INTRODUCTION

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Life Cycle and General Aspects of Immunity

In the intermediate host, after infection of intestinal epithelial cells, the infective stages (oocysts or bradyzoites) transform into tachyzoites, which display rapid multiplication by endodyogeny within an intracellular parasitophorous vacuole. When the cells become packed with tachyzoites, the host cell plasma membrane ruptures and parasites are released into the extracellular milieu. The free tachyzoites can then infect virtually any nucleated cell they encounter, and they continue intracellular replication, spreading throughout host tissues (Fig. 1). If not controlled by the immune system, tachyzoites are highly virulent and cause a generalized toxoplasmosis which is always fatal (57). Indeed, many studies show that normally avirulent strains of T. gondii are highly virulent in T-lymphocyte-deficient animals (71, 133). Therefore, induction of T-cell-mediated immune responses and resistance to the tachyzoite stage is a key step in the T. gondii life cycle, determining the survival of the intermediate host and the parasite itself.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   T. gondii life cycle. During acute infection, initiated by ingestion of cysts or oocysts, tachyzoites invade and proliferate within virtually any nucleated cell, resulting in host cell lysis and reinfection of more cells. Concurrent with the development of immunity, tachyzoites transform into slow-growing bradyzoites, which reside within cysts in tissues of the muscles and CNS. This chronic infection can persist for the life of the host, but in patients with immunodeficiency, cysts may rupture, leading to reinitiation of an acute infection. Within the gut of the cat, ingested parasites undergo differentiation into male and female gametes, which results in the formation of oocysts. These are shed in the feces and can remain infectious for many months.

After development of immunity, the tachyzoite stage is cleared from host tissues, and bradyzoites, the slowly multiplying, essentially dormant and harmless forms of the parasite, persist. The bradyzoites survive within cysts and are effectively isolated from the host immune system by the cyst wall, which is composed mainly of host tissue-derived products (Fig. 2A). The ability of bradyzoites to escape the host immune response and persist in a quiescent form within the host is therefore another key event in the T. gondii life cycle. The bradyzoites are infective for either definitive or intermediate hosts and are largely responsible for parasite transmission to different species of mammals and birds (Fig. 1).

View larger version (104K): [in this window] [in a new window]   FIG. 2.   (A) Cyst in an immunocompetent mouse chronically infected with T. gondii. (B and C) Satellite cysts (B) and clusters of free tachyzoites (arrows) (C) in the brains of chronically infected mice subsequently treated with anti-TNF- or anti-IFN- MAb. These sections were stained with hematoxylin and eosin.

Although bradyzoites are apparently harmless, sequestered within dormant cysts, a persistent immunity to T. gondii is required to avoid reemergence of the tachyzoite stage and accompanying pathologic changes. Indeed, the latter is often observed in chronically infected immunocompromised hosts (139, 162, 213). Bradyzoites are found in proportionately larger numbers in the central nervous system (CNS); indeed, cyst reactivation most often occurs in the brain. This fact is well illustrated by the high incidence of encephalitis induced by T. gondii as a major cause of morbidity and mortality in patients with AIDS (139, 162).

Two hypotheses have been put forward regarding control of parasite replication during chronic toxoplasmosis. According to the first, the host immune response actively induces tachyzoite transformation into the bradyzoite stage and is crucial in maintaining T. gondii in the latter developmental form (Fig. 3A). Indeed, recent in vitro studies indicate that NO, an important effector molecule produced by activated macrophages, induces both parasite stasis and expression of a subset of bradyzoite-specific antigens (Ag) (14, 208).

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Alternative hypotheses for the control of T. gondii tachyzoite replication in tissues of immunocompetent hosts. (A) The cellular immune response plays an active role in driving encystment of the parasite. Recent evidence suggests that production of RNI may promote tachyzoite-bradyzoite transformation. (B) Initiation of cyst formation occurs independently of host immunity. Once cysts are established, a low rate of conversion of bradyzoites to tachyzoites occurs continuously, but these reemergent parasites are effectively controlled by components of type 1 cytokine-based immunity such as RNI.

