This trend was less clearly observed for CD8+ T cells due to the overall lower responses (Figure 5D)

This trend was less clearly observed for CD8+ T cells due to the overall lower responses (Figure 5D). auricular lymph nodes harvested 3 days later for analysis by flow cytometry. Organs were diced into small pieces and digested at room temperature for 30 minutes in PBS made up of 0.3 mg/ml collagenase-dispase (Roche) and 0.02 mg/ml DNAse. For initial characterization of MVA vaccinated mice, LNs were surface stained for CD80-FITC, CD11c PE, CD8 PerCPCy5.5, B220-PECy7, F4/80 Pacific Blue, CD103-biotin, streptavidin qDot 565, CD4-qDot 655, CD11b Lauric Acid Alexa-700, MHC Class II APC-Alexa780 and Live-Dead Aqua prior to fixation and intracellular staining with CD207-APC (eBioscience) in perm-wash buffer. A) FACS plots demonstrate the gating strategy to classify the population of CD11c+B220?CD11b+MHC IIint DCs relative to plasmacytoid DCs (pDCs), lymph node (LN) resident DCs, or skin migrating DCs isolated from the draining auricular nodes (top panel) or non-draining inguinal and popliteal nodes (bottom panel) three days after id vaccination. CD11b+MHC IIint DCs were overlayed over the population of LN resident DCs to investigate expression of CD4 and CD8, or overlayed over the population of skin migrating DCs to investigate the expression of CD103 and CD207. Histograms represent the expression of either F4/80 (left) or CD80 (right) on LN resident (grey filled line), skin migrating (black dashed line) or CD11b+MHC IIint (black bold line) DCs. Graphs represent the percentage of live cells (B) or total number (C) of B cells (B220+CD11c?), CD4 (CD4+CD8?), CD8 (CD8+CD4?), DCs (CD11c+) and CD11c?CD11b+. CD11c+ MHC II+ cells were further subdivided into plasmacytoid DCs (pDCs) (B220+), CD11b+ MHC IIint, LN resident (CD11b?, MHC IIint) or skin migrating (MHC IIhi) with graphs representing the percentage (D) or total number of each populace (E). In a separate experiment, auricular LNs from Balb/c mice immunized id with 106 PFU of either MVA-GFP (triangle), MVA-Ag85A (circles), MVA-Ag85A IMX313 (square) or unvaccinated (diamonds) were analyzed at day 3 days by flow cytometry. Digested lymph nodes were surface stained for CD80-FITC, CD11c-PE, CD8-PerCPCy5.5, B220-PeCy7, CD4-qDot650, CD11b-Alexa700, MHC Class II APC with the addition of DAPI prior to acquisition of samples around the LSR II. Graphs represent the percentage (F) and total number (G) of CD11b+MHCint cells or surface expression level of CD80 (H) or MHC Class II fluorescence (I) on CD11b+MHC IIint cells. In all graphs, bars represent the Hoxa2 mean per group with each individual animal displayed as a single point.(TIF) pone.0033555.s002.tif (459K) GUID:?D663FA7F-771E-4BB1-962B-1726B8EE55C0 Figure S3: Expression of activation markers on IFN-g+ CD4+ or CD8+ T cells 3 days after immunization. Balb/c mice were vaccinated id with 106 PFU MVA85A or MVA85A-IMX313 and the draining auricular lymph node harvested 3 days later. After 6 hours of restimulation with the total 85A peptide pool, cells Lauric Acid were surface stained with CD69-FITC, CD44-PE, CD8-PerCPCy5.5, CD62L-PECy7, CD127 Pacific Blue, CD122-bi followed by streptavidin dQot565 (Invitrogen), CD4-eFluro 650 and CD25-Alexa700 prior to fixation and intracellular staining for IFN- Alexa647. Cells were subdivided into either CD4+ T cells (A) or CD8+ T cells (B) prior to analysis of IFN- and surface marker expression. Data was analyzed with a two-way analysis of variance and no statistically significant difference was observed for any marker investigated.(TIF) pone.0033555.s003.tif (132K) GUID:?F146CDB3-399F-4DE3-BA36-CF72A2ABF75A Table S1: Ag85A peptide pools. The table lists the separation of the 66 individual 20 mer peptide (overlapping by 10) spanning the length of Ag85A into the 7 peptide pools labelled A to G.(DOCX) pone.0033555.s004.docx (51K) GUID:?7FB8A1B3-E341-4CED-88FF-90FECDB758C9 Table S2: Amino acid sequences of C4bp oligomerization domains. Using a Clustal V Method Lauric Acid of alignment (MegAlign, DNASTAR Lasergene software), bold letters represents homologous amino acids between chicken, IMX313 and human protein sequences. Grey letters represent the amino acid changes between human and the predicted rhesus macaque sequence (accession no. XP_002801994). Italic letters represent the major sequence change.