Penetration of CD8+ Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii, Leads to Its Elimination
2019; Elsevier BV; Volume: 189; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2019.04.018
ISSN1525-2191
AutoresAshish Tiwari, Rancie Hannah, Jenny Lutshumba, Eri Ochiai, Louis M. Weiss, Yasuhiro Suzuki,
Tópico(s)Herpesvirus Infections and Treatments
ResumoCD8+ cytotoxic T cells kill target cells through direct cell-cell contact. However, it remains unclear how these T cells eliminate a target of large mass. We investigated how CD8+ T cells remove tissue cysts of Toxoplasma gondii, which can grow to the size of >50 μm in diameter within infected cells. Notably, immunohistologic analyses in the brains of infected mice visualized the presence of numbers of CD8+ immune T cells that had migrated halfway through the cyst wall as well as T cells located fully within the cysts. Perforin was required for their invasion and cyst elimination. Cysts invaded by the T cells displayed morphologic deterioration and destruction. Within these deteriorated cysts, granular structures intensely positive for granzyme B were detected in association with T. gondii bradyzoites. Furthermore, the bradyzoites within the destroyed cysts were located within accumulated ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia and Ly6C+ macrophages, suggesting that these phagocytes had phagocytosed those organisms for their eradication. The present study uncovered a previously unappreciated capability of CD8+ cytotoxic T cells to penetrate into a large target, T. gondii cysts, for their elimination. This invasive capability of CD8+ cytotoxic T cells in collaboration with phagocytes appears to be a powerful effector mechanism that functions against not only T. gondii cysts but also other large targets, including solid cancers. CD8+ cytotoxic T cells kill target cells through direct cell-cell contact. However, it remains unclear how these T cells eliminate a target of large mass. We investigated how CD8+ T cells remove tissue cysts of Toxoplasma gondii, which can grow to the size of >50 μm in diameter within infected cells. Notably, immunohistologic analyses in the brains of infected mice visualized the presence of numbers of CD8+ immune T cells that had migrated halfway through the cyst wall as well as T cells located fully within the cysts. Perforin was required for their invasion and cyst elimination. Cysts invaded by the T cells displayed morphologic deterioration and destruction. Within these deteriorated cysts, granular structures intensely positive for granzyme B were detected in association with T. gondii bradyzoites. Furthermore, the bradyzoites within the destroyed cysts were located within accumulated ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia and Ly6C+ macrophages, suggesting that these phagocytes had phagocytosed those organisms for their eradication. The present study uncovered a previously unappreciated capability of CD8+ cytotoxic T cells to penetrate into a large target, T. gondii cysts, for their elimination. This invasive capability of CD8+ cytotoxic T cells in collaboration with phagocytes appears to be a powerful effector mechanism that functions against not only T. gondii cysts but also other large targets, including solid cancers. Toxoplasma gondii is an obligate intracellular protozoan parasite that can establish a chronic infection in humans. One-third of the human population in the world is estimated to be infected with this parasite.1Montoya J.G. Liesenfeld O. Toxoplasmosis.Lancet. 2004; 363: 1965-1976Google Scholar The basis of the persistent chronic infection is the cysts, which can contain hundreds to thousands of bradyzoites surrounded by the cyst wall,2Dubey J.P. Lindsay D.S. Speer C.A. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts.Clin Microbiol Rev. 1998; 11: 267-299Google Scholar, 3Sims T.A. Hay J. Talbot I.C. An electron microscope and immunohistochemical study of the intracellular location of Toxoplasma tissue cysts within the brains of mice with congenital toxoplasmosis.Br J Exp Pathol. 