Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection, with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10-producing cells in the lung during acute RSV infection were CD4(+) T cells. The IL-10-producing CD4(+) T cells included Foxp3(+) regulatory T cells, Foxp3(-) CD4(+) T cells that coproduce IFN-γ, and Foxp3(-) CD4(+) T cells that do not coproduce IFN-γ. RSV infection of IL-10-deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared with control mice. We also observed an increase in the magnitude of the RSV-induced CD8(+) and CD4(+) T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4(+) T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4(+) T cells and a concomitant decrease in Foxp3(+) regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.     

Natural Killer (NK) Cells in Antibacterial Innate Immunity: Angels or Devils?

Natural killer (NK) cells were first described as immune leukocytes that could kill tumor cells and soon after were reported to kill virus-infected cells. In the mid-1980s, 10 years after their discovery, NK cells were also demonstrated to contribute to the fight against bacterial infection, particularly because of crosstalk with other leukocytes. A wide variety of immune cells are now recognized to interact with NK cells through the production of cytokines such as interleukin (IL)-2, IL-12, IL-15 and IL-18, which boost NK cell activities. The recent demonstration that NK cells express pattern recognition receptors, namely Toll-like and nucleotide oligomerization domain (NOD)-like receptors, led to the understanding that these cells are not only under the control of accessory cells, but can be directly involved in the antibacterial response thanks to their capacity to recognize pathogen-associated molecular patterns. Interferon (IFN)-γ is the predominant cytokine produced by activated NK cells. IFN-γ is a key contributor to antibacterial immune defense. However, in synergy with other inflammatory cytokines, IFN-γ can also lead to deleterious effects similar to those observed during sepsis. Accordingly, as the main source of IFN-γ in the early phase of infection, NK cells display both beneficial and deleterious effects, depending on the circumstances.     

MicroRNA-deficient NK cells exhibit decreased survival but enhanced function

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.     

Preexposure to CpG Protects against the Delayed Effects of Neonatal Respiratory Syncytial Virus Infection

Severe respiratory viral infection in early life is associated with recurrent wheeze and asthma in later childhood. Neonatal immune responses tend to be skewed toward T helper 2 (Th2) responses, which may contribute to the development of a pathogenic recall response to respiratory infection. Since neonatal Th2 skewing can be modified by stimulation with Toll-like receptor (TLR) ligands, we investigated the effect of exposure to CpG oligodeoxynucleotides (TLR9 ligands) prior to neonatal respiratory syncytial virus (RSV) infection in mice. CpG preexposure was protective against enhanced disease during secondary adult RSV challenge, with a reduction in viral load and an increase in Th1 responses. A similar Th1 switch and reduction in disease were observed if CpG was administered in the interval between neonatal infection and challenge. In neonates, CpG pretreatment led to a transient increase in expression of major histocompatibility complex class II (MHCII) and CD80 on CD11c-positive cells and gamma interferon (IFN-γ) production by NK cells after RSV infection, suggesting that the protective effects may be mediated by antigen-presenting cells (APC) and NK cells. We conclude that the adverse effects of early-life respiratory viral infection on later lung health might be mitigated by conditions that promote TLR activation in the infant lung.     

IL-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice

Interleukin (IL-) 10 is a pleiotropic cytokine with broad immunosuppressive functions, particularly at mucosal sites such as the intestine and lung. Here we demonstrate that infection of BALB/c mice with respiratory syncytial virus (RSV) induced IL-10 production by CD4(+) and CD8(+) T cells in the airways at later time points (e.g. day 8); a proportion of these cells also co-produced IFN-γ. Furthermore, RSV infection of IL-10(-/-) mice resulted in more severe disease with enhanced weight loss, delayed recovery and greater cell infiltration of the respiratory tract without affecting viral load. In addition, IL-10(-/-) mice had a pronounced airway neutrophilia and heightened levels of pro-inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid. Notably, the proportion of lung T cells producing IFN-γ was enhanced, suggesting that IL-10 may act in an autocrine manner to dampen effector T cell responses. Similar findings were made in mice treated with anti-IL-10R antibody and infected with RSV. Therefore, IL-10 inhibits disease and inflammation in mice infected with RSV, especially during recovery from infection.     

Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.     

Sequential Involvement of NK Cells and CD8+ T Cells in Granuloma Formation of Rhodococcus aurantiacus-Infected Mice

We investigated the effect of in vivo administration of antibodies against T-cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN-γ) production and granuloma formation in Rhodococcus aurantiacus-infected mice. High titers of endogenous IFN-γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN-γ production was reduced by in vivo administration of anti-NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1⁺ cells. Endogenous IFN-γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN-γ at 1 and 3 weeks was reduced by in vivo administration of anti-CD8 MAb, but not by anti-CD4 MAb or anti-NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti-IFN-γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8⁺ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN-γ is attributable to NK cells in the early phase of the infection and CD8⁺ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8⁺ T cells through the secretion of endogenous IFN-γ.     

