concomitant with parasites undergoing stage conversion from a primarily encysted, quiescent bradyzoite to a fast-replicating, highly motile tachyzoite. Thus, reduced cyst burden is one of the early hallmarks of disease recrudescence. This was further validated by depleting gamma interferon (IFN-γ), a cytokine known to control latent toxoplasmosis, in chronically infected prerecrudescent mice. Since CD8⁺ T cells (an important source of IFN-γ) lose their functionality during the later phases of chronic toxoplasmosis, we next examined if adoptive transfer of functional CD8⁺ T cells from acutely infected donors to the chronically infected prerecrudescent hosts could impede parasite de-encystation and rescue exhausted CD8⁺ T cells. While the transfer of immune CD8⁺ T cells temporarily restricted the breakdown of cysts, the exhausted endogenous CD8⁺ T cell population was not rescued. Over time, the donor population got deleted, resulting in parasite de-encystation and host mortality. Considering that donor CD8⁺ T cells fail to become long-lived, one of the cardinal features of memory CD8⁺ T cells, it bears the implication that memory CD8 differentiation is impaired during chronic toxoplasmosis. Moreover, our data strongly suggest that while adoptive immunotherapy can prevent parasite de-encystation transiently, reduced antigen burden in the chronic phase by itself is insufficient for rescue of exhausted CD8⁺ T cells. The conclusions of this study have profound ramifications in designing immunotherapeutics against chronic toxoplasmosis.     

Immune CD8+ T Cells Prevent Reactivation of Toxoplasma gondii Infection in the Immunocompromised Host

Toxoplasma gondii remains a serious cause of morbidity and mortality in individuals that are immunosuppressed, patients with AIDS in particular. The cellular immune response, especially by gamma interferon (IFN-gamma)-producing CD8(+) T cells, is an essential component of protective immunity against the parasite. In the present study the role of CD8(+) T cells during the reactivation of Toxoplasma infection in an immunocompromised murine model was evaluated. Chronically infected mice were challenged with LP-BM5 virus, and the kinetics of CD8(+) T-cell function was studied. At 10 weeks after viral infection, mice showed obvious signs of systemic illness and began to die. At this stage, CD8(+) T cells were unresponsive to antigenic stimulation and unable to kill Toxoplasma-infected targets. IFN-gamma production by the CD8(+) T cells from dual-infected animals reached background levels, and a dramatic fall in the frequency of precursor cytotoxic T lymphocytes was observed. Histopathological analysis of the tissues demonstrated signs of disseminated toxoplasmosis as a result of reactivation of infection. However, treatment of the dual-infected animals with immune CD8(+) T cells at 5 weeks post-LP-BM5 challenge prevented the reactivation of toxoplasmosis, and mice continued to live. Our study for the first time demonstrates a therapeutic role for CD8(+) T cells against an opportunistic infection in an immunocompromised state.     

Depletion of L3T4+ (CD4+) T lymphocytes prevents development of resistance to Toxoplasma gondii in mice.

The role of L3T4+ (CD4+) T lymphocytes in the resistance of mice of different haplotypes to Toxoplasma gondii was examined, using the monoclonal antibody GK 1.5. Outbred Swiss-Webster or inbred CBA/Ca (H-2k), BALB/c (H-2d), and C57BL/6 (H-2b) mice injected with monoclonal antibody GK 1.5 24 h before and 24 h and 7, 15, and 21 days after oral inoculation with 10 live cysts of the low-virulence strain ME49 of T. gondii exhibited an almost complete abrogation of their antibody and cell-mediated immune responses to the parasite. Delayed-type hypersensitivity response and lymphocyte stimulation assays showed significantly reduced values compared with those of control mice inoculated with ME49 cysts but not treated with the monoclonal antibody. The number of T. gondii cysts in the brains of GK 1.5-treated mice was significantly higher than in controls. Challenge of the GK 1.5-treated and chronically infected mice with the virulent RH strain of T. gondii resulted in 100% mortality, whereas 100% of chronically infected controls survived the infection. These results suggest that L3T4+ (CD4+) T lymphocytes actively participate in the development of resistance to T. gondii and in the mechanisms controlling the formation of cysts of the parasite in the brains of mice.     

NK cells help to induce CD8(+)-T-cell immunity against Toxoplasma gondii in the absence of CD4(+) T cells

CD8(+) T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4(+) T-cell help was needed for launching in vivo CD8(+) T-cell activity against these pathogens and tumors. However, recently CD4(+) T-cell-independent CD8 responses during several microbial infections including those with Toxoplasma gondii have been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4(+) T cells, T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8(+) T-cell immunity. Depletion of NK cells inhibited the generation of CD8(+) T-cell immunity in CD4(-/-) mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8(+) T-cell immunity against T. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8(+) T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4(+) T cells, NK cells can play an important role in induction of primary CD8(+) T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection.     

