Article Title: Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection
Figure Lengend Snippet: Mice infected with H5N1 (2:6) mount robust T cell responses but show delayed viral clearance. (A-E) C57BL/6 mice (n=3-4/group) were infected with 100 PFU of H1N1 or H5N1 (2:6) and on day 8 pi, T cells from the lungs were isolated and evaluated in various assays. (A-C) Comparative analysis of lung CD8+ T cells from H5N1 (2:6) or H1N1 infected mice by NP or PA tetramer staining. (A) Representative FACS plots for NP or PA tetramer staining. (B) Relative frequency of tetramer positive CD8+ T cells. (C) Absolute numbers of virus specific CD8+ T cells. (D-E) Comparative analysis of cytokine production in T cells isolated from the lungs of H5N1 (2:6) or H1N1 infected mice. T cells were co-cultured with BMDC either infected with X31 (H3N2) or pulsed with NP peptide, and the frequencies of IFNγ and GrB producing T cells were analyzed by flow cytometry. (D) Relative frequency of cytokine producing CD8+ T cells. (E) Relative frequency of cytokine producing CD4+ T cells stimulated with NP peptide. (F) Evaluation of viral loads in the lungs of infected mice. C57BL/6 mice were infected with 100 PFU of H1N1 or H5N1 (2:6) and at various times pi, viral loads in the lungs were measured by standard plaque assay. (G-H) Ex vivo analysis of cytotoxic T cell functions. CFSE labeled splenocytes pulsed with NP peptide were co-cultured with lung CD8+ T cells for 8hrs, followed by staining with 7-AAD. Ex vivo cytotoxic effects of CD8+ T cells were evaluated by analyzing 7-AAD positive splenocytes. (G) Representative FACS plots for 7-AAD positive cells and (H) relative level of killing by T cells shown as percentage of 7-AAD positive cells. (I-J) In vivo analysis of cytotoxic T cell functions. (I) Representative FACS plots for in vivo killing of adoptively transferred NP pulsed splenocytes in H1N1 or H5N1 (2:6) virus infected mice and (J) relative level of kiiling by T cells. The values are expressed as mean ± SD. *, **, *** denotes significance of
Article Snippet: Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen).
Techniques: Mouse Assay, Infection, Isolation, Staining, FACS, Cell Culture, Flow Cytometry, Plaque Assay, Ex Vivo, Labeling, In Vivo