Listeria monocytogenes infection affects a subset of Ly49-expressing NK cells in the rat

NK cells are protective against certain bacterial and viral infections, and their production of IFN-γ is important for the early innate immune defence against L. monocytogenes. We have previously shown that depletion of NK cells in rats leads to increased bacterial burden upon L. monocytogenes infection, and that a subset of NK cells encompassing the majority of Ly49 receptors (Ly49s3+ NK cells) contributed to this effect. In this study, we have further investigated how the Ly49s3+ NK cell subset is affected by L. monocytogenes infection. We observed an increased percentage of Ly49s3+ NK cells in the spleen and a reduction in the bone marrow within the first 48 hrs of L. monocytogenes infection. Concomitantly, we observed increased expression levels of the inflammatory chemokine receptors CCR5 and CXCR3 by Ly49s3+ bone marrow NK cells, as compared to Ly49s3- NK cells, suggesting involvement of Ly49s3+ NK cells in the early phase of infection. However, NK cell production of IFN-γ was independent of Ly49 receptor expression. Furthermore, we observed increased expression levels of MHC class I molecules on both macrophages and NK cells during the first 48 hrs of infection, paralleled by a reduction in the surface expression of Ly49s3 on NK cells. In conclusion, L. monocytogenes infection modulates the tissue distribution of Ly49s3+ NK cells, and induces increased MHC class I expression and hence reduced surface expression of Ly49 receptors on NK cells. These changes indicate that L. monocytogenes infection may have multiple effects on NK cells in vivo, and suggests the involvement of Ly49-expressing NK cells in the immune responses towards L. monocytogenes.     

NK cells require type I IFN receptor for antiviral responses during genital HSV-2 infection

Type I interferon (IFN) signalling, NK cells and NK cell-derived IFN-γ are critical in the early control of genital HSV-2 infection. We have recently reported that NK cells are the source of early IFN-γ in the genital tract in response to HSV-2. However, the response of NK cells to genital HSV-2 infection is not well defined in the context of type I IFN signalling. Here we show that HSV-2 replication was significantly higher in mice deficient in the type I IFN receptor or NK cells compared to wild type controls. There was no detectable IFN-γ production in the genital washes from IFN-α/βR^−/− mice or NK cell depleted mice in response to HSV-2 infection compared to control mice. Absence of the type I IFN receptor does not alter homing of NK cells to the genital mucosa. Moreover, the absence of IL-12 had no significant effect on NK cell-derived IFN-γ. Surprisingly, IFN-α/βR^−/− mice had more IL-15 positive cells in the genital mucosa in response to HSV-2 infection compared to control mice. We then examined the expression of IL-15 receptors on NK cells. There was no significant differences in the levels of IL-15 receptor expression on NK cells from IFN-α/βR^−/− or control mice. Our data clearly suggest that type I IFN receptor signalling is essential for NK cell activation in response to genital HSV-2 infection, and propose that NK cell activation by IL-15 may involve type I IFNs.     

NK Cells Regulate CD8+ T Cell Priming and Dendritic Cell Migration during Influenza A Infection by IFN-γ and Perforin-Dependent Mechanisms

An effective immune response against influenza A infection depends on the generation of virus-specific T cells. NK cells are one of the first-line defenses against influenza A infection. We set out to delineate the role of NK cells in T cell immunity using a murine model of influenza A infection with A/PR/8/34. We show that early T cell recruitment mainly occurs in the posterior mediastinal lymph node (pMLN). Depletion of NK cells significantly impaired both dendritic cell (DC) and T cell recruitment into the pMLN. A similar reduction of T cell recruitment was observed when migration was blocked by pertussis toxin, suggesting that migration of pulmonary NK cells and DCs regulates cell recruitment to the pMLN. T cell recruitment was dependent on IFN-γ, and transfer of IFN-γ-competent naive NK cells into IFN-γ(-/-) mice restored T cell recruitment, whereas IFN-γ-deficient NK cells failed to do so. In addition, NK cell depletion reduced the uptake and transport of influenza A virus by DCs, and significantly impaired the virus-specific T cell response. Both IFN-γ(-/-) and perforin(-/-) mice showed reduced viral Ag transport by DCs, suggesting that the ability of NK cells to influence virus transport depends on IFN-γ and perforin. In summary, our data suggest that NK cells play a critical role in the initiation and shaping of the T cell response after influenza A infection.     

