Journal: PLoS Pathogens
Article Title: Impairment of Immunoproteasome Function by β5i/LMP7 Subunit Deficiency Results in Severe Enterovirus Myocarditis
doi: 10.1371/journal.ppat.1002233
Figure Lengend Snippet: (A) Quantitative real-time PCR: mRNA expression of indicated cytokines (first row) and antiviral molecules of the innate immune response (second row) was determined in the hearts of β5i/LMP7 +/+ and β5i/LMP7 -/- mice at d4 and d8 p.i. Data shown are mean of n≥5 mice±SEM. (B)+(C) Flow cytometric analysis was completed on splenocytes isolated from β5i/LMP7 +/+ and β5i/LMP7 -/- mice at d4 and d8 p.i. (n = 6 mice, representative for 3 independent experiments). (B) Cell numbers of B cells (CD19 + , B220 + ) are illustrated as % of each cell population in comparison to the total number of cells from each spleen (left panel). CVB3-specific IgG levels were determined in sera collected at indicated time points, and titers of virus-specific IgG antibodies were determined by a CVB3-specific ELISA (n≥8 mice, right panel). (C) Ratios of CD4 + T cells (CD4 + , CD3 + ) and CD8 + T cells (CD8 + , CD3 + ) are illustrated. (D) For T cell transfer studies splenic CD8 T cells were separated by MACS (Miltenyi Biotec) from 1×10 7 splenocytes . Left panel: To assess T cell survival after adoptive transfer, CD8 T cells were transferred from CVB3-infected β5i/LMP7 +/+ (CD45.1) into β5i/LMP7 -/- (CD45.2) mice and from β5i/LMP7 -/- mice (CD45.2) into β5i/LMP7 +/+ mice (CD45.1). Recipient mice were infected with CVB3 and sacrificed at d8 p.i. T cell survival was determined in splenocytes by flow cytometry. Middle panel: Prior to adoptive T cell transfer, β5i/LMP7 +/+ and β5i/LMP7 -/- mice were infected with CVB3 and myocarditis scores were determined at d8 p.i. (n = 5 mice). Right panel: Then, splenic CD8 T cells were isolated from these CVB3-infected mice at d8 p.i. as described above; 1–2×10 6 CD8 + T cells from one donor were injected i.v. through the tail vein into one recipient. Recipient mice were then infected with CVB3 i.p. and sacrificed at d8 p.i. Myocardial damage was evaluated in HE staining as described above. Data shown are mean for n = 5 mice and are representative for two independent experiments.
Article Snippet: CVB3-specific IgG antibody titers were determined with Enterovirus ELISA Kit (Genzyme Diagnostics) according to the manufacture's instructions [alternative secondary antibody (POX anti-mouse IgG, Dianova) was used].
Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Comparison, Virus, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Infection, Flow Cytometry, Injection, Staining