1989; 70: 317-325Google Scholar, 4Boothroyd J.C. Black M. Bonnefoy S. Hehl A. Knoll L.J. Manger I.D. Ortega-Barria E. Tomavo S. Genetic and biochemical analysis of development in Toxoplasma gondii.Philos Trans R Soc Lond B Biol Sci. 1997; 352: 1347-1354Google Scholar in various organs, especially the brain. This chronic infection can reactivate in immunocompromised individuals, such as those with AIDS, neoplastic diseases, and organ transplants, resulting in life-threatening toxoplasmic encephalitis.1Montoya J.G. Liesenfeld O. Toxoplasmosis.Lancet. 2004; 363: 1965-1976Google Scholar Even in immunocompetent individuals, recent epidemiologic studies shed light on the pathogenic effects of this widespread chronic infection by reporting a higher incidence of multiple types of cancers in individuals seropositive to this parasite.5Cong W. Liu G.H. Meng Q.F. Dong W. Qin S.Y. Zhang F.K. Zhang X.Y. Wang X.Y. Qian A.D. Zhu X.Q. Toxoplasma gondii infection in cancer patients: prevalence, risk factors, genotypes and association with clinical diagnosis.Cancer Lett. 2015; 359: 307-313Google Scholar, 6Thomas F. Lafferty K.D. Brodeur J. Elguero E. Gauthier-Clerc M. Misse D. Incidence of adult brain cancers is higher in countries where the protozoan parasite Toxoplasma gondii is common.Biol Lett. 2012; 8: 101-103Google Scholar, 7Vittecoq M. Elguero E. Lafferty K.D. Roche B. Brodeur J. Gauthier-Clerc M. Misse D. Thomas F. Brain cancer mortality rates increase with Toxoplasma gondii seroprevalence in France.Infect Genet Evol. 2012; 12: 496-498Google Scholar Current chemotherapy is effective only against tachyzoites. Therefore, there is an urgent need to develop technique(s) capable of eradicating the cyst stage of T. gondii from chronically infected individuals. Therefore, development of an immunologic intervention capable of attacking and eradicating the cysts is a valuable approach to fight against this widespread infection. Although information exists on the molecular mechanisms of the interferon (IFN)-γ–mediated protective immunity to control proliferation of T. gondii tachyzoites (the acute stage form),8Suzuki Y. Sa Q. Gehman M. Ochiai E. Interferon-gamma- and perforin-mediated immune responses for resistance against Toxoplasma gondii in the brain.Expert Rev Mol Med. 2011; 13: e31Google Scholar, 9Munoz M. Liesenfeld O. Heimesaat M.M. Immunology of Toxoplasma gondii.Immunol Rev. 2011; 240: 269-285Google Scholar the mechanisms of the host immunity against the cyst stage of the parasite are not well understood. Our recent studies revealed that an adoptive transfer of CD8+ immune T cells from chronically infected mice to infected immunodeficient [athymic nude or severe combined immunodeficiency (SCID)] animals, which have already established large numbers of cysts in their brains, is able to markedly reduce numbers of the cysts in the brains of the recipients.10Suzuki Y. Wang X. Jortner B.S. Payne L. Ni Y. Michie S.A. Xu B. Kudo T. Perkins S. Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells.Am J Pathol. 2010; 176: 1607-1613Google Scholar Notably, in contrast to the protective immunity against tachyzoites, the capability of CD8+ T cells to produce IFN-γ is dispensable for their activity to reduce the cyst numbers.10Suzuki Y. Wang X. Jortner B.S. Payne L. Ni Y. Michie S.A. Xu B. Kudo T. Perkins S. Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells.Am J Pathol. 2010; 176: 1607-1613Google Scholar Of interest, perforin was found to be required for the activity of CD8+ T cells to reduce cyst numbers in the brains of infected mice.10Suzuki Y. Wang X. Jortner B.S. Payne L. Ni Y. Michie S.A. Xu B. Kudo T. Perkins S. Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells.Am J Pathol. 