The second hypothesis suggests that an immune response controls tachyzoite replication but does not, in itself, exert an effect on bradyzoites, which are essentially harmless to the host (Fig. 3B). Instead, it is suggested that parasites are continually released from cysts in chronically infected hosts, resulting in constant boosting of the immune system. Tachyzoite replication itself is controlled by the immune system in immunocompetent animals. Strong evidence for this model comes from the finding that in animals chronically infected with T. gondii, treatment with neutralizing doses of antibody (Ab) against either gamma interferon (IFN-) or tumor necrosis factor alpha (TNF-) results in the emergence of free tachyzoites and an increased number of cysts (Fig. 2B and C).

It is noteworthy that in nature, mice are a major intermediate host of T. gondii. This would suggest that the mouse immune response is well adapted, in evolutionary terms, to cope optimally with this particular parasite. Of further note, the life cycle of the parasite in mice closely resembles that in humans. Toxoplasma is simple to maintain in vitro as tachyzoites and in vivo as either tachyzoites or bradyzoites. Thus, T. gondii infection in inbred mice has become a major model to elucidate the basis of protective immunity against intracellular pathogens in general and to examine the regulation of immunopathologic changes elicited by such infectious agents (62, 67, 200). The knowledge acquired from these studies has provided, and should continue to provide, new insights into designing more effective vaccines based on induction of cell-mediated immunity (CMI), as well as new strategies for immunotherapy of opportunistic infections in immunodeficient hosts.

A series of early studies have pointed away from Ab and toward T cells as the major effectors of resistance to T. gondii. First, passive transfer of serum from chronically infected animals to naive recipients fails to protect the latter against virulent-parasite challenge (56). In addition, mice treated with antibodies against the µ heavy chain of immunoglobulin M (IgM) can control infection despite a lack of B lymphocytes and antibodies (177). Consistent with these observations is the fact that T. gondii lacks an extracellular stage, in contrast to some trypanosomatids such as Trypanosoma brucei, against which Ab have been shown to play a crucial protective role (127, 185). However, it is important to note that some studies indicate the importance of IgA as an element of mucosal immunity to oral infection with Toxoplasma cysts (30). Antibodies of this isotype may be important in avoiding reinfection with T. gondii, and therefore induction of IgA is a major strategy for vaccine development.

Studies showing the importance of T cells in resistance against T. gondii are nonequivocal. Thus, athymic nude mice, which lack functional T cells, are extremely susceptible to both virulent and avirulent parasite strains (71, 133). More importantly, adoptive transfer of immune T cells (in particular the CD8⁺ subset) to naive mice protects animals against challenge with virulent T. gondii strains (56, 168, 220, 221). Immunogenetic studies also point to a major influence of major histocompatibility complex (MHC) class I and II on resistance and susceptibility to the parasite, consistent with the idea that T lymphocytes are crucial in determining the outcome of infection (15, 16, 147, 149).

Virtually all mouse strains develop a strong Th1 immune response to T. gondii, regardless of whether they possess resistant or susceptible MHC haplotypes (61, 70). Thus, cytokines such as IFN- and TNF- (which activate macrophage functions) are important for controlling tachyzoite replication during both acute and chronic phases of infection (61, 68, 110, 192, 213). In contrast, interleukin-10 (IL-10) and IL-12 appear to be crucial at the initial phase of infection and less important during chronic toxoplasmosis (75, 76).

While IL-12 is clearly important in initiating a strong and effective CMI against T. gondii tachyzoites, IL-10 appears to modulate both IL-12 and IFN- synthesis in vivo, avoiding an excessive immune response that could cause extensive inflammation and host tissue damage (76, 163). Thus, IL-10 and IL-12 are two major antagonists involved in regulating IFN- synthesis during the initial phase of infection. Whereas NK cells and CD4⁺ and CD8⁺ T lymphocytes appear to be major sources of IFN- at the early stages of infection T lymphocytes are the dominant source of this cytokine during the chronic phase (58, 61, 70, 71, 75, 99, 117, 210).

