No abstract

Proliferation of pancreatic b-cells has long been known to reach its peak in the neonatal stages and decline during adulthood. However, b-cell proliferation has been studied under the assumption that all b-cells constitute a single, homogenous population. It is unknown whether a subpopulation of b-cells retains the capacity to proliferate at a higher rate and thus contributes disproportionately to the maintenance of mature b-cell mass in adults. We therefore assessed the proliferative capacity and turnover potential of virgin b-cells, a novel population of immature b-cells found at the islet periphery. We demonstrate that virgin b-cells can proliferate but do so at rates similar to those of mature b-cells from the same islet under normal and challenged conditions. Virgin b-cell proliferation rates also conform to the agedependent decline previously reported for b-cells at large. We further show that virgin b-cells represent a long-lived, stable subpopulation of b-cells with low turnover into mature b-cells under healthy conditions. Our observations indicate that virgin b-cells at the islet periphery can divide but do not contribute disproportionately to the maintenance of adult b-cell mass. More than 30 million Americans currently have diabetes, which results from progressive b-cell loss caused by either autoimmune attack or lifestyle and genetic factors (1). Because b-cells are the only cells in the body capable of secreting insulin, a major therapeutic goal for diabetes has been to replace b-cells lost during disease. Islet or wholepancreas transplantation has had some success in achievement of insulin independence, but limited donor availability, immunological complications, and transplant survival make this process not a viable option for the majority of patients with diabetes (2). Thus, a great interest continues in strategies that promote the regeneration of b-cells from various cell sources (3,4). Despite these efforts, clinically meaningful restoration of b-cell mass has not yet been achieved, illustrating the ongoing need for strategies to regenerate b-cells. During late pancreas development, b-cell mass expands rapidly by self-replication of young b-cells. Seminal experiments by Dor et al. (5) established, by pulse-chase labeling of b-cells, that self-replication is the main mechanism to maintain b-cell mass-a conclusion that is noncontroversial (6,7). However, b-cell proliferation declines rapidly with age in mice (8-10), with even lower proliferation rates observed in human islets (11), which complicates the challenge of restoring b-cell mass secondary to diabetesassociated b-cell loss. Against this backdrop, numerous reports of insulin-positive multipotent precursors (12,13), transdifferentiation of aand d-cells to b-cells (14-16), transdifferentiation of exocrine cells to b-cells (17,18), and, most recently, the existence of a population of protein C receptor (ProCr)-positive islet-resident stem cells ( 19) continue to raise the prospect that alternative paths to generation of b-cells exist and coul...

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)–based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.