Delayed engraftment remains a major hurdle following cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the BM microenvironment. Since fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the BM microvasculature can occur, a simple 30 minute ex vivo incubation of CB HPC with fucosyltransferase (FT) - VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB HPC hypo-fucosylation was investigated in vivo in NOD-SCID IL-2Rγnull (NSG) mice. By isolating fucosylated and non-fucosylated CD34+ cells from CB we show that only fucosylated CD34+ cells are responsible for engraftment in NSG mice. Further, since the proportion of CD34+ cells that are fucosylated in CB is significantly less than in BM and PB, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed following CB transplantation. Since engraftment appears to be correlated with the fucosylation of CD34+ cells, we hypothesized that increasing the proportion of CD34+ cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase (FT)-VI significantly increases the levels of CD34+ fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells, or pattern of long-term, multi-lineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a

Regulatory T cells (Tregs) that promote tumor immune evasion are enriched in certain tumors and correlate with poor prognosis. However, mechanisms for Treg enrichment remain incompletely understood. We described a mechanism for Treg enrichment in mouse and human tumors mediated by the αvβ8 integrin. Tumor cell αvβ8 bound to latent transforming growth factor–β (L–TGF-β) presented on the surface of T cells, resulting in TGF-β activation and immunosuppressive Treg differentiation in vitro. In vivo, tumor cell αvβ8 expression correlated with Treg enrichment, immunosuppressive Treg gene expression, and increased tumor growth, which was reduced in mice by αvβ8 inhibition or Treg depletion. Structural modeling and cell-based studies suggested a highly geometrically constrained complex forming between αvβ8-expressing tumor cells and L–TGF-β–expressing T cells, facilitating TGF-β activation, independent of release and diffusion, and providing limited access to TGF-β inhibitors. These findings suggest a highly localized tumor-specific mechanism for Treg enrichment.

No abstract

Hereditary Hemorrhagic Telangiectasia (HHT) is a vascular condition caused by germline heterozygous loss-of-function mutations of ENG, AVCRL1, and occasionally SMAD4, encoding components of TGFβ/BMP signaling. Telangiectases occur in most HHT patients, and pulmonary, visceral, or cerebral arteriovenous malformations (AVMs) occur in 20-50%, but our understanding of how HHT mutations disrupt downstream signaling pathways causing clinical manifestations, and why some patients suffer more serious sequelae, is incomplete. We previously showed that genetic variation within PTPN14 at rs2936018 associates with the presentation of PAVM in HHT patients. Here we show rs2936018 is a cis-eQTL for PTPN14, with lower expression of the HHT at-risk allele, and that in primary human endothelial cells, PTPN14 physically interacts with the transcription factor, SMAD4, protecting it from ubiquitylation to support higher SMAD4 expression levels. In a panel of 69 lung samples, we find Ptpn14 RNA expression correlates with markers of angiogenesis, lymphangiogenesis, cell-cell interaction, BMP signaling, and rho kinase signaling, indicating preferential expression in endothelial cells. RNAScope in situ hybridization analysis and use of transgenic Ptpn14-reporter mice (Ptpn14.tm1(KOMP)Vlcg) show that Ptpn14 is predominantly but not exclusively expressed in lymphatic and vascular ECs of lung, heart and skin. In lung, Ptpn14, Acvrl1, Eng and SMAD4 expression are tightly correlated as well as with Flt1, Ece1, Sash1, and Mapk3k. Additionally, Ptpn14, Acvrl1, Eng and SMAD4 show strong expression correlation with components of G protein-coupled receptor (GPCR) signaling pathways impacting rho kinase, Cdc42, a regulator of cell migration. We report, for the first time, that in primary human endothelial cells PTPN14 binds SMAD4, as demonstrated by coimmunoprecipitation studies and confirmed by proximity ligation assay. In the nucleus, PTPN14 stabilizes SMAD4, protecting it from ubiquitylation and turnover and potentiating basal transcriptional activity from BMP and TGFβ responsive reporters, whereas in the cytoplasm PTPN14 sequesters phospho-TAZ (pTAZ) leading to TAZ turnover. PTPN14 therefore provides a physical link supporting BMP/ALK-1/SMAD4 signaling while inhibiting YAP/TAZ signaling in ECs to regulate a balance between these two pathways to maintain vascular stability. Polymorphisms in PTPN14 may alter tonic BMP/ALK-1/SMAD4 and TGFβ/TGFβRI/SMAD4 signaling to magnify ligand-mediated responses. In this way, genetic variation within PTPN14 may influence the clinical outcomes of HHT through its action on SMAD4.