In this paper, we review studies that have elucidated the molecular and cellular basis underlying the induction of T-cell-mediated immunity to T. gondii. We also discuss the effector mechanisms of parasite-triggered immunity, examining both beneficial and detrimental host effects that are brought into play during the encounter of this opportunistic pathogen with its mammalian host.

INITIATION AND REGULATION OF T-CELL RESPONSES DURING T. GONDII INFECTION

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Role of Macrophages and Natural Killer Cells

In addition to inducing an effective parasite-specific T-cell-mediated immune response, it is important to note that T. gondii infection elicits strong nonspecific, T-cell-independent immunity. Indeed, the latter response plays an important role in influencing the development of parasite-specific T cells. The nonspecific aspect is revealed by studies showing that T. gondii infection limits coinfection by nonrelated pathogens such as Listeria monocytogenes and Schistosoma mansoni, infection with certain viruses, and development of certain tumors (69, 90, 143, 183). In addition, early studies demonstrated the ability of T. gondii to activate cells from the innate compartment of the immune system, such as macrophages and NK cells (85, 86). Further studies demonstrated that nonspecific activation occurs at early stages of infection, and is a T-cell-independent phenomenon resulting in IFN- synthesis by NK cells and leading to macrophage microbicidal function (62, 203). This early activation of the immune system appears to play two main roles during infection with T. gondii. The first is to limit tachyzoite replication at a time prior to recruitment of T-cell-mediated immunity. The second is to direct the development of an appropriate T-cell response by driving the differentiation of Th precursor (Thp) cells into Th1 effector cells. These considerations suggest that T. gondii tachyzoites may possess adjuvant-like activity that causes generalized potentiation of CMI.

More recent studies have focused on elucidating the cytokine circuits, host cell populations, and parasite molecules involved in the adjuvanticity of tachyzoites. The use of mice with severe combined immunodeficiency (scid mice), which lack both T and B cells, and more recently the use of gene knockout (KO) mice have shed light on these processes (8, 45). NK cells have been identified as a major source of IFN- (203). The cytokine IL-12 appears to be the central mediator initiating the synthesis of NK cell IFN-, whereas other monokines, such as TNF-, IL-1 and IL-15, potentiate the effects of IL-12 on NK cells (27, 71, 97, 100, 102, 105) (Fig. 4A). A recent study also shows the importance of costimulatory molecules such as CD28 in stimulating IFN- synthesis by murine NK cells (102). A similar system has recently been shown to be operative when human NK cells and monocytes are cultured in the presence of soluble tachyzoite Ag (STAg) (124).

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Alternative pathways for induction of T-cell-independent and T-cell-dependent IFN- synthesis. (A) The T-cell-independent pathway is driven largely by IL-12, as well as TNF-, IL-1, and IL-15. These proinflammatory cytokines induce NK cell production of IFN-, which can promote macrophage activation and acquisition of microbiostatic activity, as well as Th1-cell differentiation. (B) Presentation of parasite peptide by a professional Ag-presenting cell (APC) such as a macrophage or dendritic cell to a Thp cell. This results in Th1 differentiation in the presence of T-cell-derived IL-2 and IL-12 produced by the Ag-presenting cell. (C) A less well-characterized but potentially very important pathway of early IFN- production, in which T. gondii is able to directly activate T cells in a manner that does not require IL-12.

The above-described cytokine circuit appears to be functional in vivo as well as in vitro. Thus, upon infection with T. gondii, scid mice produce high levels of IFN- and other proinflammatory cytokines such as IL-12 (97). Similar studies with scid mice show that in vivo neutralization of IL-12 or IFN- enhances their susceptibility to acute T. gondii infection (75). Conversely, administration of recombinant IL-12 prolongs the survival of scid animals, an effect which is abolished by Ab-mediated NK cell or IFN- depletion (71).