Deficient Humoral Responses Underlie Susceptibility to Toxoplasma gondii in CD4-Deficient Mice

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.     

Toxoplasma gondii Infection of Decidual CD1c+ Dendritic Cells Enhances Cytotoxicity of Decidual Natural Killer Cells

There is crosstalk between decidual natural killer (dNK) cells and decidual dendritic cells (dDCs) that promotes tolerance of trophoblast cells carrying paternally derived antigens. In the present study, we report that infection of CD1c⁺ dDCs with Toxoplasma gondii enhanced gamma interferon (IFN-γ) production by dNK cells in co-culture. The enhancement of IFN-γ production was induced by cytokine IL-12 which increased obviously in co-culture of dDCs with dNK cells following T. gondii infection, and this enhancement largely abrogated when cells were cultured in the presence of an anti-IL-12 antibody. The expression of KIR2DL4 and NKG2D on dNK cells was increased after T. gondii infection, and higher expression of NKG2D was induced by co-cultured dDCs. Neutralization of IL-12 decreased NKG2D expression on dNK cells. Furthermore, dDCs with T. gondii infection increased the cytotoxicity of co-cultured dNK cells against K562 target cells, which was mediated by activating receptor of NKG2D. Thus, T. gondii infection of dDCs enhanced dNK cell IFN-γ production and NKG2D expression, and then led to increased cytotoxicity of dNK cells. The up-regulated dNK cell cytotoxicity at the maternal–fetal interface may contribute to abnormal pregnancy outcomes caused by T. gondii infection in early pregnancy.     

Peptide epitope identification for tumor-reactive CD4 T cells

Because T lymphocytes have the capacity to recognize tumor cells, significant efforts are being devoted towards the development of T cell-based immunotherapy for cancer. Most of this work has centered in the induction of anti-tumor CD8 T cells, which exhibit cytolytic activity towards tumor cells expressing tumor-specific or tumor associated antigens. Unfortunately to this day, T cell-based immunotherapy for cancer remains suboptimal. One of the possible explanations is that these immunotherapies have ignored the role that CD4 T helper lymphocytes play in the generation and persistence of CD8 T cell responses. Thus, we believe that in order to obtain clinical benefits T cell-based immunotherapy must stimulate both CD8 and CD4 tumor-reactive T cell responses. During the past seven years our group has focused on the identification of CD4 T cell epitopes from tumor-associated and tumor-specific antigens that could be used to complement the already identified CD8 T cell epitopes to produce effective vaccination strategies against numerous tumor types. We will describe here the strategy we used that resulted in the identification and characterization of numerous CD4 T cell epitopes that are applicable to developing therapies against hematological malignancies and solid tumors.     

Role of CD4+ Foxp3+ Regulatory T Cells in Protection Induced by a Live Attenuated,Replicating Type I Vaccine Strain of Toxoplasma gondii

Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii type I and type II strains. The involvement of regulatory T cells (Tregs) in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4⁺ Foxp3⁺ T regulatory cells followed by recruitment/expansion of CD4⁺ Foxp3⁻ CD25⁺ effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Tregs were of the TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4⁺ Foxp3⁺ cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of the TH1/TH17-like type. In addition, in vivo Treg induction in RH-infected mice with interleukin-2 (IL-2)/anti-IL-2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Tregs may contribute to the protective response after vaccination with Mic1.3KO.     

Short-Lived Effector CD8 T Cells Induced by Genetically Attenuated Malaria Parasite Vaccination Express CD11c

Vaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodent Plasmodium yoelii model. Protection is dependent on CD8⁺ T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8⁺ T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8⁺ T cell phenotype and demonstrated significant upregulation of CD11c on CD3⁺ CD8b⁺ T cells in the liver, spleen, and peripheral blood. CD11c⁺ CD8⁺ T cells are predominantly CD11a^hi CD44^hi CD62L⁻, indicative of antigen-experienced effector cells. Following in vitro restimulation with malaria-infected hepatocytes, CD11c⁺ CD8⁺ T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c⁻ CD8⁺ T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8⁺ T cells. Coculture of CD11c⁺, but not CD11c⁻, CD8⁺ T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8⁺ T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c⁺ CD8⁺ T cell response, but CD11c expression was lost as the CD8⁺ T cells entered the memory phase. Further analyses showed that CD11c⁺ CD8⁺ T cells are primarily KLRG1⁺ CD127⁻ terminal effectors, whereas all KLRG1⁻ CD127⁺ memory precursor effector cells are CD11c⁻ CD8⁺ T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected hepatocytes.     

Peptide dependency of alloreactive CD4+ T cell responses

Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.     

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans.

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.