Short-term (ST) incubation with a stabilized derivative, 16,16-dimethyl-prostaglandin E2 (dmPGE2), enhanced the formation of stem cells and zebrafish marrow recovery following irradiation injury. HSC in the mouse bone marrow. Limiting dilution competitive transplantation analysis demonstrated a two- to fourfold increase in HSC number after short PGE2 exposure, without creating an impact on multilineage hematopoietic differentiation or decreasing serial transplantation and self-renewal potential.4, 5 PGE2 functions through cyclic AMP (cAMP)-mediated regulation of the Wnt signaling pathway to control HSC homing, proliferation and survival.5, 6, 7 On the basis of these findings, we performed a phase Ib pilot clinical trial of dUCBT, using one untreated and one PGE2-treated UCB unit, to determine safety and engraftment parameters (http://www.clinicaltrials.gov (“type”:”clinical-trial”,”attrs”:”text”:”NCT00890500″,”term_id”:”NCT00890500″NCT00890500)). We demonstrated a significant reduction in the median time to engraftment compared with historic controls and skewing toward long-term hematopoiesis derived from the PGE2-treated UCB unit.8 Although these previous studies investigated the effects of ST exposure of HSC to PGE2, the effect of this treatment on the T cells present in the UCB has not been examined. PGE2 has immunomodulatory effects and, when continuously present during culture, alters the differentiation program of T cells by suppressing Th1 cell differentiation9 and promoting development of Treg. 10 PGE2 can also mediate Th17 differentiation. 11 Although in other cell types PGE2 can induce direct Gsk3 phosphorylation and inactivation, thereby leading to stabilization of -catenin and activation of T cell factor (TCF)/lymphoid enhancer-binding factor (LEF)-mediated gene activation,7, 12 whether PGE2 can modulate Wnt signaling in T cells has never been examined. Wnt/-catenin signaling Btk inhibitor 1 R enantiomer hydrochloride promotes quiescence and improves survival of CD4+ T cells.13, 14 Wnt signaling controls the generation of long-lived memory CD8+ T cells and highly potent stem cell memory CD8+ T cells (TSCM), which display naive immunophenotypic features and limited expansion but potent antigen-specific function.13, 15, 16 Such effects of PGE2 might be of particular importance in UCBT where impaired immune reconstitution is characterized by T-cell skewing to a late effector CD8+ phenotype.17 Here we examined the effects of ST exposure of UCB T cells to dmPGE2. We determined that transient exposure of UCB T cells to PGE2 modified the Wnt signaling cascade through E-prostanoid (EP)2/EP4 receptors and cAMP-regulated phosphorylation of Gsk3, resulting in stabilization of -catenin and TCF/LEF-mediated transcription. As a consequence, PGE2 induced an increase in interleukin (IL)-7R and IL-2R mRNA and protein expression, enhanced survival mediated by the homeostatic cytokines IL-7 and IL-15 and protected UCB T cells against pro-apoptotic TCR-mediated signals. PGE2 also induced expression of Wnt pathway components and Wnt receptors, suggesting that PGE2 treatment of the UCB might prime UCB T cells to receive Wnt/-catenin signals after infusion into the UCBT recipients. Consistent with this hypothesis, using as a paradigm samples from four recipients of PGE2-treated UCBT recipients, we detected elevated expression of the transcription factors and PGE2 treatment improves survival and immunological properties of UCB T cells in a Wnt-dependent manner. Materials and methods Cell isolation and culture Mononuclear cells were isolated using Ficoll gradient centrifugation from fresh research umbilical cord blood units obtained from the Dana-Farber Cancer Institute (Boston, MA, USA). Umbilical cord blood units were collected according to local Institutional Review Board-approved protocols. Naive T cells were subsequently purified by negative selection using the Human Naive T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladhach, Germany). For T-cell activation and apoptosis induction, the tissue culture plates were coated overnight with 5?g/ml of rabbit anti-mouse immunoglobulins (Dako, Glostrup, Denmark) at 4?C and the following day they were.We used the established combination of CD45RO and CD62L markers to identify naive (CD45RO?CD62L+), central memory (CD45RO+CD62L+), effector memory (CD45RO+CD62L?) and late effector memory (CD45RO?CD62L?) CD8+ T-cell subsets. increase in HSC number after short PGE2 exposure, without creating an impact on multilineage hematopoietic differentiation or decreasing serial transplantation and self-renewal potential.4, 5 PGE2 functions through cyclic AMP (cAMP)-mediated regulation of the Wnt signaling pathway