Enhanced susceptibility is also the outcome when immunocompetent mice are treated at early stages of infection with anti-IL-12, anti-IFN-, anti-TNF-, or anti-NK cell Ab (75, 99, 110). Furthermore, in vivo treatment with either recombinant IL-12, IFN-, TNF-, IL-1, or IL-1 has a protective effect against T. gondii infection (28, 71, 75). For the case of recombinant IL-12 (rIL-12), protection was curtailed by treatment with either anti-NK cell or anti-IFN- Ab. The latter finding suggests that IL-12 may function through induction of NK cell IFN-; this result finds support from the observation that IFN- KO mice are susceptible to T. gondii despite maintaining an ability to produce IL-12 (192).

The T-cell-independent system is potentiated by IL-2, and in certain immunodeficiency states, this is critical to host survival. Thus, in ₂-microglobulin KO animals, which lack CD8⁺ T lymphocytes, an enormous expansion of NK cells occurs in response to infection, and these cells are responsible for controlling tachyzoite proliferation via synthesis of IFN- (43). This NK cell expansion appears to be dependent on CD4⁺ lymphocytes, probably through the production of IL-2 itself (209). More importantly, continuous treatment of AIDS patients with IL-2 leads to enhancement of NK cell IFN- synthesis, compensating (at least in part) for HIV-induced CD4⁺ T-lymphocyte deficiency (124).

The early burst of IFN- and other proinflammatory cytokines is crucial in shaping the adaptive immune response which develops against T. gondii (67). First, IFN- triggers, or potentiates, the synthesis of chemokines such as the macrophage gene induced by IFN- (MIG) and IFN--inducible protein 10, which are important in T-lymphocyte recruitment (1, 62). In addition, IFN- synergizes with IL-12 in driving the differentiation of Thp cells toward the Th1 phenotype (Fig. 4B). Thus, IFN- enhances IL-12 synthesis by macrophages exposed to tachyzoite products (71, 75, 141), induces expression of the IL-12 receptor on T cells (108), and inhibits IL-4, an important cytokine for differentiation of Thp to the Th2 phenotype (197). Engagement of the IL-12 receptor also activates the phosphorylation of signal transducer and activator of transcription 4 (STAT-4), a transcription factor involved in Th1 lymphocyte differentiation (6, 108, 197).

Interestingly, the Bcl-3 oncoprotein, a member of the IB family which functions as a positive regulator of NF-B activity, plays a critical role in the development of T-cell-mediated immunity and resistance to T. gondii (55). Most Bcl-3 KO mice infected with T. gondii do not survive beyond 1 month, whereas 100% of infected wild-type or heterozygote animals survive for over 6 months. While STAg-stimulated splenocytes (or purified T cells) from Bcl-3 KO mice produce normal levels of IFN- during early (7 days) infection, the ability to produce this cytokine (after 12 days of infection) is impaired. Cytotoxic T-lymphocyte (CTL) activity against infected target cells, but not NK cytolytic activity, is also defective in these animals. Together, these findings suggest that the Bcl-3 KO mice possess an intrinsic T-cell defect responsible for blocking the development of Th1 cells, resulting in compromised adaptive, rather than natural, immunity to T. gondii.

Because of the importance of IL-12 in the development of CMI, several groups are characterizing signalling pathways involved in IL-12 induction by microbial products. Briefly, IL-12 is a heterodimer cytokine composed of two chains, p35 and p40, linked by a disulfide bond. Whereas the p40 chain is tightly regulated in macrophages and can be induced with different microbial stimuli, the p35 chain is constitutively expressed by many different types of cells and tissues (12, 225). The signalling pathways involved in the induction of the p40 chain have been partially characterized. First, several studies have demonstrated the requirement of two signals for optimal IL-12 (p40) synthesis by macrophages. The first is a priming signal provided by IFN-, and the second comes from microbial products themselves, such as lipopolysaccharide or STAg (58, 71, 75, 141).

