(A) Quantification of CD301b+PDL2+
DCs and (B) CD103+ DCs in the dLN of Th0 or Th2
immunized WT (clear bars) or
Ccr8−/− (gray
bars) mice as determined by flow cytometry. Percentages reflect percent of
CD301b+PDL2+ or CD103+ out of total
CD11c+ cells, numbers reflect total cells per popliteal LN.
(C & D) Flow cytometry of inguinal LNs was performed 24 hrs
after immunization of photoconverted flank skin of Kaede+ transgenic
mice. (C) Total
CD11c+CD301b+Kaedered (i.e.,
photoconverted) cells per inguinal LN are shown in comparison to
Ccr7−/−Kaede+ and
(D)
Ccr8−/−Kaede+ groups.
(E) CD301b staining (white) of whole popliteal LNs of WT or
Ccr8−/− mice 24 hrs after Th0 or
Th2 immunization. Scale bar indicates 200μm. (F)
Quantification of total number CD11c+CD301b+ cells per
entire LN section or (G) percent of total
CD11c+CD301b+ within the SCS (identified by podoplanin
and CD169 immunohistochemical staining) 24 hrs after immunization.
(H) CD301b+ DCs within the SCS space of
Ccr8−/− mice, but not WT mice, 24
hrs after Th2 immunization. Panels show single stains of CD301b (red), CD11c
(blue), podoplanin (green), and overlay of those stains and CD169 (white). Scale
bars indicate 10μm. Images (E & H) are one
representative of 2-3 mice per condition and 4 independent experiments. Bar
graphs represent mean value of individual mouse data points (3-6 mice per
condition per experiment) taken from a representative of 3 independent
experiments (A, B, D, F, G, H), or 2 independent experiments
(C). Error bars represent SEM. * p<0.05, **
p<0.01, *** p<0.001, Student’s T test. See also Figure S3.