The slides were examined using light microscope. Flow cytometry analysis and Western blot Differentiation of myeloid cells and lymphoid cells were analyzed using flow cytometry. in lactation. However, PRL also regulates hematopoietic cell development and homeostasis24C28. Specifically, PRL enhances the development of myeloid and erythroid progenitors from CD34+ cells24,26. PRL also drives the maturation and activation of T cells, B cells, NK cells, neutrophils, macrophages and dendritic cells27C33. This hormone is usually released mainly by the anterior pituitary gland, although immune cells, such as myeloid cells, are non-endocrine sources of PRL27,28,34,35. PRL signals through the PRL receptor (PRLR), which is a member of the cytokine receptor superfamily36C40 because of its use of kinases and signal transduction activators of transcription (STATs)36,38,41. Apart from mammary gland tissue, decidua and uterus all of which abundantly express PRLR, immune cells also express this receptor27,34,39,42,43. Moreover, myeloid cells ortho-iodoHoechst 33258 can co-express both PRL and its receptor (PRLR), indicating the presence of both autocrine and paracrine actions of this molecule within the hematopoietic CDC25L system26,27,34,44. The expression of PRLR in a subset of human CD34+ hematopoietic stem cells (HSCs) has previously been described and suggests a role for PRL during hematopoiesis24C26,28. In line with this, PRL directly promotes hematopoietic cell differentiation, accelerating immune reconstitution after bone marrow transplant (BMT)24,28. Studies also suggest the indirect involvement of PRL during lymphoid development, but the details remain unclear28. In this study, we report that stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (FLT3L) induce the PRLR on CD34+ myeloid progenitors. We show that PRL acts on the CD34+PRLR+ myeloid progenitors resulting in the activation of pro-inflammatory ortho-iodoHoechst 33258 factors such as IL-15 that support CD56+ lymphoid lineage development45C47. Mechanistically, we demonstrate that PRL increased mothers against decapentaplegic homolog 7 (SMAD7) which inhibits transforming growth factor beta (TGF-) signaling by binding to and cleaving TGF- receptor48,49. ortho-iodoHoechst 33258 Moreover, the reduction in TGF-1 following PRL stimulation is likely consistent with prior work showing SMAD7-induced negative-feedback regulation of TGF-48C50. TGF- inhibits NK cell development and function through inhibition of various metabolic pathways, including oxidative phosphorylation, glycolytic pathways, and respiratory pathways50C53. Thus, these studies show that PRL-induced SMAD7 facilitates CD56+ lymphocyte development through TGF- repression. Results SCF and FLT3L Drive the Differentiation of HSCs into PRLR+CD34+ Myeloid Progenitors While studying differentiation of CD56+ lymphocytes from CD34+ progenitors, we noticed a minor populace of non-ILC lineage cells that differentiated early in the cultures and were CD11alow and unfavorable for ILC markers including CD56, CD94, CD336, CD117 and CD29416. We sought to both characterize these cells and to determine whether they promoted or suppressed CD56+ lymphocyte development. Interestingly, these CD11alow non-ILC cells expressed the PRLR (Supplementary Fig.?1). Freshly isolated cord blood CD34+ HSCs lacked the PRLR (Fig.?1A,B, Supplementary Fig.?2A), but ~15% of CD34+-derived cells acquire PRLR after a few days in media containing cytokines previously shown to expand HSCs (SCF, thrombopoietin (TPO), low-density lipoprotein (LDL) and FLT3L)54. Similarly, freshly isolated bone marrow and peripheral blood CD34+ HSCs lacked PRLR expression but acquired PRLR after four days of culture in media made up of SCF, TPO, LDL and FLT3L (Supplementary?2B). The proportion of PRLR expressing progenitors was stable during the first two weeks of culture (Fig.?1A,B), while the absolute number significantly increased over time (Fig.?1C). Accordingly, these PRLR expressing progenitors upregulated PRLR mRNA (Fig.?1D). To understand the factors that drive PRLR expression, CD34+ cells were cultured in various cytokine combinations and PRLR mRNA and surface protein expression was tested. As shown in Fig.?1E, FLT3L significantly enhanced PRLR mRNA expression, while SCF (either alone or in combination) significantly increased surface PRLR expression (Fig.?1F). Open in a separate window Physique 1 CD34+PRLR+ progenitors are present in cultures that favor CD56+ ILC differentiation. UCB-derived CD34+ HSCs were expanded for up to 13 days ortho-iodoHoechst 33258 and the expression of PRLR was analyzed using qPCR or flow cytometry. (A) Expression of PRLR in differentiating HSCs at various time points. Representative histograms and values show the percentage of CD34+PRLR+ cells as assessed by FACS (n?=?4). (B,C) The percentage (B) and absolute count (C) of CD34+PRLR+ progenitors in cultures at various time points is usually shown in bar graph (n?=?4/group). (D) The quantitative expression of PRLR mRNA in CD34+PRLR+ cells is usually shown relative to its expression in CD34+PRLR? cells after normalizing to GAPDH expression.