Recent studies have identified the IFN- consensus sequence binding protein (ICSBP) as an important element in IL-12(p40) responsiveness to IFN- (95, 189). The ICSBP gene is normally induced by IFN-, and mice lacking a functional ICSBP gene are highly deficient in IL-12 synthesis both in vivo and in vitro, although they produce normal levels of IL-12- independent IFN- upon mitogen stimulation. More importantly, the ICSBP KO animals are highly susceptible to T. gondii, although they express normal levels of other proinflammatory cytokines, such as IL-1, TNF-, and IL-6 (189). This result indicates that ICSBP activity is required for IL-12 production exclusively. In contrast, activation of reactivating kinase (or p38) from the mitogen-activated protein kinase-activated protein 2 pathway appears to be necessary for the induction of several monokines, including IL-1, TNF-, and IL-12(p40) itself (11, 22, 129). Molecular analysis of the IL-12(p40) gene promoter suggests a functional role for an NF-B half-site, a nucleotide sequence matched at 8 of 10 nucleotides forming the consensus NF-B sequence (141). In addition, the ets-2 element is present in the promoter region (156). The ets molecules form a large family of transcription factors, some of which are known to be important for the regulation of other inflammatory cytokine genes, such as TNF-. The induction of IL-12(p40) is modulated by cyclic AMP (22), consistent with early reports showing suppressive effects of prostaglandins on expression of this cytokine.

A central paradox has been raised by these experiments: If IFN- is required for the induction of macrophage IL-12 synthesis and if IFN- production requires IL-12, which of these cytokines is produced first during infection? Of relevance to this issue, it has recently been found that mouse dendritic cells produce high levels of IL-12 in response to T. gondii stimulation, even in the absence of IFN- (175). In addition, STAg-induced IL-12 synthesis occurs independently of CD40-CD40L interactions. This observation suggests lack of a requirement for T-cell signalling to dendritic cells through CD40-CD40L interaction (175), the latter of which has been shown to be necessary in several other systems (39, 198, 204). However, as mentioned above, mice without a functional ICSBP gene are deficient in IL-12 synthesis and are highly susceptible to T. gondii infection, although their macrophage effector functions remain normal (189). Thus, this study suggests that IFN- is necessary for optimal production of IL-12 and development of CMI in vivo. It is also important to note that in contrast to Th precursor cells, resting NK cells express the IL-12 receptor and can therefore respond to IL-12 even in the absence of IFN-.

Thus, during T-cell differentiation, it is likely that the NK cells provide the initial IFN- source, which functions to further enhance IL-12 synthesis initiated by microbial stimuli. Nevertheless, recent evidence also indicates that Toxoplasma displays the capability of activating T cells in an IL-12-independent manner (Fig. 4C) (191). This may also relate to studies in humans and mice (described below), which suggest that the parasite can act as a T-cell-specific and possibly a V-specific mitogen (41, 211).

Thus, during the earliest stages of infection, Toxoplasma triggers several components of the innate immune system (Fig. 4). Macrophages, NK cells, and dendritic cells and neutrophils (144, 175) respond by releasing cytokines such as IL-12, TNF-, and IFN-. Within this milieu of proinflammatory cytokines, components of the acquired immune system important for control of infection, namely, T lymphocytes, recognize the appropriate combination of parasite Ag, MHC, and costimulatory molecules. Viewed in this regard, it is clear that innate immune responses exert a strong influence on developing T-cell immunity, and it should not be surprising that T. gondii drives an extremely powerful Th1 response.

T lymphocytes. Through the use of T-cell receptor (TCR) KO mice and depleting monoclonal Ab mAb, T cells are now known to play a protective role against several bacterial and protozoan pathogens (32, 118, 150, 151, 182, 226). The protective effects of T cells are often (although not always [25, 226]) associated with early disease stages rather than later in infection or during reinfection models. Indeed, -T-cell numbers are elevated during early infection with bacterial pathogens such as Listeria monocytogenes (165). These findings have led to the general view that T cells find their major role as members of the armamentarium providing the "first line of defense" against infection.

