Supplementary MaterialsS1 Fig: Identification of DbM187 and KdM282 T cells by flow cytometry

Supplementary MaterialsS1 Fig: Identification of DbM187 and KdM282 T cells by flow cytometry. inflammatory site, there are other factors that determine the hierarchy[23]. The Ki-67 expression showed that KdM282 T cells were more proliferative than DbM187 T cells (Fig 1C), consistent with the numerical dominance. Open in a separate window Fig 1 Numerical dominance of KdM282 T cell response is associated with expansion capacity.(a) Magnitude of DbM187 and KdM282 T cell response at 7 dpi. Frequency of DbM187 and KdM282 T cells in CD8 T cell population were quantitatively assessed with flow cytometry. Data represent 5 independent experiments (n = 5/group/experiment). (b) The DbM187 and KdM282 T cells infiltrate into lung parenchyma following RSV infection. Lung lymphocytes were isolated from RSV- infected mice at 7 dpi. T cells in vasculature were pre-labeled by intravenous anti-Thy1.2 staining prior to euthanizing mice. Proportion of DbM187 and KdM282 T cells in lung parenchyma were quantitatively assessed with flow cytometry. Data represent 3 independent experiments (n = 5/group/experiment). (c) Lymphocytes isolated from spleens at 7 and 9 dpi were studied for Ki-67 expression by flow cytometry. The frequencies of Ki-67(+) cells in DbM187 and KdM282 T cell subsets are shown. Data represent 3 independent experiments (n = 4/group/experiment). All data are shown as mean with independent data point and compared by Student t-test. Each symbol represents one mouse. DbM187 T cells have superior cytolytic activity To functionally evaluate DbM187 and KdM282 T cell cytotoxicity were up-regulated in DbM187 T cells, while genes related to cell division (eg. and (Fig 4A) that have been associated with effector-memory differentiation[25, 26]. In contrast, KdM282 T cells had elevated expression of and and (and and was also one of the top 25 up-regulated genes in DbM187 T cells (S5B Fig). Flow cytometry analysis showed that KdM282 T cells were more likely to be CP-409092 stained with Annexin V than DbM187 T cells, which is an early indication of apoptotic change (Fig 6B). In contrast, DbM187 T cells were more likely to express Bcl-2 than KdM282 T cells. Both CD8 T cell subsets from lung were more apoptotic than those from spleen (p 0.0001, DbM187 and KdM282 T cells). The pattern of pro- and anti-apoptotic molecule expression at both transcriptional and protein levels suggests that a large proportion of the CD8 T cells, particularly KdM282 T cells, progress towards apoptosis at the site of inflammation. Open in a separate window Fig 6 Activated KdM282 T cells are apoptotic. (a) Transcriptional expression of genes encoding pro- and anti-apoptosis molecules with FC 1.3, p 0.05 and FDR 0.25 were listed. Data were pooled from 10 or 11 individual mice in each group. (b) Apoptotic cells were identified by Annexin V staining, and post-transcriptional expression of Bcl-2 was identified by monoclonal antibody with flow cytometry at 7 dpi. The frequencies are shown as mean with independent data point and compared by Students T cell labeling Na?ve or RSV-infected mice were injected intravenously with Cy5PE-conjugated anti-Thy1.2 antibody purchased from BioLegend (San Diego, CA). CP-409092 Mice were sacrificed 10 min. later. Lymphocytes from lung and spleen were isolated and stained for lineage differentiation and pMHC specificity following standard procedures, and then applied to flow cytometry analysis[21, 22]. Cytotoxic activity assays To evaluate cytotoxicity of DbM187 and KdM282 T cells em in vivo /em , we setup an assay CP-409092 based on previous described[24]. The targets were prepared from na?ve spleen cells that were loaded with M187-195 (M187) or M282-95 (M282) peptides (Anaspec Inc, San Jose, CA) at a concentration of 10 g/ml, then stained with 10 uM carboxyfluorescein succinimidyl ester (CFSE) or 10 uM CellTracker Red CMTPX (both from Invitrogen) respectively; controls were prepared from na?ve spleen cells that were loaded with OVA257-264 (OVA257) peptide (Anaspec Inc) at a concentration of 10 g/ml, then stained with either 0.1 uM CFSE or 0.2 uM CellTracker Red CMTPX respectively. These four populations, including M187-CFSEhigh, M282-CMTPXhigh, OVA257-CFSElow and OVA257-CMTPXlow, each at 2 GDF7 x 106, were co-transferred into RSV-infected or na?ve mice via tail vein. Cells from the lung and spleen were collected 3 hours later from the recipients. Donor cells were identified by fluorochromes spectra and distinguished by fluorescence intensity. The specific lysis were calculated using formula: ratio = (percentage fluorochromelow/percentage fluorochromehigh). Specific lysis (%) = [1 ? (ratio CP-409092 naive/ratio infected) 100]. Frequency of pMHC-specific CD8 T cells was assessed CP-409092 simultaneously in the same cell preparation recovered. The cytolytic unit per pMHC specific CD8 T effector cell was calculated by dividing the percent of.