Background Multisystemic Langerhans cell histiocytosis (mLCH) is an aggressive disease characterized by the accumulation of mononuclear phagocytes with immunohistochemical features of dendritic cell (histiocytes)¹. Histiocytes infiltrate mostly the skin, bone marrow (BM), lung and spleen, and produce high levels of proinflammatory cyto/chemokines, leading to organ dysfunction^2,3. In patients, around 10% of cells in lesions carries an oncogenic mutation in the MAPK pathway, mostly BRAF^V600E (70% of cases). BRAF^V600E can be detected also in monocytes and BM progenitors (HSPC) from these patients, whereas only a fraction of them carries a mutation in B cells and none in T cells⁴.

Objectives To study the role of BRAF^V600E in the pathogenesis of mLCH, we set up a humanized mouse model of mLCH based on the transplantation into immunodeficient mice (NSG) of human HSPC expressing BRAF^V600E.

Methods We isolated HSPC from human cord blood and transduced them at two different levels (50% and 20%) with lentiviral vectors that ubiquitously express BRAF^V600E, BRAF^WT or GFP.

Results All BRAF^V600E mice manifested severe weight loss within 7 weeks with a median of 3 and 5 weeks for 50% and 20% transduction groups, respectively. Mice showed dysplastic bone marrow (BM) with infiltration of histiocytes; lesions were present also in lungs, kidneys, CNS, spleen and liver. Immunophenotype of infiltrating histiocytes closely resembles mLCH, staining positive for CD14, CD68, S-100 and langerin. None of the control mice lost weight nor displayed organ alteration. Flow cytometry analyses of BM showed 5- to 7-fold reduction in engraftment of human cells in BRAF^V600E vs GFP groups (p<0,001). Percentage of myeloid cells increased by 3- to 4-fold in BRAF^V600E vs GFP groups (p<0,001). On the contrary, percentage of B cells was reduced by 3- to 6,5-fold in BRAF^V600E vs GFP groups (p<0,001). Moreover, there was no difference in the percentage of GFP-positive cells between myeloid cells, whole BM and in vitro sample, suggesting a non-cell autonomous mechanism underlying this myeloid phenotype.

Conclusions In summary, we generated for the first time a human xenogeneic transplantation mouse model of mLCH, showing that BRAF^V600E in human HSPC promotes myeloid skewing rather than proliferation.

References

1. Wilejto et al. Curr Opin Rheumatol 2012. DOI: 10.1097/BOR.0b013e32834db53e.

2. Donadieu et al. Histiocytic Disorders of Children and Adults 2005.

3. Kannourakis et al. Br J Cancer 1994, Sep;23:S37–40.

4. Berres et al. J Exp Med 2014, DOI: 10.1084/jem.20130977.

References

Disclosure of Interest None declared