OTME-14. TGF-beta signaling in microtube formation of glioblastoma

Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas. By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

Functional architecture of the pancreatic islets: First responder cells drive the first-phase [Ca2+] response

Insulin-secreting β-cells are functionally heterogeneous. Subpopulations of β-cells can control islet function and the regulation of hormone release, such as driving the second (oscillatory) phase of free-calcium ([Ca2+]) following glucose elevation. Whether there exists a subpopulation that drives the first-phase response, critical for effective insulin secretion and disrupted early in diabetes, has not been examined. Here, we examine a ‘first responder’ cell population, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation. We record [Ca2+] dynamics in intact mouse islets, via β-cell specific expression of the [Ca2+] indicator GCamP6s. We identify multiple β-cell subpopulations based on signatures of their [Ca2+] dynamics and investigate the role of ‘first responder’ cells in islet function by means of 2-photon laser ablation. We further characterize the functional properties of ‘first responder’ cells by NAD(P)H autofluorescence, fluorescent recovery after photobleaching, glibenclamide stimulation, and network analysis. We also investigate which functional characteristics of these cells are critical by a computational model of islet electrophysiology. Based on the dynamics of [Ca2+] responses, first responder cells are distinct from previously identified functional subpopulations. The first-phase response time of β-cells is spatially organized, dependent on the cell’s distance to the first responder cells, and consistent over time up to ~24 h. First responder cells showed characteristics of high membrane excitability and slightly lower than average coupling to their neighbors. When first responder cells were ablated, the first-phase [Ca2+] diminished, compared to ablating a random cell. We also observed a hierarchy of the first-phase response time, where cells that were next earliest to respond often take over the role of the first responder cell upon ablation. In summary, we discover and characterize a distinct first responder β-cell subpopulation, based on [Ca2+] response timing, which is critical for the islet first-phase response to glucose.

Human CD4+CD25+ regulatory T Cells Exhibit Dual Mechanisms of Action in Suppressing in Vitro Alloreactivity

There is extensive evidence from murine models supporting a role for CD4+CD25+ regulatory T cells in suppressing alloreactivity. However, clinical evidence regarding the role of regulatory T cells in human alloresponses is conflicting and may reflect the difficulty in defining and isolating naturally occurring human CD4+CD25+ regulatory T cells. Moreover, the mechanism of action of Foxp3+ regulatory T cells remains somewhat controversial with a number of proposed modes of suppression described, including cell contact-dependent inhibition, cytokine mediated suppression and cytokine consumption. We have previously used peripheral blood monocyte-derived dendritic cells (DCs) as antigen presenting cells (APCs) in allogeneic mixed lymphocyte reactions (MDCLRs), to assess the in vitro suppressive function of human CD4+CD25+ T cells. Healthy volunteer donor MACS isolated CD4+CD25+ regulatory T cells demonstrated anergy and significant dose-dependent suppression of responder cell proliferation, to physiological frequencies, in allogeneic MDCLRs (p<0.005). In this study we investigated the in vitro mechanism of action of human CD4+CD25+ T cells in suppressing alloreactivity in MDCLRs. Co-culture of CD4+CD25+ regulatory T cells with autologous CD4+ responder cells in allogeneic MDCLRs, at a ratio of 1:4, resulted in suppression of proliferation by 68%. Peak suppression of CD4+ responder cell proliferation, observed on day five of co-culture, was accompanied by significant suppression of co-culture supernatant IL-5 concentration (p<0.05), and preceded by inhibition of IL-2. Intracellular cytokine staining confirmed that CD4+ responder cell intracellular IL-2 was reduced by 60% on co-culturing with CD4+CD25+ regulatory T cells in the allogeneic MDCLR. CD4+CD25+ regulatory T cells mediated suppression of proliferation and cytokine responses across transwell membranes, demonstrating a cell contact-independent mechanism and a potential soluble factor in the mode of action of CD4+CD25+ regulatory T cells in suppressing human alloresponses. This was confirmed by suppression of responder cell proliferation and cytokine responses by supernatant transfer from DCs co-cultured with allogeneic CD4+CD25+ regulatory cells. We surveyed possible candidate molecules responsible for mediating the cell contact-independent suppression; in our hands, neither IL-10 nor TGF-β was identified as the soluble factor. Next, we examined the effect of CD4+CD25+ regulatory T cells on APCs in the allogeneic MDCLR. Immunophenotypic characterization of DCs recovered from MDCLRs in the presence of CD4+CD25+ regulatory T cells showed down-regulation of HLA-DR, CD83, and co-stimulatory molecules, CD80 and CD86, compared with DCs cultured with CD4+CD25− T cells or cytokines, IL-4 and GM-CSF, alone. The suppression of DC activation by CD4+CD25+ regulatory T cells was not mediated across transwell membranes, demonstrating a cell contact-requirement. Moreover, downregulation of DC activation markers was not accompanied by IL-12 suppression, implying no role for IL-10 in mediating the suppressive effect of the regulatory cells on the APCs. Our results demonstrate both a cell contact-independent mode of action in suppressing CD4+ responder cell proliferation, Th1 and Th2 cytokine responses, independent of IL-10 and TGF-β, and a cell contact-requirement in the suppression of allogeneic DC maturation and activation. In summary, our findings suggest that multiple mechanisms of action contribute to the in vitro suppressive effects of human CD4+CD25+ regulatory T cells on alloreactivity.