Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: Aging limits T RM establishment within the brain. 8 week. adult or 18 months aged C57BL/6 mice were inoculated with 10 3 pfu MHV‐A59 i.n., CD8 + T cells harvested from the spleen 7 DPI and then adoptively transferred intravenously (i.v.) via the tail vein into either 8 week. or 18 months old animals that were infected with (a,b) 10 4 pfu or (c,d) 10 3 pfu MHV‐A59 1 day prior. (a, c) Survival and (b, d) weight change were monitored for 30 DPI. (e) Representative flow cytometry plots of lymphocytes stained for CD8, then CD45.1 and CD45.2 to distinguish between host and donor cells isolated from the brains of 8 week. or 18 months old animals at 30 DPI following infection with 10 3 pfu MHV‐A59 and adoptive transfer of 8 week. or 18 months cells as indicated. (f) Frequency and (g) total number of total CD8 + cells and total transferred cells. (h) Representative flow cytometry histograms of CD103 expression by CD8 + T cells present in the brain of 8 week. or 18 months animals 30 DPI following infection and adoptive transfer as described. (i) Quantification of CD103 MFI by host or donor cells within the same host. Data are representative of 1 independent experiment with each data point representing an individual animal. Survival assessed by Log‐rank Mantel‐Cox assessment, weight change and flow cytometry assessment conducted according to two‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, Adoptive Transfer Assay, Expressing

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: CD8 + T cells mediate neuronal apoptosis following MHV‐A59 infection. (a) Representative IHC of DAPI, NeuN, and CD8a in the DG and cortex of 8 week or 18 months old animals at (a) 12 DPI and (b) 30 DPI. (c) Representative flow cytometry plots of CD45 + CD8 + cells stained for IFN‐γ isolated from the brains of 8 week or 18 months old, mock or MHV‐A59 infected animals at 30 DPI following 4 h. PMA/ionomycin stimulation. (d) Quantification of percent CD8 + CD44 + cells positive for IFN‐γ. (e) Representative immunocytochemical (ICC) staining for NeuN and TUNEL of primary cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without coculture with CD8 + T cells purified from the spleen of an 8 week old animal at 7 DPI with 10 3 pfu MHV‐A59. Quantification of ICC images: (f) total number NeuN + neuronal nuclei, (g) total number TUNEL + NeuN + neuronal nuclei, (h) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. (i) Representative ICC of NeuN and TUNEL staining of primary embryonic cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without naïve or PMA/ionomycin stimulated CD8 + T cells purified from the spleen of an 8 week old uninfected animal. Quantification of ICC images: (j) total number NeuN + neuronal nuclei, (k) total number TUNEL + NeuN + neuronal nuclei, and (l) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. Data are representative of three independent experiments with each data point representing an individual sample. All images were taken at 40X magnification and scale bar = 100 μm. Three images were captured per sample and averaged. NeuN + and TUNEL + NeuN + neuronal nuclei quantified in ImageJ software using the Cell Counter plugin. Statistics according to unpaired one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, TUNEL Assay, Purification, Software