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Effect of pomalidomide on T cell polarization is mediated through epigenetic modifcations

AuthorsAlvarez Laderas, Isabel; Piruat, José I.; Ceballos, María L. de ; Reyes, José C. ; Pérez Simón, Jose Antonio
Allogeneic hematopoietic stem cell transplant
Issue Date2017
Citation22nd Congress of EHA (2017)
Abstract[Background]: There is conflicting evidence regarding the potential use of IMIDs and particularly pomalidomide after allogeneic stem cell transplantation (allo-HSCT). It has been well described that IMIDs polarize naïve T cells towards a Th1 phenotype increasing IFN-γ cytokine production via the augmentation of T-bet transcription factor. This effect might increase the risk of GvHD after allo-HSCT. Nevertheless, a recent trial has reported a potential benefit on the use of pomalidomide as GVHD treatment. [Aims]: In the current study, we have analyzed the effect of pomalidomide in the polarization of CD45RA+ cells and the epigenetic mechanisms that might be involved in this effect. [Methods]: Isolated CD45RA+ T cells from healthy donor’s Buffy Coats werestimulated with anti-CD3 plus anti-CD28 in the presence of several cytokines to polarize towards Th1 (IL-12, INF-γand anti-IL4) or Th2 (IL-4, IL-2, anti-IFN-γ and anti-IL-12) for 5 days. Pomalidomide at two different doses (10 and 100 nM) were added into the culture and the effect on T cells polarization was analyzed by flow cytometry after staining with anti-CD25, anti-IFNγ, anti-CD4 and anti-IL2 for Th1 cell polarization and anti-CD25, anti-IL10, anti-CD3 and anti-IL4 for Th2 cell polarization. In addition, the release of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) in cell culture supernants were measured with BD Human Th1/Th2 Cytokine CBA kit (BDBiosciences) and T-bet and GATA-3 expression were analyzed by Western Blot. Chromatin immunoprecipitation (ChIP) assays were performed to assess the trimethylation of H3K4 (associated with gene activation) and the trimethylation of H3K27 (associated with gene repression) in the TBET and GATA-3 gene promoters. [Results]: Pomalidomide increased the expression of INF-γ and IL-2 as determined by flow cytometry in Th1 cell culture conditions. By contrast, in the presence of Th2 promoting cytokines, we observed an increase for both IL-10 and IL-4 upon adding pomalidomide to the culture. In addition, the exposure to pomalidomide increased the levels of TNF-α, INF-γ and IL-2 in the Th1 polarizing culture while, under Th2 promoting conditions, an increased concentration of IL-4 and IL-2 in supernatant was observed after exposure to pomalidomide. Furthermore, exposure to pomalidomide led to an increased expression of T-bet as assessed by western-blot in naïve CD45RA+ cells activated with anti-CD3 plus anti-CD28 and supplemented with IL-12, INF-γ and anti-IL4. By contrast, in Th2 polarization conditions, pomalidomide increased GATA-3 expression.We next studied whether or not the effect of pomalidomide in T cell polarization might be mediated by epigenetic mechanisms: in the presence of Th1 promoting conditions there was a significant increase of the activation marker H3K4me3 at the TBET promoter and a significant decrease in H3K27me3 upon exposure to the drug while, under Th2 promoting conditions, a significant increase in H3K4me3 at the promoter of GATA-3 gene was observed among T cells exposed to pomalidomide. [Conclusion]: Pomalidomide favours both Th1 and Th2 cell differentiation of CD45RA+ cells depending on the cytokines present in the medium. Treatment of naïve T cells with pomalidomide induces epigenetic modifications during T cell polarization which might favour the process of differentiation of the naïve T cells.
DescriptionResumen del póster presentado al 22nd Congress of the European Hematology Association, celebrado en Madrid (España) del 22 al 25 de junio de 2017.-- Alvarez Laderas, Isabel et al.
Appears in Collections:(CABIMER) Comunicaciones congresos
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