NK cell immunophenotypic and genotypic analysis of infants with severe respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants. Reduced numbers of NK cells have been reported in infants with severe RSV infection; however, the precise role of NK cells during acute RSV infection is unclear. In this study the NK and T cell phenotypes, LILRB1 gene polymorphisms and KIR genotypes of infants hospitalized with RSV infection were analyzed. Compared to controls, infants with acute RSV infection showed a higher proportion of LILRB1+ T cells; in addition, a subgroup of infants with RSV infection showed an increase in LILRB1+ NK cells. No differences in NKG2C, NKG2A, or CD161 expression between RSV infected infants and controls were observed. LILRB1 genotype distribution of the rs3760860 A>G, and rs3760861 A>G single nucleotide polymorphisms differed between infants with RSV infection and healthy donors, whereas no differences in any of the KIR genes were observed. Our results suggest that LILRB1 participates in the pathogenesis of RSV infection. Further studies are needed to define the role of LILRB1+ NK in response to RSV and to confirm an association between LILRB1 polymorphisms and the risk of severe RSV infection.     

The Chemokine MIP1α/CCL3 Determines Pathology in Primary RSV Infection by Regulating the Balance of T Cell Populations in the Murine Lung

Background

CD8 T cells assist in the clearance of respiratory syncytial virus (RSV) infection from the lungs. However, disease after RSV infection is in part caused by excessive T cell activity, and a balance is therefore needed between beneficial and harmful cellular immune responses. The chemokine CCL3 (MIP1α) is produced following RSV infection and is broadly chemotactic for both T cells and natural killer (NK) cells. We therefore investigated its role in RSV disease.

Methodology/Principal Findings

CCL3 was produced biphasically, in both the early (day 1) and late (day 6–7) stages of infection. CCL3 depletion did not alter the recruitment of natural killer (NK) cells to the lungs during the early stage, but depletion did affect the later adaptive phase. While fewer T cells were recruited to the lungs of either CCL3 knockout or anti-CCL3 treated RSV infected mice, more RSV-specific pro-inflammatory T cells were recruited to the lung when CCL3 responses were impaired. This increase in RSV-specific pro-inflammatory T cells was accompanied by increased weight loss and illness after RSV infection.

Conclusions/Significance

CCL3 regulates the balance of T cell populations in the lung and can alter the outcome of RSV infection. Understanding the role of inflammatory mediators in the recruitment of pathogenic T cells to the lungs may lead to novel methods to control RSV disease.     

Unconventional repertoire profile is imprinted during acute chikungunya infection for natural killer cells polarization toward cytotoxicity

Chikungunya virus (CHIKV) is a worldwide emerging pathogen. In humans it causes a syndrome characterized by high fever, polyarthritis, and in some cases lethal encephalitis. Growing evidence indicates that the innate immune response plays a role in controlling CHIKV infection. We show here that CHIKV induces major but transient modifications in NK-cell phenotype and function soon after the onset of acute infection. We report a transient clonal expansion of NK cells that coexpress CD94/NKG2C and inhibitory receptors for HLA-C1 alleles and are correlated with the viral load. Functional tests reveal cytolytic capacity driven by NK cells in the absence of exogenous signals and severely impaired IFN-γ production. Collectively these data provide insight into the role of this unique subset of NK cells in controlling CHIKV infection by subset-specific expansion in response to acute infection, followed by a contraction phase after viral clearance.     

A new experimental murine model for lipopolysaccharide-mediated lethal shock with lung injury

We have recently established a new experimental murine model for lipopolysaccharide (LPS)-mediated lethal shock with lung-specific injury. Severe lung injury is induced by administration of LPS into α-galactosylceramide (α-GalCer)-sensitized mice; the mice died with acute lung injury and respiratory distress within 24 h. α-GalCer activates natural killer T (NKT) cells in the lungs and liver, and induces the production of interferon (IFN)-γ. However, IFN-γ signaling is only triggered in the lungs and makes them susceptible to LPS. On the other hand, IFN-γ signaling is inhibited in liver and results in few hepatic lesions. Unlike liver NKT cells, lung NKT cells fail to produce interleukin (IL)-4, which down-regulates the IFN-γ signaling, in response to α-GalCer. The differential cytokine profile between lung and liver NKT cells may lead to organ-specific lung lesions. The experimental system using α-GalCer sensitization could be a useful experimental model for clinical endotoxic or septic shock as it presents respiratory failure, a typical manifestation in severe septic patients. In this review, key evidence and the introducuction of the detailed mechanism of LPS-mediated lung-specific injury in α-GalCer-sensitized mice is provided. In particular, the molecular background of organ-specific development of lung injury in the model is focused on.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

Negative Impact of IFN-γ on Early Host Immune Responses to Retroviral Infection

The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFN-α and IFN-γ. LDV-induced IFN-α had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-γ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-γ signaling indirectly modulated FV-specific CD8(+) T cell responses. Second, intrinsic IFN-γ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-γ production can have detrimental effects on early adaptive immune responses and virus control.     

