B. conceived and carried out subcellular studies of GABA-ergic components in islet cells. D.M. and A.C. conceived and identified GABA release from islets in pulses and pioneered the biosensor cell technique for analyzing the dynamics of islet GABA release. E.A.P. conceived and identified the role of VRAC and TauT in GABA release and uptake. D.W.H. and E.A.P. analyzed the genetic models for LRRC8A −/− MIN6 cells, βc-LRRC8A −/− murine islets, and knock-down LRRC8A-shRNA human islets; D.M., D.W.H., and E.A.P. performed experiments to detect GABA, taurine, and serotonin/insulin secretion; J.M. and J.A. performed hormone assay experiments and ELISAs; J.A. conducted NPY-pHluorin experiments to measure exocytosis; H.Y.G. generated adeno-NPY-pHluorin vectors; R.S. generated genetic models for LRRC8A knockout MIN6 cells and LRRC8A fl/fl murine islets; C.K. isolated and shipped LRRC8A fl/fl murine islets; M.W.B. isolated rodent islets and performed Western blot analyses; C.C. prepared cultures of primary rat hippocampal neurons; P.

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Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3–glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits (“Dimer,” “Trimer,” “Tetramer,” “Pentamer,” “Hexamer”) demonstrated glycan-binding properties and cell death–inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

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Extracellular vesicles (EVs) can affect immune responses through antigen presentation and costimulation or coinhibition. We generated designer EVs to modulate T cells in the context of type 1 diabetes, a T cell–mediated autoimmune disease, by engineering a lymphoblast cell line, K562, to express HLA-A*02 (HLA-A2) alongside costimulatory CD80 and/or coinhibitory programmed death ligand 1 (PD-L1). EVs presenting HLA-A2 and CD80 activated CD8 + T cells in a dose, antigen, and HLA-specific manner. Adding PD-L1 to these EVs produced an immunoregulatory response, reducing CD8 + T cell activation and cytotoxicity in vitro. EVs alone could not stimulate T cells without antigen-presenting cells. EVs lacking CD80 were ineffective at modulating CD8 + T cell activation, suggesting that both peptide-HLA complex and costimulation are required for EV-mediated immune modulation. These results provide mechanistic insight into the rational design of EVs as a cell-free approach to immunotherapy that can be tailored to promote inflammatory or tolerogenic immune responses.