T lymphocytes. Infection with T. gondii also provides a direct and potent stimulus for T-lymphocyte activation, and as a result, T-cell-derived IFN- production occurs early during acute infection. In part, early T-cell production of IFN- may be explained by the ability of host Ag-presenting cells to activate T lymphocytes in a milieu of inflammatory cytokines. While such an early type 1 cytokine response may be expected to serve a protective role for the host, under certain conditions, T. gondii-induced inflammatory cytokines may contribute to the pathologic findings of the disease.

In vivo evidence for early -T-cell activation comes from a variety of reports. -T-cell production of IFN- during acute infection (7 days postinoculation) has been linked to host protection during intraperitoneal infection with the cystogenic ME49 parasite strain (

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Nonimmune CD4+ and CD8+ T lymphocytes proliferate in response to T. gondii and Ag-presenting cells. T lymphocytes from normal C57BL/6 mice (purified by passage over anti-mouse Ig columns) were subjected to treatment with rabbit complement and anti-CD4 MAb (to obtain CD8+ T cells) or anti-CD8 MAb (to obtain CD4+ T cells). The resulting populations were cultured with irradiated splenic adherent cells (SAC) that were either untreated, preincubated with T. gondii soluble Ag extract, or preinfected with RH strain tachyzoites. For some CD8+ populations, recombinant murine IL-2 was included. Proliferation was measured by incorporation of [3H]thymidine (3H-TdR), added 72 h after culture initiation.

Modulation of T-cell activation. While early production of inflammatory cytokines is required for the subsequent parasite-induced protective T-cell-mediated immune response, if uncontrolled this response can lead to severe immunopathologic changes and even to death. Thus, under normal circumstances, the strength of the host CMI triggered by T. gondii must be tightly regulated to limit infection, on the one hand, and to avoid immunopathologic changes, on the other. Elucidation of the molecular mechanisms underlying regulation of the protective immune response has provided important clues for understanding the basis of successful long-term interaction between parasites and their vertebrate host. At the same time that macrophages induce strong type 1 cytokines, these cells also promote the regulation of CMI through production of IL-10 and transforming growth factor  (TGF-), which regulate the expression and function of IL-12 and other monokines (62). A recent study also suggests that IL-4 may display a similar regulatory role during acute toxoplasmosis (179). In addition, upon stimulation with microbial products, macrophages produce high levels of NO, which displays potent antiproliferative effects on cells from the lymphocytic lineage (62).

Role of regulatory cytokines. The cytokine IL-10 was first identified by its ability to inhibit IFN- synthesis by Th1 lymphocytes and was shown to be produced by Th2 cells (53). Similar effects of IL-10 were also observed on in vitro IFN- synthesis by STAg-stimulated NK cells from scid mice (203). Follow-up studies indicated that IL-10 is produced by a large variety of cells in addition to Th2 lymphocytes, including B cells and macrophages. Indeed, the latter cell population, itself, appears to be the main target of IL-10 suppressive action (153). Thus, IL-10 inhibits the synthesis of a wide variety of proinflammatory monokines by macrophages and is therefore an important modulator of macrophage effector functions against different pathogens, including T. gondii (54, 74). The main method by which IL-10 inhibits IFN- synthesis by NK and Th1 lymphocytes is through inhibition of macrophage IL-12 synthesis (37, 96).

Role of nitric oxide. Another important mechanism by which macrophages regulate the immune response during experimental acute toxoplasmosis is through generation of NO (109). After priming with IFN-, macrophages exposed to microbial products or TNF- produce high levels of reactive nitrogen intermediates (RNI). These compounds were first recognized for their importance as effector molecules responsible for microbicidal and microbiostatic functions displayed by activated murine macrophages (see below). Experiments performed both in vivo and in vitro also reveal an immunosuppressive activity associated with RNI, in particular during the early phase of infection with T. gondii (23).

