B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease

Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or 'LCH cells'. Badalian-Very et al. recently reported the presence of a canonical (V600E)B-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using 'next generation' pyrosequencing. In 9 cases the mutation identified was (V600E)B-RAF. In 2 cases novel polymorphisms were identified. A somatic (600DLAT)B-RAF insertion mimicked the structural and functional consequences of the (V600E)B-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The (600DLAT)B-RAF and (V600E)B-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%-2% relative mutation abundance. A novel germ line (T599A)B-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, (T599A)B-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.Our data confirmed presence of the (V600E)B-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that (V600E)B-RAF and (600DLAT)B-RAF mutations are somatic mutants enriched in LCH CD1a(+) cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line (T599A)B-RAF allele

Retinoic Acid is essential for Th1 cell lineage stability and prevents transition to a th17 cell program

SummaryCD4+ T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4+ T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells

Analysis of B-RAF mutant.

<p><b>A.</b> Sequence alignment, results from 454 pyrosequencing of granuloma cells from patients 1–10 and 16. <b>B.</b> ‘Sanger’ sequencing of patient 16 blood; A/G transition at nucleotide 1795. <b>C.</b> Pedigree of patient 16. Both the patient and his mother carry a ^T599AB-RAF allele, while his father is ^wtB-RAF. <b>D–E.</b> Comparison between ^wtB-RAF 5P_15056 (D, purple), ^V600EB-RAF structure (E, cyan) and the modeled mutant ^600DLATB-RAF (F, grey). In D, Val600 (yellow) forms a hydrophobic contact with Phe468 (red arrow). In E and F charged residues Asp and Glu (in orange) disrupt the hydrophobic network of interactions, stabilising the active conformation of the P-loop. In F, insertion Asp-Leu-Ala-Thr shifted Val600 and disrupt the hydrophobic cluster. <b>G, H.</b> MEK phosphorylation in 293 T cells. 293 T cells were transiently transfected with with mock or B-RAF mutant expressing vectors (WT, V600E, T599A, 600DLAT, D594A, G596R), and with (H) or without (G) wtC-RAF. Twenty-four hours after transfection, the medium was changed to serum-free DMEM, followed by further 18 hours culture. Total cell lysates were immunoblotted with the indicated antibodies.</p

Age, sex, clinical features, and molecular findings in 22 patients with available blood samples at the time of diagnosis or relapse.

<p>Age, sex, clinical features, and molecular findings in 22 patients with available blood samples at the time of diagnosis or relapse.</p

Presence and relative abundance of B-RAF mutant clones identified in granuloma and blood from patients with LCH.

<p>Presence and relative abundance of B-RAF mutant clones identified in granuloma and blood from patients with LCH.</p

Analysis of ^T599AB-RAF.

<p>(<b>A, B</b>) Comparison between models of ^WTB-RAF 5P_15056 (A, violet) and ^T599AB-RAF (B, gold). ^T599AB-RAF substitutes a polar uncharged residue with a hydrophobic residue, causing the loss of short-ranged interactions with residues D576 and D594. <b>C</b>. Analysis of MEK and ERK phosphorylation in THP1 cell lines stably transfected with ^WTB-RAF-FLAG and ^T599AB-RAF-FLAG. Experiment was repeated twice with similar results. (<b>D–F</b>) Analysis of MEK and ERK phosphorylation and IL-8 production in U937 cell lines stably transfected with ^WTB-RAF, ^T599AB-RAF, and ^D594AB-RAF. Experiment was repeated twice with similar results.</p

Age, sex, and clinical characteristics of patients 1 to 16.

<p>Age, sex, and clinical characteristics of patients 1 to 16.</p