Figure 2.

BRAFV600E abrogates CCR7 function and DC migration and trap DCs in tissues. (A–D) Tracing DC migration to the tissue dLN in BRAFV600E^CD11c mice. (A) Cartoon depicts the experimental procedure to trace migDCs. The skin of BRAFV600E^CD11c or control mice was painted with FITC to promote the migration of skin FITC⁺ DCs to the skin dLN. (B) Histograms show FITC uptake by skin DCs including epidermal LC and dermal DCs. (C) Flow cytometry pseudo-color dot plots show the representative frequency and bar graphs show absolute numbers of FITC⁺CD3⁻B220⁻CD11c⁺MHCII^high migDCs that have migrated to the skin dLN in control and BRAFV600E^CD11c mice (*, P = 0.0104; unpaired t test). (D) Flow cytometry plots and bar graphs show the quantification of CD11c^intMHCII^high migDCs (*, P = 0.0104; unpaired t test) and CD11c^highMHCII^int lymphoid-resident DCs (P = 0.0328, unpaired t test) in the skin dLN of BRAFV600E^CD11c or control mice. Data shown are representative of at least two experiments ± SEM (n = 3–4 per group). (E) Transwell migration assay in which control and BRAFV600E BMDCs were exposed to CCL19 chemokine gradient. Representative data of at least three experiments with three biological replicates are shown ± SEM (**, P = 0.0014; unpaired t test). (F) BRAFV600E^CD11c BMDCs were subjected to transwell migration assay as in E ± BRAF inhibitor (****, P < 0.00001; unpaired t test). (G) Heat map summarizes the chemokine receptor expression profile measured by genechip arrays on ex-vivo FACS-sorted DC subsets (CD103⁺ lung DC, CD11b⁺ lung DC, and CD11b⁺ liver DC) and BMDCs from control versus BRAFV600E^CD11c mice. (H) CCR7 surface protein levels measured by flow cytometry staining of control (red) and BRAFV600E (blue) BMDCs. Isotype staining control is depicted in gray. Representative of at least five experiments with three technical replicates each. (I and J) CCR7 protein expression levels measured by flow cytometry in control and BRAFV600E BMDCs (***, P < 0.0001; unpaired t test) stimulated overnight with 100 ng/ml TNFα or 100 ng/ml IL-1β. Data representative of at least twp independent experiments with triplicate technical replicates are shown ± SEM. (J) BRAFV600E BMDC unstimulated (***, P < 0.0003; unpaired t test), stimulated with TNFα (***, P < 0.0001; unpaired t test), or stimulated with IL-1β (P = 0.0778, unpaired t test) as in I overnight ± 100 nM GSK1120212 MEKi. (K) Quantitative real-time PCR analysis of CCR7 mRNA expression in BRAF-WT and BRAFV600E lesion relative to healthy skin. CCR7 expression was normalized to CD207 expression in each lesion to normalize for DC numbers. Units are expressed in log2 format to express fold-change relative to healthy skin. Data represent 3 tissue samples per group. (***, P < 0.0001; unpaired t test). (L) Chemokine receptor expression profile analyzed by Affymetrix genechip of purified CD207⁺ cells isolated from four BRAFV600E⁺ human LCH lesions untreated or treated with Vemurafenib BRAF inhibitor or Trametinib MEKi for 12 h. Error bars indicate SEM.