Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells.

BACKGROUND AIMS: Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity. METHODS: We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs. RESULTS: We showed that Tregs could be enriched to 87- 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136-732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A. CONCLUSIONS: This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

Acute in vitro exposure to environmentally relevant atrazine levels perturbs bovine preimplantation embryo metabolism and cell number.

Atrazine is a widely used herbicide known to negatively alter endocrine systems and perturb metabolism. Preimplantation exposure to pesticides may adversely affect long-term health, however few studies examine the effect of environmental levels and whether specific periods of development are particularly sensitive. In this study, the effect of acute, preimplantation atrazine exposure (days 3.5-7.5 post-fertilization) at levels detected and deemed safe in drinking water (0.02 and 20 µg/L respectively) on in vitro bovine embryo development, quality, metabolism, and gene expression was investigated. Atrazine exposure had no effect on development or quality, but significantly reduced blastocyst total cell numbers, attributable to a decrease in trophectoderm cells. Notably, atrazine (20 µg/L) markedly increased carbohydrate metabolism. Therefore, short-term exposure to environmentally relevant atrazine concentrations perturbs bovine preimplantation embryo metabolism and cell number, highlighting a potential mechanism by which atrazine can mediate embryo viability and health.