2010; 176: 1607-1613Google Scholar However, how CD8+ T cells reduce T. gondii cyst burden using their perforin-mediated activity remains to be elucidated. In the present study, we determined that perforin-mediated cyst burden reduction is not due to indirect effects of inhibiting tachyzoite proliferation but is due to the direct removal of pre-existing cysts. Furthermore, immunohistochemical studies visualized that CD8+ immune T cells are capable of invading into cysts during the anticyst immune process in the brains of infected mice. The T-cell–invaded cysts displayed morphologic deterioration and destruction in association with condensed granular structures positive for granzyme B. An accumulation of large numbers of ionized calcium binding adaptor molecule 1 (Iba1)+ microglia and Ly6C+ inflammatory macrophages was always detected within and around those destroyed cysts, and destroyed individual organisms were detectable within these infiltrated phagocytes. These studies uncovered a superior and aggressive effector capability of CD8+ cytotoxic T cells to penetrate into T. gondii cysts and induce their elimination in collaboration with microglia and macrophages. CBA/J, BALB/c, and BALB/c-background SCID mice were obtained from the Jackson Laboratories (Bar Harbor, ME). BALB/c-background athymic nude mice and Swiss-Webster mice were from Taconic (Germantown, NY). BALB/c-background perforin-knockout (Prf1−/−) mice11White D.W. MacNeil A. Busch D.H. Pilip I.M. Pamer E.G. Harty J.T. Perforin-deficient CD8+ T cells: in vivo priming and antigen-specific immunity against Listeria monocytogenes.J Immunol. 1999; 162: 980-988Google Scholar were bred in our animal facility. Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with established institutional guidance and approved protocols from the Institutional Animal Care and Use Committee. Female mice were used for all studies. There were three to seven mice in each experimental group. The ME49 strain of T. gondii was maintained in vivo by infecting Swiss-Webster mice with 10 cysts intraperitoneally.12Kang H. Suzuki Y. Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain.Infect Immun. 2001; 69: 2920-2927Google Scholar, 13Suzuki Y. Orellana M.A. Wong S.Y. Conley F.K. Remington J.S. Susceptibility to chronic infection with Toxoplasma gondii does not correlate with susceptibility to acute infection in mice.Infect Immun. 1993; 61: 2284-2288Google Scholar In the experiments described in the present study, cysts were obtained from brains of chronically infected Swiss-Webster mice and all experimental mice were infected with 10 or 20 cysts orally by gavage. SCID and nude mice were treated with sulfadiazine in the drinking water (400 mg/L) beginning at 9 to 11 days after infection for the entire period of the experiments to inhibit the proliferation of tachyzoites and establish a chronic infection in their brains.12Kang H. Suzuki Y. Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain.Infect Immun. 2001; 69: 2920-2927Google Scholar, 14Wang X. Claflin J. Kang H. Suzuki Y. Importance of CD8+Vβ8+ T cells in IFN-gamma-mediated prevention of toxoplasmic encephalitis in genetically resistant BALB/c mice.J Interferon Cytokine Res. 2005; 25: 338-344Google Scholar Prf1−/− mice received sulfadiazine beginning at 4 weeks after infection. CBA/J mice were divided into two groups after infection, and one group received sulfadiazine treatment beginning at 3 weeks after infection. CD8+ immune T cells were purified from the spleens of chronically infected BALB/c and Prf1−/− mice using magnetic bead–conjugated anti-mouse CD8 (53-6.7) monoclonal antibodies (Miltenyi Biotech, Auburn, CA), as described previously.12Kang H. Suzuki Y. Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain.Infect Immun. 2001; 69: 2920-2927Google Scholar, 14Wang X. Claflin J. Kang H. Suzuki Y. Importance of CD8+Vβ8+ T cells in IFN-gamma-mediated prevention of toxoplasmic encephalitis in genetically resistant BALB/c mice.J Interferon Cytokine Res. 2005; 25: 338-344Google Scholar CD8+ normal T cells were also purified from the spleens of uninfected BALB/c mice in the same manner. A total of 2.1 to 4.2 × 106 CD8+ T cells were injected intravenously from a tail vein into sulfadiazine-treated SCID or nude mice at 3 weeks after infection.12Kang H. Suzuki Y. Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain.Infect Immun. 