Dan S. Luciani

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Live-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software, it is challenging to accurately extract features from dense multilayered mitochondrial networks, such as those in insulin-secreting pancreatic β-cells. Motivated by this, we developed a comprehensive pipeline and associated ImageJ plugin that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse β-cells and by extension other similarly challenging cell types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photolabeling and unsupervised cluster analysis and is capable of morphological and functional analyses on a per-organelle basis, including in 4D ( xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function and provides a valuable resource to accelerate mitochondrial research in health and disease.

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1+/– mice compared with Pdx1+/+ littermate controls. Glucose sensing and islet Ca2+ responses were also normal. Depolarization-evoked exocytosis and Ca2+ currents in single Pdx1+/– cells were not different from controls, arguing against a ubiquitous β cell stimulus-secretion coupling defect. However, isolated Pdx1+/– islets and dispersed β cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1+/+ islets. BclXL and Bcl-2 expression were reduced in Pdx1+/– islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both β cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and β cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

OBJECTIVE: Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca(2+) release channels in the ER stress-associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP(3)Rs) and the ryanodine receptors (RyRs) on the induction of beta-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS: Kinetics of beta-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca(2+) was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca(2+) in ER and mitochondria. RESULTS: Neither RyR nor IP(3)R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca(2+) and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP(3)Rs and RyRs. Conversely, stimulation of ER Ca(2+) release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization. CONCLUSIONS: This study demonstrates that the activity of ER Ca(2+) channels regulates the susceptibility of beta-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in beta-cell apoptosis associated with dysfunctional beta-cell ER Ca(2+) homeostasis and ER stress.