Toxoplasma is a strong inducer of Ag-specific CD4⁺ and CD8⁺ T lymphocytes, indicating that parasite peptides are efficiently targeted to the appropriate cellular pathways of Ag presentation during infection. In general, processing for MHC class I presentation requires Ag entry into the cytoplasm, followed by proteolytic generation of peptides by proteasomes, transporter associated with antigen presentation-mediated transport across the endoplasmic reticulum, association with MHC class I heavy chain and ₂-microglobulin, and exocytosis to the cell surface (237). Presentation in the context of MHC class II molecules generally requires soluble Ag endocytosis, proteolysis within the phagolysosome, trafficking to MHC class II-containing endosomes, association with MHC class II, and transport to the cell surface (35, 232).

For T. gondii Ag, it is not clear where peptide loading onto MHC occurs. In one model, Ag-MHC interaction could occur at the cell surface, as a result of either Ag secretion by extracellular tachyzoites or deposition on the cell surface during invasion. This model would require proteolytic degradation of parasite Ag outside of host cells and would additionally require that parasite Ag displace already bound peptide. In an alternate model, presentation of parasite Ag would depend upon phagocytosis or receptor-mediated uptake of dead or dying tachyzoites or soluble parasite Ag. Parasites entering the cell in this manner are trafficked through the endosomal-lysomal pathway. Although this route classically favors MHC class II presentation, it is now clear that peptides can access the cytosolic presentation pathway by this route (111, 154). In this regard, murine bone marrow macrophages are extremely efficient at processing and presenting to soluble Ag cytolytic CD8⁺ T cells in a class I-restricted manner (42, 47).

A final model would involve the transport of Ag from within the parasitophorous vacuole to the cytosolic and possibly endocytic presentation pathway. The parasitophorous vacuole is believed to function as a molecular sieve, allowing free diffusion of molecules smaller than 1,300 to 1,900 Da between the host cytoplasm and parasitophorous vacuolar space (196). This property alone would exclude macromolecular Ag from accessing the host cell cytoplasm but instead suggests that antigenic peptides are formed within the parasitophorous vacuole and that this is followed by passive diffusion into the host cell cytoplasm. Nevertheless, intact antigenic proteins could also potentially be actively transported across the parasitophorous vacuole membrane into the host cell cytoplasm for subsequent trafficking to the conventional MHC presentation pathways.

CD8⁺ CTL. CTL are best known for their ability to kill virus-infected and transformed target cells. It is now clear that several intracellular protozoans are also effective at stimulating CD8⁺ CTL function against infected target cells, in part through their ability to traffic antigenic peptides into the MHC class I presentation pathway. Vaccination of mice with Plasmodium berghei and P. falciparum sporozoites generates circumsporozoite-specific CD8⁺ T cells capable of killing Ag-specific target cells and transferring protection to naive recipients (128, 181). Infection with Trypanosoma cruzi likewise results in the presentation of parasite-derived peptide with cell surface MHC class I glycoproteins, in turn inducing CD8⁺ CTL effector function (59, 164). The ability of these parasites to stimulate CTL function is most probably related to their capacity of evading the lysomal pathway during cell invasion, residing instead either directly in the cytoplasm (T. cruzi) or within a parasitophorous vacuole (Plasmodium). This may also explain why Leishmania spp., which find their niche within the macrophage phagolysosome, are relatively poor at inducing CD8⁺ CTL function (167, 230).

View larger version (12K): [in this window] [in a new window]   FIG. 6.   Both 2-microglobulin and A KO mice, which lack MHC class I-restricted CD8+ and class II-restricted CD4+ T lymphocytes, respectively, are susceptible to normally nonlethal T. gondii infection. Animals were infected by intraperitoneal injection of 20 cysts of the low-virulence ME49 parasite strain, and survival was monitored. Strain C57BL/6 mice (wild-type counterparts to the KO strains) do not begin to succumb to infection until approximately 100 days postinfection (48).