2001; 69: 2920-2927Google Scholar, 15Wang X. Kang H. Kikuchi T. Suzuki Y. Gamma interferon production, but not perforin-mediated cytolytic activity, of T cells is required for prevention of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease.Infect Immun. 2004; 72: 4432-4438Google Scholar The brains of infected SCID mice were obtained after perfusion with 20 mL of phosphate-buffered saline (PBS; pH 7.2) at 7 days after a transfer of CD8+ T cells, and RNA was isolated from a half of each brain, as described previously.16Singh J. Graniello C. Ni Y. Payne L. Sa Q. Hester J. Shelton B.J. Suzuki Y. Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites in the brain during the chronic stage of infection.Microbes Infect. 2010; 12: 1252-1257Google Scholar, 17Hester J. Mullins J. Sa Q. Payne L. Mercier C. Cesbron-Delauw M.F. Suzuki Y. Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection.Infect Immun. 2012; 80: 3611-3620Google Scholar The total RNAs were pretreated with DNase I (Invitrogen, Carlsbad, CA), to remove genomic DNA contaminating the RNA preparations, and then applied for cDNA synthesis.16Singh J. Graniello C. Ni Y. Payne L. Sa Q. Hester J. Shelton B.J. Suzuki Y. Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites in the brain during the chronic stage of infection.Microbes Infect. 2010; 12: 1252-1257Google Scholar, 17Hester J. Mullins J. Sa Q. Payne L. Mercier C. Cesbron-Delauw M.F. Suzuki Y. Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection.Infect Immun. 2012; 80: 3611-3620Google Scholar Real-time PCR for mouse β-actin, bradyzoite-specific BAG1, cyst wall glycoprotein (CST)1, and SAG2c, tachyzoite-specific SAG1, the T-cell marker CD3δ, IFN-γ, and the effector molecules (guanylate-binding protein 1, immunity-related GTPases M3, and inducible nitric oxide synthase 2) of IFN-γ–mediated protective immunity against tachyzoites was performed with the cDNA using the reagents from Applied Biosystems with the StepOnePlus real-time PCR system (Applied Biosystems, Branchburg, NJ).18Wen X. Kudo T. Payne L. Wang X. Rodgers L. Suzuki Y. Predominant interferon-gamma-mediated expression of CXCL9, CXCL10, and CCL5 proteins in the brain during chronic infection with Toxoplasma gondii in BALB/c mice resistant to development of toxoplasmic encephalitis.J Interferon Cytokine Res. 2010; 30: 653-660Google Scholar The primers and probes for BAG1 and SAG1 were described previously.19Sa Q. Ochiai E. Tiwari A. Perkins S. Mullins J. Gehman M. Huckle W. Eyestone W.H. Saunders T.L. Shelton B.J. Suzuki Y. Cutting Edge: IFN-gamma produced by brain-resident cells is crucial to control cerebral infection with Toxoplasma gondii.J Immunol. 2015; 195: 796-800Google Scholar The primers and probe for CST1 and SAG2c are as follows: 5′-CTTGTTACTGTTCCGCCTTTCTG-3′ (forward), 5′-CGTCAAAGTCTTTACATCGTTGCA-3′ (reverse), and 5′-TCCGGTCCAAGAAACC-3′ (probe) for CST1; and 5′-CGCACAGTCATTCAACCAAAAAGTT-3′ (forward), 5′-TGGAGGTGACCGCTACAGT-3′ (reverse), and 5′-TTGTGTCGTTCAGATAAATG-3′ (probe) for SAG2c. Expression levels of mRNA for the molecules of interest were determined by relative ratios to mouse β-actin mRNA levels. Sagittal sections (4 μm thick) of the brains were deparaffinized and rehydrated using xylene, ethanol, and then water. Heat-induced epitope retrieval (HIER) was performed in citrate buffer (pH 6) within a microwave for 5 minutes. The slides were then treated with 3% H2O2 for 15 minutes and then blocked with 5% bovine serum albumin in Tris-buffered saline with 0.5% Tween 20 for 2 hours. All primary antibodies and secondary antibodies used for staining were diluted in 5% bovine serum albumin/Tris-buffered saline with 0.5% Tween 20, and the incubation with these antibodies was for 1 or 2 hours at room temperature or overnight at 4°C. Staining of slides for T. gondii and CD3 was performed as described previously.20Ochiai E. Sa Q. Brogli M. Kudo T. Wang X. Dubey J.P. Suzuki Y. CXCL9 is important for recruiting immune T cells into the brain and inducing an accumulation of the T cells to the areas of tachyzoite proliferation to prevent reactivation of chronic cerebral infection with Toxoplasma gondii.Am J Pathol. 