to control HSC homing, proliferation and survival.5, 6, 7 On the basis of these findings, we performed a phase Ib pilot clinical trial of dUCBT, using one untreated and one PGE2-treated UCB unit, to determine safety and engraftment parameters (http://www.clinicaltrials.gov (“type”:”clinical-trial”,”attrs”:”text”:”NCT00890500″,”term_id”:”NCT00890500″NCT00890500)). We demonstrated a significant reduction in the median time to engraftment compared with historic controls and skewing toward long-term hematopoiesis derived from the PGE2-treated UCB unit.8 Although these previous studies investigated the effects of ST exposure of HSC to PGE2, the effect of this treatment on the T cells present in the UCB has not been examined. PGE2 has immunomodulatory effects and, when continuously present during culture, alters the differentiation program of T cells by suppressing Th1 cell differentiation9 and promoting development of Treg.10 PGE2 can also mediate Th17 differentiation.11 Although in other cell types PGE2 can induce direct Gsk3 phosphorylation and inactivation, thereby leading to stabilization of -catenin and activation of T cell factor (TCF)/lymphoid enhancer-binding factor (LEF)-mediated gene activation,7, 12 whether PGE2 can modulate Wnt signaling in T cells has never been examined. Wnt/-catenin signaling promotes quiescence and Btk inhibitor 1 R enantiomer hydrochloride improves survival of CD4+ T cells.13, 14 Wnt signaling controls the generation of long-lived memory CD8+ T cells and highly potent stem cell memory CD8+ T cells (TSCM), which display naive immunophenotypic features and limited expansion but potent antigen-specific function.13, 15, 16 Such effects of PGE2 might be of particular importance in UCBT where impaired immune reconstitution is characterized by T-cell skewing to a late effector CD8+ phenotype.17 Here we examined the effects of ST exposure of UCB T cells to dmPGE2. We determined that transient exposure of UCB T GluA3 cells to PGE2 modified the Wnt signaling cascade through E-prostanoid (EP)2/EP4 receptors and cAMP-regulated phosphorylation of Gsk3, resulting in stabilization of -catenin and TCF/LEF-mediated transcription. As a consequence, PGE2 induced an increase in interleukin (IL)-7R and IL-2R mRNA and protein expression, enhanced survival mediated by the homeostatic cytokines IL-7 and IL-15 and protected UCB T cells against pro-apoptotic TCR-mediated signals. PGE2 also induced expression of Wnt pathway components and Wnt receptors, suggesting that PGE2 treatment of the UCB might prime UCB T cells to receive Wnt/-catenin signals after infusion into the UCBT recipients. Consistent with this hypothesis, using as a paradigm samples from four recipients of PGE2-treated UCBT recipients, we detected elevated expression of the transcription factors and PGE2 treatment improves survival and immunological properties of UCB T cells in a Wnt-dependent manner. Materials and methods Cell isolation and culture Mononuclear cells were isolated using Ficoll gradient centrifugation from fresh research umbilical Btk inhibitor 1 R enantiomer hydrochloride cord blood units obtained from the Dana-Farber Cancer Institute (Boston, MA, USA). Umbilical cord blood units were collected according to local Institutional Review Board-approved protocols. Naive T cells were subsequently purified by negative selection using the Human Naive T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladhach, Germany). For T-cell activation and apoptosis induction, the tissue culture plates were coated overnight with 5?g/ml of rabbit anti-mouse immunoglobulins (Dako, Glostrup, Denmark) at 4?C and the following day they were washed three times and were incubated with anti-CD3 (0.7?g/ml; OKT3, eBioscience, San Diego, CA, USA) for 1?h at room temperature. After three washes, cells were added to the plates and were incubated for the indicated time periods. Where indicated, soluble human recombinant IL-7 and IL-15 were added Btk inhibitor 1 R enantiomer hydrochloride at a final concentration of 50?ng/ml. Short, transient PGE2 treatment was carried out by culturing naive T cells (5 106 cells per ml) with dmPGE2 (10?M) for 2?h at 37?C. Cells were subsequently gently cleaned onetime with culture moderate and useful for the indicated research. cAMP dimension Naive T cells had been incubated with PGE2 for different time factors (2C120?min). Cell pellets had been collected and removal was made by Btk inhibitor 1 R enantiomer hydrochloride using Amersham cAMP Biotrak EIA program (GE Healthcare, Small Chalfont, UK). cAMP focus in cell components was dependant on enzyme immunoassay based on the manufacturer’s guidelines. PKA assays Naive T cells (1 106 cells per test) were.