CD4⁺ CTL. While CD4⁺ lymphocytes are not generally considered major cytotoxic effectors, several reports indicate that Toxoplasma infection in humans results in generation of CTL of the helper T-cell phenotype (24, 36, 152, 173). Indeed, some groups report that CD4⁺ CTL are easier to isolate in vitro than CD8⁺ CTL, although the explanation for this phenomenon is presently unknown (173). Interestingly, human CTL lines display a predominant V7 TCR usage, possibly indicating the presence of an immunodominant Ag (152).

View larger version (19K): [in this window] [in a new window]   FIG. 7.   Dual role of CD8+ T lymphocytes as effectors of immunity to T. gondii. Priming of resting CD8+ cells, which requires parasite Ag, CD4+ T cells, and IL-2, results in anti-parasite effectors. These cells display MHC class I-restricted cytolytic activity toward Toxoplasma-infected target cells and release high levels of IFN- in response to parasite stimulation. Based upon studies in IFN- and perforin KO mouse strains, cytokine secretion is the major effector activity of CD8+ cells in acute and vaccine models of infection. IFN- acts, at least in part, through its ability to induce macrophage microbicidal functions such as NO production. Nevertheless, CTL function appears to play a secondary functional role in protection during chronic infection, as measured by increased mortality and incidence of brain cysts in perforin KO animals.

As discussed above, infection with T. gondii induces strong CMI, characterized by a highly polarized Th1-cell response (62, 67, 200). The acute phase of infection is marked by elevated levels of IFN- and IL-12, as well as other proinflammatory cytokines such as TNF-, granulocyte-macrophage colony-stimulating factor, IL-6, and IL-1. Nevertheless, IL-10, an anti-inflammatory cytokine, is also induced at this stage of infection. Macrophages and/or dendritic cells are generally considered the major source of proinflammatory cytokines, as well as the anti-inflammatory mediator IL-10, during acute T. gondii infection. In addition, neutrophils, like macrophages, are capable of producing both proinflammatory and anti-inflammatory cytokines during early infection (144, 180). In the first days following inoculation with T. gondii, NK cells and T lymphocytes provide a major source of IFN-. Perhaps not surprisingly, given the cross-regulatory properties of type 1 and type 2 cytokines (199), infection is associated with low levels of IL-4 and IL-5 (75).

As the chronic stage of infection progresses in mice, levels of proinflammatory cytokines such as IL-1, IL-6, TNF-, and IFN- decrease (as measured by presence of gene transcripts in the brain) while the level of the anti-inflammatory cytokine IL-10 increases (68). Interestingly, IL-4 can also be briefly detected early in chronic disease (days 10 to 15 after infection), but its expression rapidly falls to background levels (103). During the chronic phase, both CD4⁺ and CD8⁺ T lymphocytes are required to prevent reactivation of toxoplasmosis (61). When the latter cells are stimulated in vitro with parasite Ag, they produce high levels of both IFN- and IL-2. Unlike acute-stage disease, NK cells do not appear to contribute significantly to cytokine production during the persistant period of infection (75). This general pattern of a polarized type 1 cytokine response associated with chronic toxoplasmosis has also been reported in human patients (63, 142).

In susceptible mouse strains (such as C57BL/6), a moderate inflammation characterized by the presence of gene transcripts for IFN-, TNF-, granulocyte-macrophage colony-stimulating factor, IL-6, IL-1, and IL-10, is found in the CNS of chronically infected animals (68, 103, 104). Interestingly, the inflammatory infiltrate appears to be dominated largely by CD8⁺ and CD4⁺ T lymphocytes (101, 193). The moderate inflammatory process may gradually increase in severity (depending upon the mouse strain and parasite dose), resulting in a high rate of mortality over a period of 2 to 6 months. Both in vitro and in vivo studies show that cytokines such as IFN- and TNF- are key mediators in triggering the effector functions against T. gondii during both acute and chronic stages of infection. However, if uncontrolled, this T. gondii-induced CMI response can lead to host tissue damage, pathologic changes, and, sometimes, death.