Figure 1.
Lack of influenza specific TCD8+ responses in Batf3o/o mice.
(A) Pre-gating strategy to identify DC (B–C) DC from the inguinal and mediastinal lymph nodes of wildtype or Batf3o/o mice on a B6 background were analyzed for their expression of either (B) CD8 and CD205 or (C) CD8 and CD103. Representative plots from 3-pooled mice from two independent experiments are shown. (D–E) B6 and Batf3o/o mice were infected with PR8. On day 10, the absolute number of influenza specific T cells specific for defined peptide sequences were measured in the spleen (D) or BAL (E). The specific T cell response was elucidated following stimulation without peptides (Nil) or the peptides NP366–374, PA224–233, PB1-F262–70, or PB1703–711. Shown is the absolute number of IFNγ+ CD8+ T cells, calculated using the following equation: cell count x%PI− x%CD8+ x%IFNγ+. Average is taken from between 5–6 mice per group over two independent experiments and the error shows the SEM.
Figure 2.
PA224–33 TCD8+ can be generated in Batf3o/o mice.
B6 or Batf3o/o mice were inoculated intraperitoneally with 2.5×106 LPS-treated, PA224–233 peptide-pulsed B6 bone-marrow-derived DC. 7 days later, the number of PA224–233 responding TCD8+ present in the spleen was determined by ICS. Average is taken from 6 mice per group over two independent experiments and the error shows the SEM.
Figure 3.
Antigen presentation by DC subsets after influenza infection.
B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8+ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c+CD8− cells (upper left; left gate) were segregated into CD103+CD11b− (upper right; top gate) and CD103−CD11b+ (upper right; lower gate); and finally CD11c− cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2Db restricted influenza epitope NP366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP366–374 T cells (5×104) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.
Figure 4.
DC subset upregulation of MHC II during acute influenza infection.
B6 mice were inoculated i.n. with PKH26 or carrier 16 hours before i.n. infection with influenza virus. One to three days post infection, individual lung draining mediastinal lymph nodes were harvested and DC isolated. Enriched DC were stained with anti-CD11c, anti-CD8, anti-CD103, anti-CD11b, and anti-MHC II and analyzed on a flow cytometer. (A) Representative FACS plots of – Dye (left) and + Dye (middle) treated animals are shown. In addition, a representative histogram overlay from day 1 post infection (right) shows expression of MHC II on the CD11c+CD8+ DC (Gate I-filled) and PKH26+ DC (Gate II-open) subsets. (B) Representative dot plot of pKH26+ cells expression of CD103 and CD11b. (C–D) Upper histograms are an overlay showing expression of MHC II on PKH26+ cells from naïve (dotted line) or PR8 infected (open) mice day 2 (C) or day 3 (D) post infection. Lower histograms are identical with exception they represent CD8+ DC from naïve (dotted line) or PR8 infected (filled) mice. (E–F) Similar to C–D, with the exception expression of CD86 is shown. (G) Dot plot shows CD8+ DC (red dots) from naïve (left) or D3 PR8 infected (right) mice as compared to total CD11c+ DC from naïve mice. The numbers in each plot represent the frequency of CD8+ DC in the MHC IIhigh population. Representative histograms from 1 of 5 mice from two independent experiments are shown.