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Introduction: Bone morphogenetic protein 7 (BMP-7), a protein with BMP-activating and TGF-β inactivating properties, induces the proliferation of a unique population of progenitor-like cells characterized by the expression of PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). These cells can be sorted using ALK3 and the purinergic receptor P2Y1 (P2RY1) as a novel surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7 expandable colonies. Upon BMP-7 withdrawal, they differentiate into cells of all three lineages (endocrine, acinar and ductal) of the adult pancreas. PDX1+/ALK3+ cells absent from islets are represented in the major pancreatic ducts and pancreatic duct glands. Preliminary evidence indicates that these cells are also present in the murine pancreas. We hypothesized that islet β-cell regeneration could be studied in real time using live murine pancreatic slices transduced with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus. If new β-cells arise from preexisting insulin-expressing-cells they would remain green. However, if neogenesis occurs from non-β-cells, there is a window of time during which the degrading red and the new green protein co-localize in the cell. Allowing us to discriminate between neogenesis and β-cell proliferation. Methods: Low melting point (LMP) agarose-embedded, vibratome-sectioned live pancreatic slices were generated from CD-1 mice. Transduction with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus was done in slices cultured atop optically clear membranes. Slices were treated for 4 days with an ALK3 agonist (THR-123) or saline at the same time of the co-transduction. Bright field and fluorescence images of them were taken every 24h for 7 days. Conclusions: This is the first report on the use of live pancreatic slices as a novel tool for the study of pancreatic regeneration. Our preliminary results suggest that there are quantifiable β-cell neogenic events in response to BMP-7-like agents. Disclosure F. Qadir: None. J. Weitz: None. S. Alvarez-Cubela: None. D. Klein: None. G. Lanzoni: None. C.A. Garcia-Santana: Employee; Self; Ophysio Incorporated. A. Montalvo: None. C. Ricordi: None. L. Inverardi: None. A. Caicedo: None. R. Pastori: None. J. Domínguez-Bendala: Stock/Shareholder; Self; Ophysio.

Natural regulatory T cells (nTreg) can suppress different immune-cell responses and maintain the balance between tolerance and immunity in the individual. These cells are defined by CD4+ , CD25hi, and FOXP3+ expression, although a variety of other nTreg-associated markers have been reported to be expressed at different levels (e.g., HLA-DR, CTLA-4, GARP, Helios, CD39, etc.), presumably reflecting different functional stages of the heterogeneous nTreg population. Several markers show low/negative expression (i.e., CD127, CD49d, and CD26), but none of these markers are specific to nTreg. CD25hi expression has been a useful surface marker to identify/isolate nTreg; however, CD25 is also expressed on "adaptive" or "induced" Treg, as well as in activated conventional T cells. In addition, the fact that FOXP3 is also found in CD25 low/negative CD4+ T cells, and in a subset of CD8+ T cells, further complicates the definition of a specific nTreg marker. Although CD4+, CD25hi, and CD127low/negative markers characterize the majority of nTreg, it is still imperative to find additional surface-marker combinations that improve the identification/isolation of nTreg and their subsets. Herein, we present evidence that CD4+ CD25hi CD6(lo/-) nTreg have high expression of FOXP3and exhibit in vitro suppressive activity on CD8+ T-cell proliferation. Dot-plot analyses of CD4+ cells, with CD6, CD127, CD49d, or CD26 reveal that a higher enrichment yield of CD25hi FOXP3+ cells can be achieved in the combined CD6(lo/-) CD127(lo/-) population. We conclude that FOXP3+ nTreg cells are characterized by CD6(lo/-) expression, providing a new tool for the identification of nTreg cells without recourse to intracellular staining, and for the purification of these cells by negative selection.

Natural regulatory T cells (nTreg) can suppress different immune-cell responses and maintain the balance between tolerance and immunity in the individual. These cells are defined by CD4+ , CD25hi, and FOXP3+ expression, although a variety of other nTreg-associated markers have been reported to be expressed at different levels (e.g., HLA-DR, CTLA-4, GARP, Helios, CD39, etc.), presumably reflecting different functional stages of the heterogeneous nTreg population. Several markers show low/negative expression (i.e., CD127, CD49d, and CD26), but none of these markers are specific to nTreg. CD25hi expression has been a useful surface marker to identify/isolate nTreg; however, CD25 is also expressed on "adaptive" or "induced" Treg, as well as in activated conventional T cells. In addition, the fact that FOXP3 is also found in CD25 low/negative CD4+ T cells, and in a subset of CD8+ T cells, further complicates the definition of a specific nTreg marker. Although CD4+, CD25hi, and CD127low/negative markers characterize the majority of nTreg, it is still imperative to find additional surface-marker combinations that improve the identification/isolation of nTreg and their subsets. Herein, we present evidence that CD4+ CD25hi CD6(lo/-) nTreg have high expression of FOXP3and exhibit in vitro suppressive activity on CD8+ T-cell proliferation. Dot-plot analyses of CD4+ cells, with CD6, CD127, CD49d, or CD26 reveal that a higher enrichment yield of CD25hi FOXP3+ cells can be achieved in the combined CD6(lo/-) CD127(lo/-) population. We conclude that FOXP3+ nTreg cells are characterized by CD6(lo/-) expression, providing a new tool for the identification of nTreg cells without recourse to intracellular staining, and for the purification of these cells by negative selection.