FMNL1 Is Involved in Immune Response and Hematopoietic Differentiation in MDS

Abstract 1735

One of the models of the pathophysiology of myelodysplastic syndromes (MDS) suggests that the transformation of hematopoietic cells induces an autoimmune response of T cells with bone marrow (BM) becoming the target organ. Evidence has shown that T-cell mediated marrow suppression is the cause of cytopenia in approximately 20–30% of MDS patients. Higher frequencies of cytotoxic CD8⁺ cells has been shown in low-risk MDS, as compared to high-risk MDS and these cells mediate the cytotoxicity of BM precursors. On the other hand, the suppressed immune response observed in high-risk MDS results from increased numbers of regulatory T (Treg) cells. FMNL1 belongs to a conserved family of formin-related proteins, indispensable for many fundamental actin-dependent processes, including migration, morphogenesis and cytokinesis. FMNL1 is restrictedly expressed in lymphoid hematopoietic-lineage-derived cells and overexpressed in malignant hematopoeitic cells. Depletion of FMNL1 in cytotoxic lymphocytes was recently reported to abrogate cell-mediated killing. The aim of this work was to study the role of FMNL1 in the immune system of MDS. For this, we characterized FMNL1 expression in peripheral blood CD3⁺ cells of patients with MDS and normal donor and we evaluated the CD4: CD8 T-cell ratios and molecular markers for Treg cells. We also assessed FMNL1 expression in MDS and normal BM cells, and during hematopoietic cell differentiation, using cell line models. A total of eighty patients with a diagnosis of MDS, receiving no treatment, and forty-seven samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. FMNL1 expression levels were determined by quantitative PCR (q-PCR) or Western blotting in cell lines, CD3⁺ cells (obtained by Ficoll-Hypaque followed by magnetic selection), or total BM cells. CD3⁺ cell counts and CD4: CD8 T-cell ratios were determined by flow cytometry. Q-PCR was performed to determine IL-10, TGFB1, and CTLA4 expression in CD3⁺ cells. Megakaryocytic differentiation was obtained by treating K562 with 20nM of PMA for 4 days. For granulocytic differentiation, NB4 was treated with 10⁻⁶ M of ATRA for 4 days. FMNL1 expression was significantly higher in CD3⁺ cells of the low-risk MDS group (according to FAB, WHO and IPSS classification), compared to normal donors (P <0.03), with no change in CD3⁺ cell absolute number between these groups. CD4: CD8 T-cell ratios were significantly higher in high-risk MDS, compared to the normal donor group (P <0.03). IL-10 expression levels were significantly higher in the high-risk group, when compared to both low-risk and normal donor groups (P <0.02); TGFB1 and CTLA4 showed no differences. Regarding FMNL1 expression in BM samples, there was a significantly lower expression in MDS, compared with normal donor cells (P =0.04), especially in the high risk group (P =0.02). Using cell line models for hematopoietic differentiation, a fifteen-fold increase and a five-fold increase on FMNL1 expression were observed during megakaryocytic (P =0.002) and granulocytic (P =0.05) differentiation, respectively. Western blot analysis corroborated these findings. The higher expression of FMNL1 in low-risk MDS CD3⁺ lymphocytes may be related to clonal or oligo-clonal T cell activation, since FMNL1 is important for the cytotoxic function of these cells, which are known to contribute to peripheral blood cytopenia. Indeed, it has already been showed that activation of CD3⁺ peripheral cells resulted in increased FMNL1 expression. The rise in IL10 transcripts in the CD3⁺ cells of the high-risk group may be reflecting the increase in Treg, which could be involved in the CD4: CD8 imbalance observed in this group. The lower FMNL1 expression in MDS BM, characterized by disturbed differentiation, could reflect the role of this protein in cell differentiation, as indicated by our cell line studies. Taken together, these results suggest that FMNL1 may be involved in hematopoietic differentiation and in the immune response in MDS.