Bited attenuated antigen presenting activity [24]. We previously showed that LMP7 plays

Bited attenuated antigen presenting activity [24]. We previously showed that LMP7 plays a crucial role in inducing antigen-specific CD8+ T cells, and LMP7-deficient mice were more susceptible to tumors [25] and protozoan infection [26,27], where CD8 T cells mainly function as effector cells. Malaria remains a crucial threat to public health worldwide. It is well accepted that antibodies and CD4+ T cells play critical roles in protection against blood-stage malaria that can be acquired during natural or experimental infection [28?1]. In addition, innate immunity attributed to macrophages, NK cells andMalaria Resistance in LMP7-Deficient Micedendritic cells (DCs) is also important. Especially, phagocytosis exerted by macrophages residing in the reticuloendothelial system is crucial for the elimination of parasitized red blood cells (pRBCs). In contrast, the contribution of CD8+ T cells to protective immunity against blood-stage malaria is controversial. KDM5A-IN-1 Although RBCs are exceptional cells that express no MHC class I molecules, CD8+ T cells are activated during blood-stage malaria [32,33]. Furthermore, activation of CD8+ T cells is required for the development of experimental cerebral malaria [34]. We recently found that CD8 T cells are important for immunity against bloodstage malaria [35], leading us to hypothesize that LMP7-deficiency impairs resistance to infection with blood-stage malaria. In this study, we observed that LMP7-deficient mice were partially resistant to infection with rodent malaria parasites, Plasmodium yoelii. We examined immune responses in LMP7deficient mice in detail and found no explainable difference in innate and adaptive immunity including CD8 T cell responses. However, we found that pRBCs from LMP7-deficient mice were highly phagocytosed.San Jose, CA), and the list data were analyzed using CellQuest Pro software (BD Biosciences).Quantitative real-time PCRmRNA quantification of IFN-c was performed with a real-time PCR system (Applied Biosystems, Foster City, CA), using SYBR 15900046 Green I double-strand DNA binding dye. Total RNA extracted from 16107 splenocytes from an uninfected or infected mouse (5 days after infection) was reverse-transcribed followed by PCR. For IFN-c, the sense and antisense primers were 59-AGCGGCTGACTGAACTCAGATTGTAG-39 and 59-GTCACAGTTTTCAGCTGTATAGGG, respectively. Fluorescence data collected after each extension step were analyzed using an ABI Prism 7000 SDS software. The relative ratio of mRNA encoding IFN-c in each sample was normalized to the relative quantity of b-actin.Phagocytosis of MacrophagesPeritoneal macrophages were collected from WT and LMP7deficient mice 4 days after injection with 0.5 ml thioglycollate solution. RBCs (107 cell/ml) were incubated with 10 mM carboxyfluorescein succinimidyl ester (CFSE) in PBS for 15 min at 37uC. CFSE 6R-Tetrahydro-L-biopterin dihydrochloride staining was stopped by addition excess complete medium (fetal bovine serum-supplemented RPMI1640) and washing cells three times with complete medium. Macrophages (56105 or 46105 cells/well) were cultured with 56106 CFSElabeled RBCs at a final volume of 200 ml for 1 h at 37uC. After coculture, non-ingested RBCs were removed by hemolysis with NH4Cl lysing buffer. The remaining macrophages were washed twice with complete 1326631 medium, and then stained with PEconjugated anti-mouse CD11b Ab before flow cytometric analysis.Materials and Methods Ethics StatementAll experiments that involved mice were reviewed and approved by the Committee for Ethics on Animal Expe.Bited attenuated antigen presenting activity [24]. We previously showed that LMP7 plays a crucial role in inducing antigen-specific CD8+ T cells, and LMP7-deficient mice were more susceptible to tumors [25] and protozoan infection [26,27], where CD8 T cells mainly function as effector cells. Malaria remains a crucial threat to public health worldwide. It is well accepted that antibodies and CD4+ T cells play critical roles in protection against blood-stage malaria that can be acquired during natural or experimental infection [28?1]. In addition, innate immunity attributed to macrophages, NK cells andMalaria Resistance in LMP7-Deficient Micedendritic cells (DCs) is also important. Especially, phagocytosis exerted by macrophages residing in the reticuloendothelial system is crucial for the elimination of parasitized red blood cells (pRBCs). In contrast, the contribution of CD8+ T cells to protective immunity against blood-stage malaria is controversial. Although RBCs are exceptional cells that express no MHC class I molecules, CD8+ T cells are activated during blood-stage malaria [32,33]. Furthermore, activation of CD8+ T cells is required for the development of experimental cerebral malaria [34]. We recently found that CD8 T cells are important for immunity against bloodstage malaria [35], leading us to hypothesize that LMP7-deficiency impairs resistance to infection with blood-stage malaria. In this study, we observed that LMP7-deficient mice were partially resistant to infection with rodent malaria parasites, Plasmodium yoelii. We examined immune responses in LMP7deficient mice in detail and found no explainable difference in innate and adaptive immunity including CD8 T cell responses. However, we found that pRBCs from LMP7-deficient mice were highly phagocytosed.San Jose, CA), and the list data were analyzed using CellQuest Pro software (BD Biosciences).Quantitative real-time PCRmRNA quantification of IFN-c was performed with a real-time PCR system (Applied Biosystems, Foster City, CA), using SYBR 15900046 Green I double-strand DNA binding dye. Total RNA extracted from 16107 splenocytes from an uninfected or infected mouse (5 days after infection) was reverse-transcribed followed by PCR. For IFN-c, the sense and antisense primers were 59-AGCGGCTGACTGAACTCAGATTGTAG-39 and 59-GTCACAGTTTTCAGCTGTATAGGG, respectively. Fluorescence data collected after each extension step were analyzed using an ABI Prism 7000 SDS software. The relative ratio of mRNA encoding IFN-c in each sample was normalized to the relative quantity of b-actin.Phagocytosis of MacrophagesPeritoneal macrophages were collected from WT and LMP7deficient mice 4 days after injection with 0.5 ml thioglycollate solution. RBCs (107 cell/ml) were incubated with 10 mM carboxyfluorescein succinimidyl ester (CFSE) in PBS for 15 min at 37uC. CFSE staining was stopped by addition excess complete medium (fetal bovine serum-supplemented RPMI1640) and washing cells three times with complete medium. Macrophages (56105 or 46105 cells/well) were cultured with 56106 CFSElabeled RBCs at a final volume of 200 ml for 1 h at 37uC. After coculture, non-ingested RBCs were removed by hemolysis with NH4Cl lysing buffer. The remaining macrophages were washed twice with complete 1326631 medium, and then stained with PEconjugated anti-mouse CD11b Ab before flow cytometric analysis.Materials and Methods Ethics StatementAll experiments that involved mice were reviewed and approved by the Committee for Ethics on Animal Expe.