Fig 1.
Phenotypic identification of cDC and L-DC in spleen.
Representative flow cytometric analysis outlines the method used for delineation of subsets. This profile is reflective of multiple similar isolations involving individual mice. Splenocytes were prepared by red blood cells lysis followed by T and B cell depletion. Cells were then stained with antibodies specific for CD11b, CD11c, CD8, Ly6C, Ly6G, CD43 and Siglec-F. Prior to flow cytometry, cells were stained with propidium iodide (PI, 1μg/ml) to delineate live (PI-) cells. L-DC and myeloid cells were delineated on the basis of CD11b versus CD11c expression. Myeloid cells were gated as CD11bhiCD11c- cells, and further delineated on the basis of Ly6C and Ly6G expression to reveal subsets of inflammatory monocytes (Infl mono) as Ly6ChiLy6G- CD43+Siglec-F- cells, neutrophils (Neutro) as Ly6C+Ly6G+CD43+Siglec-F- and eosinophils (Eosino) as Ly6C+Ly6G-CD43+Siglec-F+ cells. Resident monocytes (Resi mono) were gated as Ly6C+Ly6G-CD43hi/+Siglec-F- cells, while L-DC were gated as Ly6C-Ly6G-CD43+Siglec-F- cells. Conventional DC (cDC) were initially gated on the basis of side scatter (SSC) and CD11c expression. CD8+ cDC were gated as CD11b-CD8+Ly6C-Ly6G- cells, while CD8- cDC were gated as CD11b+CD8-Ly6C-Ly6G- cells. Gates were set based on fluorescence minus one controls, and numbers in gates represent % specific binding.
Fig 2.
Comparison of endocytic ability of cDC subsets and L-DC subsets.
The ability of cells to endocytose antigen was measured by uptake of FITC-conjugated ovalbumin (OVA-FITC) and FITC-conjugated mannan (mannan-FITC). C57BL/6J mice were given A) OVA-FITC (iv; 1mg per mouse), and B) mannan-FITC (iv; 0.5mg per mouse) at 1, 3 and 6 hours prior to euthanasia for spleen collection. Control mice were given PBS. Splenocytes were prepared by RBC lysis, and enrichment for dendritic and myeloid cells via T, B cell and red blood cell depletion. Cells were stained with antibodies and sorted to give L-DC, CD8+ cDC and CD8- cDC subsets as shown in Fig 1. Uptake of antigen was assessed in terms of % FITC staining cells. Data are representative of 2 similar timed experiments.
Fig 3.
Activation of CD4+ T cells by splenic DC.
Antigen presenting ability of DC subsets purified from spleens of Act-mOVA mice was compared. L-DC, CD8+ and CD8- cDC, and neutrophils as a control, were sorted as described in Fig 1 following enrichment of splenocytes by depletion of red blood cells, and T and B lymphocytes using magnetic bead technology. Diluting numbers of APC were plated following treatment with and without LPS (10 μg/ml) for 2 hours. This was followed by addition of 105 CFSE-labelled OT-II (TCR-tg) CD4+ T cells, purified from mouse spleen through depletion of B cells, CD8+ T cells, DC and myeloid cells using magnetic bead protocols. Cells were cultured at T cell:APC ratios of 33:1, 100:1, 300:1 and 1000:1 for 72 hours. CD4+ OT-II T cells were then gated as PI-Thy1.2+Vα2+CD8- cells, and assessed flow cytometrically for CFSE dilution as an indicator of T cell proliferation. OT-II T cells cultured alone served as controls (con). Graphs show % proliferating OT-II cells. Three independent replicate experiments were conducted.
Fig 4.
Cross-presentation ability of DC subsets.
Cross-presentation of antigen was investigated for splenocytes harvested from Act-mOVA mice and prepared by red blood cell lysis and T/B cell depletion. Splenocyte subsets were stained and gated as described in Fig 1. Diluting numbers of DC were plated as APC followed by treatment with or without LPS (10 μg/ml) for 2 hours prior to the addition of 105 CFSE-labelled OT-I (TCR-tg) CD8+ T cells, purified from OT-I mouse spleen through depletion of B cells, CD4+ T cells, DC and myeloid cells using magnetic bead protocols. A) CD8+ cDC, CD8- cDC, L-DC and neutrophils, and B) CD8+ cDC, CD8- cDC and L-DC were cocultured with APC in T cell:APC ratios of 33:1, 100:1, 300:1 and 1000:1, respectively. After 72 hours, CD8+ OT-I T cells were gated as PI-CD11b-Thy1.2+Vα2+ cells, and assessed flow cytometrically for CFSE dilution as an indicator of T cell proliferation. OT-I T cells cultured alone served as controls (con). Graphs show % proliferating OT-I cells. C) The T cell/APC ratio required to generate 50% maximum proliferation of OT-I cells was compared in the presence and absence of LPS across 5 replicate experiments.
Fig 5.
Effect of cytochrome c treatment on cross-presentation capacity.
A) The in vivo killing effect of cytochrome c on APC was investigated in C57BL/6J mice. Cytochrome c (5mg/mouse) was delivered iv 6 hours prior to euthanasia for spleen collection. Control mice were given PBS. Splenocytes were prepared, stained and gated as described in Fig 1. Individual mice were analysed. Cell number is presented as % amongst the total dendritic and myeloid cell population. Mean and standard error (S.E.) are shown by cross bars. B) The effect of cytochrome c on cross-presentation of antigen was investigated using splenocytes harvested from Act-mOVA mice and prepared and sorted as described in Fig 1. Diluting numbers of APC were plated followed by treatment with or without cytochrome c (6 mg/ml) for 2 hours prior to the addition of 105 CFSE-labelled OT-I (TCR-tg) CD8+ T cells, purified from OT-I mouse spleen through depletion of B cells, CD4+ T cells, DC and myeloid cells using magnetic bead protocols. Cells were cocultured with APC in T cell:APC ratios of 33:1, 100:1, 300:1 and 1000:1. After 72 hours, CD8+ OT-I T cells were gated as PI-CD11b-Thy1.2+Vα2+ cells, and assessed flow cytometrically for CFSE dilution as an indicator of proliferation. OT-I T cells alone served as controls (con). The T cell/APC ratio required to generate 50% maximum proliferation of OT-I cells was compared in the presence and absence of cytochrome c across 4 replicate experiments.
Fig 6.
Subsets of CD8+ cDC, CD8- cDC and L-DC were sorted from C57BL/6J mice using the antibody staining and gating strategy described in Fig 1. RNA was extracted from sorted subsets and converted to cDNA for label preparation, prior to hybridisation to Murine Gene ST1.0 genechips (Affymetrix). Data were analysed using Partek to give signal values and p values. ANOVA was employed to determine genes up- and down- regulated ≥ 3-fold in pairwise comparison. A) Number of genes upregulated ≥ 3-fold in one of two subsets assessed in pairwise comparison. B) Genes upregulated in only one of three subsets.
Fig 7.
Specifically expressed genes which identify cDC or L-DC.
ANOVA analysis was used to make pairwise comparison of gene expression between A) CD8+ cDC and L-DC, and B) CD8- cDC and L-DC. Genes specifically expressed in one of two subsets were selected using the criteria of signal value in one subset ≤ 50, and signal value in the other ≥150. Comparison of CD8+ cDC and L-DC gave a dataset of 139 genes but the complete gene list is shown in supplementary data (S1 Fig). Only genes showing ≥ 15-fold difference in signal value are shown here. Comparison of CD8- cDC and L-DC gave a dataset of 71 genes which are shown in S2 Fig. Only genes showing ≥ 8-fold difference in signal value are shown here.