IFN-γ Receptor-Deficient Donor T Cells Mediate Protection from Graft-versus-Host Disease and Preserve Graft-versus-Tumor Responses after Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. It has been previously reported that lung GVHD severity directly correlates with the expansion of donor Th17 cells in the absence of IFN-γ. However, the consequence of Th17-associated lung GVHD in the presence of IFN-γ has not been well characterized. In the current study, T cells from IFN-γ receptor knockout (IFN-γR(-/-)) mice, capable of producing IFN-γ but unable to signal in response to IFN-γ, have been used to elucidate further the role of IFN-γ in GVHD. We found the transfer of donor T cells from either IFN-γR(-/-) or IFN-γ knockout (IFN-γ(-/-)) mice resulted in significant increases in donor Th17 cells in the lung. Marked increases in IL-4-producing Th2 cells infiltrating the lungs were also observed in the mice of donor IFN-γR(-/-) T cells. Notably, despite the presence of these cells, these mice did not show the severe immune-mediated histopathological lung injury observed in mice receiving donor IFN-γ(-/-) T cells. Increases in lung GVHD did occur in mice with donor IFN-γR(-/-) T cells when treated in vivo with anti-IFN-γ demonstrating that the cytokine has a protective role on host tissues in GVHD. A survival benefit from acute GVHD was also observed using donor cells from IFN-γR(-/-) T cells compared with control donors. Importantly, tumor-bearing mice receiving IFN-γR(-/-) T cells versus wild-type donor T cells displayed similar graft-versus-tumor (GVT) effects. These results demonstrate the critical role of IFN-γ on host tissues and cell effector functions in GVHD/GVT.     

Susceptibility to acute mouse adenovirus type 1 respiratory infection and establishment of protective immunity in neonatal mice

There is an incomplete understanding of the differences between neonatal immune responses that contribute to the increased susceptibility of neonates to some viral infections. We tested the hypothesis that neonates are more susceptible than adults to mouse adenovirus type 1 (MAV-1) respiratory infection and are impaired in the ability to generate a protective immune response against a second infection. Following intranasal infection, lung viral loads were greater in neonates than in adults during the acute phase but the virus was cleared from the lungs of neonates as efficiently as it was from adult lungs. Lung gamma interferon (IFN-γ) responses were blunted and delayed in neonates, and lung viral loads were higher in adult IFN-γ(-/-) mice than in IFN-γ(+/+) controls. However, administration of recombinant IFN-γ to neonates had no effect on lung viral loads. Recruitment of inflammatory cells to the airways was impaired in neonates. CD4 and CD8 T cell responses were similar in the lungs of neonates and adults, although a transient increase in regulatory T cells occurred only in the lungs of infected neonates. Infection of neonates led to protection against reinfection later in life that was associated with increased effector memory CD8 T cells in the lungs. We conclude that neonates are more susceptible than adults to acute MAV-1 respiratory infection but are capable of generating protective immune responses.     

The immune system utilizes two distinct effector mechanisms of T cells depending on two different life cycle stages of a single pathogen,Toxoplasma gondii,to control its cerebral infection

Toxoplasma gondii takes two different life cycle stages within intermediate hosts including humans. Tachyzoites proliferate during the acute stage, and they transform into cysts to establish a chronic infection preferentially in the brain. IFN-γ production by infiltrated CD4⁺ and CD8⁺ T cells is required for the prevention of cerebral tachyzoite growth. IFN-γ production by brain-resident cells, most likely microglia, plays a key first line defense role to facilitate both innate and T cell-mediated protective immunity to control the tachyzoite growth. IFN-γ produced by brain-resident cells activates cerebral expression of IFN-dependent effector molecules to suppress tachyzoite growth during the early stage of infection. Their IFN-γ production also induces an expression of CXCL9 and CXCL10 chemokines to recruit immune T cells into the brain, and upregulates cerebral expression of MHC class I and II molecules for antigen presentation to the recruited T cells to activate their IFN-γ production. CD8⁺ T cells also have the activity to remove T. gondii cysts from the brains of infected hosts. Of interest, the anti-cyst activity of CD8⁺ T cells does not require their IFN-γ but does require perforin. Notably, we discovered that CD8⁺ cytotoxic T cells penetrate in the cysts in a perforin-mediated manner, which induces morphological deterioration and destruction of the cysts and an accumulation of microglia and macrophages for their elimination. Thus, the immune system employs two distinct effector mechanisms mediated by IFN-γ or perforin depending on two different life cycle stages of a single pathogen, T. gondii, to control its cerebral infection.