2015; 185: 314-324Google Scholar Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab)2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight. The secondary antibody was horseradish peroxidase–conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). Three-dimensional images were obtained by Zeiss Axio Imager M1 microscope (Zeiss, Oberkochen, Germany) using ZEN 2.0.0.0 pro software for regular light microscopy (Zeiss). Staining for T. gondii, Iba1, and Ly6C was performed as follows. After the HIER and blocking, the slides were then incubated with goat anti-Iba1 antibody (Abcam, Cambridge, MA). The slides were washed in Tris-buffered saline with 0.5% Tween 20 and incubated with alkaline phosphatase–conjugated donkey anti-goat IgG antibody (Invitrogen). Color was developed using Vulcan Fast Red Chromogen (Biocare Medical, Pacheco, CA). The slides were then incubated with polyclonal rat anti–T. gondii serum,20Ochiai E. Sa Q. Brogli M. Kudo T. Wang X. Dubey J.P. Suzuki Y. CXCL9 is important for recruiting immune T cells into the brain and inducing an accumulation of the T cells to the areas of tachyzoite proliferation to prevent reactivation of chronic cerebral infection with Toxoplasma gondii.Am J Pathol. 2015; 185: 314-324Google Scholar and then after washing, they were incubated with horseradish peroxidase–conjugated donkey anti-rat IgG antibody (Jackson ImmunoResearch Laboratories). The slides were then washed, and color was developed with diaminobenzidine (Vector Laboratories, Burlingame, CA). The slides were then subjected to two more rounds of HIER to facilitate removal of the prebound antibodies used for the staining for Iba1 and T. gondii.21Lan H.Y. Mu W. Nikolic-Paterson D.J. Atkins R.C. A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens.J Histochem Cytochem. 1995; 43: 97-102Google Scholar The slides were then reblocked and incubated with rat anti-Ly6C antibody (Abcam) and then the horseradish peroxidase–conjugated anti-rat IgG antibody. Thereafter, the slides were incubated with Vina Green Chromogen (Biocare Medical) for color development. Staining for CD3 (red), T. gondii (brown), and granzyme B (green) was performed in the same manner. Rabbit anti–granzyme B antibody was from Abcam. Staining for T. gondii, CD3, glial fibrillary acidic protein, and neuronal nuclei (NeuN) was performed as follows. The staining of T. gondii and CD3 was performed as described earlier, with a slight modification by using alkaline phosphatase–conjugated goat anti-rabbit IgG antibody (Invitrogen). After the color development, the slides were subjected to two rounds of HIER to remove prebound antibodies, followed by reblocking, as described in the previous paragraph. The slides were then incubated with rabbit anti-NeuN antibody (Abcam), followed by incubation with the alkaline phosphatase–conjugated goat anti-rabbit IgG antibody. Color was developed using alkaline phosphatase substrate Vector Blue (Vector Laboratories). The slides were then incubated with goat anti–glial fibrillary acidic protein antibody (Abcam), followed by incubation with the horseradish peroxidase–conjugated donkey anti-goat IgG antibody (Invitrogen) and color development with Vina Green Chromogen. Immunofluorescence staining of a combination of T. gondii and granzyme B or CD3 was as follows. After HIER, the slides were washed in PBS with Tween 20, blocked in PBS with Tween 20 with 1% normal goat serum (Jackson ImmunoResearch) and 2.5% bovine serum albumin for 2 hours, and incubated simultaneously with a combination of rat anti–T. gondii and rabbit anti–granzyme B (Abcam) or rabbit anti-CD3 antibodies at 4°C overnight. The slides were washed in PBS with Tween 20 and incubated with the AlexaFluor-488–conjugated goat anti-rat and AlexaFluor-594–conjugated goat anti-rabbit antibodies (Jackson ImmunoResearch) at room temperature for 1 hour. After washing in PBS with Tween 20 and then in PBS, the slides were mounted with ProLong Diamond Antifade Mountant (Invitrogen). The aforementioned mountant was cured overnight at room temperature before fluorescent visualization. Confocal images were obtained by a Nikon A1R microscope (Nikon, Tokyo, Japan) using NIS Elements AR 4.50.00 software (Nikon). Four to five sections from the brain of each of four infected CBA/J mice were stained for T. gondii and CD3, and a digital image of each of the cysts detected on the sections was taken to measure the diameter of each cyst using NIS-Elements BR acquisition 3.2 (Nikon). Levels of significance between experimental groups were determined by t-test or U-test (IBM SPSS, Armonk, NY). When there were more than two groups in the comparison, the corrected P value was calculated by multiplying each P value by the number of comparisons performed among the groups. Levels of differences in frequencies of destroyed cysts in total cyst populations and those with and without association with granzyme B and Ly6C between groups were determined by the Fisher exact test (QuickCalcs 2x2; GraphPad Software, San Diego, CA). Differences that had P values or corrected P values (when these values were applied) < 0.05 were considered significant. CD8+ immune T cells have a potent capability to reduce numbers of T. gondii cysts in the brains of mice through their perforin-mediated activity.10Suzuki Y. Wang X. Jortner B.S. Payne L. Ni Y. Michie S.A. Xu B. Kudo T. Perkins S. Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells.Am J Pathol. 2010; 176: 1607-1613Google Scholar However, the perforin-mediated activity of the CD8+ T cells might be attributed to inhibiting proliferation of tachyzoites and, thereby, reduced numbers of cysts. Therefore, it was first addressed whether the reduction of cyst numbers induced by perforin-mediated activity of CD8+ immune T cells is due to indirect effects from inhibiting cerebral tachyzoite growth. CD8+ T cells purified from the spleens of infected wild-type (WT) BALB/c and Prf1−/− mice were intravenously injected into infected, sulfadiazine-treated SCID mice that lack T cells in the same manner as previously described.12Kang H. Suzuki Y. Requirement of non-T cells that produce gamma interferon for prevention of reactivation of Toxoplasma gondii infection in the brain.Infect Immun. 2001; 69: 2920-2927Google Scholar, 15Wang X. Kang H. Kikuchi T. Suzuki Y. Gamma interferon production, but not perforin-mediated cytolytic activity, of T cells is required for prevention of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease.Infect Immun. 2004; 72: 4432-4438Google Scholar Seven days after the cell transfer, markedly and significantly decreased amounts of mRNA for bradyzoite (cyst)-specific BAG1, CST1 (Figure 1A), and SAG2C (Supplemental Figure S1) were detected in the brains of the animals that had received the WT CD8+ T cells but not of those that had received the Prf1−/− CD8+ T cells, when compared with the control mice that had received no T cells (P < 0.001). In addition, the mRNA levels for BAG1, CST1, and SAG2C in the Prf1−/− CD8+ T-cell recipients were significantly greater than those of the WT T-cell recipients (P < 0.05) (Figure 1A and Supplemental Figure S1). To address whether Prf1−/− CD8+ T cells efficiently migrated into the brains of the recipients, the amounts of mRNA for CD3 were measured in the brains of recipients. In the control mice that had not received any T cells, cerebral CD3 mRNA levels were low and close to the detectable limit, as expected (Figure 1B). In contrast, large amounts of CD3 mRNAs were detected in the brains of both WT and Prf1−/− T-cell recipients in a similar manner (P < 0.001 and P < 0.01, respectively) (Figure 1B). Thus, Prf1−/− CD8+ T cells had migrated into the brains of the recipients as efficiently as the WT CD8+ T cells. This point is further supported by the evidence that CD3+ T cells were detectable in the parenchyma (Figure 1D), including perivascular areas (Figure 1E), of the brains of infected nude mice at 2 to 3 days after a systemic transfer of Prf1−/− CD8+ immune T cells. These results confirmed that perforin is the major mediator of anticyst activity of CD8+ immune T cells to reduce bradyzoite burden in the brains of recipient mice. However, because cyst burden in the Prf1−/− T-cell recipients tended to be less than that of the control mice without any T-cell transfer (Figure 1A and Supplemental Figure S1), possible involvement of other mechanisms contributing to a lesser extent to the anticyst immune process, such as that mediated by chitinase-dependent activity of alternatively activated macrophages, cannot be excluded.22Nance J.P. Vannella K.M. Worth D. David C. Carter D. Noor S. Hubeau C. Fitz L. Lane T.E. Wynn T.A. Wilson E.H. Chitinase dependent control of protozoan cyst burden in the brain.PLoS Pathog. 