Fig. 5. Phenotypic and functional characterization of AS DCs and “pure”
pDCs. (A) Heat map reporting scaled expression (log TPM values) of gene sets
common between AS DCs (DC5) and cDCs (clusters DC1 to DC4), and genes
uniquely expressed in pDCs (DC6). Gene sets were generated through K-means
clustering using the doKmeans function in the Seurat package. (B) Morphology
of pDCs, CD1C+ DCs, CLEC9A+ DCs, AXL+SIGLEC6+CD123+CD11C–/lo, and
AXL+SIGLEC6+CD123loCD11C+ by Giemsa-Wright stain. Scale bar, 10 mm.
(C) IFN-a (left panel) and IL-12p70 (right panel) concentration in culture
supernatant 24 hours after CpG and LPS stimulation (n = 8) or after LPS,
R848, and poly(I:C) stimulation (n = 4) of CD14+CD16– monocytes, pDCs,
CLEC9A+ DCs, CD1C+ DCs, AXL+SIGLEC6+CD123+CD11C–/lo cells (1, pink),
AXL+SIGLEC6+CD123loCD11C+ cells (2, blue), and CD100hiCD34int cells (3, beige).
Composite data from four to eight donors is shown (mean ± SEM; **P <
0.01, ***P < 0.001, Mann-Whitney U test). (D) Proliferation of allogeneic CD4+
and CD8+ T cells 5 days after coculture with pDCs contaminated with
AXL+SIGLEC6+ cells compared with pDCs devoid of AXL+SIGLEC6+ cells, in
the context of LPS or LPS + R848 stimulation. Top: Representative pseudo-
color dot plot. Bottom: Bar graphs of composite data (n = 4, mean ± SEM, *P <
0.05, paired t test). (E) Proliferation of allogeneic CD4+ and CD8+ Tcells 5 days
after coculture with CD14+CD16– monocytes, pDCs, CLEC9A+ DCs, CD1C+ DCs,
AXL+SIGLEC6+CD123+CD11C–/lo (1, pink), AXL+SIGLEC6+CD123loCD11C+
(2, blue) cells, and CD100hiCD34int (3, beige) cells. Top: Representative
pseudocolor dot plot. Bottom: Bar graphs of composite data (n = 7, mean ±
SEM, **P < 0.01, paired t test). (F) Top: Immunohistochemical staining
of human tonsil with AXL (brown), CD123 (purple), and CD3 (green).
Brown arrows depict AXL+CD123+ cells adjacent to CD3+ T cells. Data shown
are representative of four donors. Scale bar, 50 mm. Middle: Frequency of
AXL+SIGLEC6+CD123+ and CD123lo/– cells in human tonsil determined by
flow cytometry analysis, as a percentage of CD45+LIN(CD3, CD19, CD20,
CD56, CD161)–HLA-DR+ cells (mean ± SEM of three donors shown;
AXL+SIGLEC6+CD123+, 0.7 ± 0.2%; AXL+SIGLEC6+CD123lo/–, 1.7 ± 0.2%).
Bottom: Representative pseudocolor dot plot of AXL+SIGLEC6+CD123+
(pop. 1, pink) and AXL+SIGLEC6+CD123lo/– (pop. 2, blue) cells in human tonsil by
flow cytometry analysis (n = 3).