2012; 8: e1002990Google Scholar To address whether the absence of perforin affected the activity of CD8+ immune T cells to prevent cerebral tachyzoite growth, amounts of mRNA were compared for tachyzoite-specific SAG1 in the brains of infected SCID mice that had received the WT or Prf1−/− CD8+ T cells.10Suzuki Y. Wang X. Jortner B.S. Payne L. Ni Y. Michie S.A. Xu B. Kudo T. Perkins S. Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells.Am J Pathol. 2010; 176: 1607-1613Google Scholar, 20Ochiai E. Sa Q. Brogli M. Kudo T. Wang X. Dubey J.P. Suzuki Y. CXCL9 is important for recruiting immune T cells into the brain and inducing an accumulation of the T cells to the areas of tachyzoite proliferation to prevent reactivation of chronic cerebral infection with Toxoplasma gondii.Am J Pathol. 2015; 185: 314-324Google Scholar, 23Sa Q. Ochiai E. Sengoku T. Wilson M.E. Brogli M. Crutcher S. Michie S.A. Xu B. Payne L. Wang X. Suzuki Y. VCAM-1/alpha4beta1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis.Infect Immun. 2014; 82: 2826-2839Google Scholar In contrast to BAG1 mRNA levels, tachyzoite-specific SAG1 mRNA did not differ between the recipients of WT and Prf1−/− CD8+ T cells (Figure 1B). Furthermore, the amounts of SAG1 mRNA were 45 and 41 times less than those of BAG1 mRNA in both WT and Prf1−/− CD8+ T-cell recipients, respectively (Figure 1A). These results indicate that both WT and Prf1−/− CD8+ T cells effectively prevented tachyzoite growth in the brains of recipients to the same extent and that differences in cyst numbers were not attributable to differences in tachyzoite proliferation. Consistent with these findings, markedly increased mRNA levels for IFN-γ and the effector molecules (inducible nitric oxide synthase 2, immunity-related GTPases M3, and guanylate-binding protein 1) in the IFN-γ–mediated protective immunity to prevent tachyzoite growth24Ling Y.M. Shaw M.H. Ayala C. Coppens I. Taylor G.A. Ferguson D.J. Yap G.S. Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages.J Exp Med. 2006; 203: 2063-2071Google Scholar, 25Selleck E.M. Fentress S.J. Beatty W.L. Degrandi D. Pfeffer K. Virgin HWt Macmicking J.D. Sibley L.D. Guanylate-binding protein 1 (Gbp1) contributes to cell-autonomous immunity against Toxoplasma gondii.PLoS Pathog. 2013; 9: e1003320Google Scholar, 26Divanovic S. Sawtell N.M. Trompette A. Warning J.I. Dias A. Cooper A.M. Yap G.S. Arditi M. Shimada K. Duhadaway J.B. Prendergast G.C. Basaraba R.J. Mellor A.L. Munn D.H. Aliberti J. Karp C.L. Opposing biological functions of tryptophan catabolizing enzymes during intracellular infection.J Infect Dis. 2012; 205: 152-161Google Scholar were detected in the brains of both of these two recipient groups in the same manner (Figure 1C). These results indicate that the reduction of T. gondii cyst burden by perforin-mediated activity of CD8+ T cells is due to direct removal of pre-existing T. gondii cysts, rather than indirect effects from inhibiting tachyzoite proliferation. To visualize the direct interactions of CD8+ T cells with T. gondii cysts, immunohistochemical studies were performed on the brains of infected mice. As the first step, to have better chances to detect the interactions of the T cells with cysts, a strain (CBA/J) of mice that forms large numbers of brain cysts after infection was used.27Suzuki Y. Joh K. Orellana M.A. Conley F.K. Remington J.S. A gene(s) within the H-2D region determines the development of toxoplasmic encephalitis in mice.Immunology. 1991; 74: 732-739Google Scholar, 28Brown C.R. McLeod R. Class I
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