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.

Flavobacterium commune sp. nov., isolated from freshwater and emended description of Flavobacterium seoulense.

A Gram-stain-negative, yellow, facultatively-anaerobic, short, rod-shaped, non-spore-forming bacterium, designated PK15T, was isolated from freshwater. Growth was observed at 4-40 °C (optimum, 30 °C), pH 6-9 (optimum, 8), and in the presence of 0-0.8 % (w/v) NaCl (optimum, 0.4 %). Strain PK15T exhibited both catalase and oxidase activities and was able to reduce nitrate. On the basis of 16S rRNA gene sequence similarities, strain PK15T was shown to belong to the genus Flavobacterium with close similarities to Flavobacterium palustre S44T (97.9 %) and Flavobacterium seoulense EM1321T (97.7 %). Menaquinone-6 (MK-6) was the major respiratory quinone, while the G+C content of the genomic DNA was 35.5 (±0.9) mol%. The major polar lipids were phosphatidylethanolamine, three unidentified aminolipids, one unidentified aminophospholipid and three unidentified polar lipids. The predominant cellular fatty acids (≥10 %) were anteiso-C15 : 0 (17.3 %), a summed feature comprising C16 : 1ω7c and/or C16 : 1ω6c (15.1 %) and iso-C15 : 0 (10.0 %). Chemotaxonomic data supported the affiliation of strain PK15T to the genus Flavobacterium. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain PK15T from strains of closely related species. It was, therefore, evident that PK15T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium commune sp. nov. is proposed with strain PK15T (=KCTC 52562T=JCM 32115T) as the type strain. Based on the results of the chemotaxonomic characterization in the present study, an emended description of Flavobacterium seoulense is also proposed.

Flavobacterium keumense sp. nov., isolated from freshwater.

A yellow-pigmented, oxidase-positive, catalase-negative, Gram-stain-negative, rod-shaped, aerobic and non-motile bacterial strain designated K3R-10T was isolated from a freshwater source. The strain grew over a temperature range from 4 to 35 °C (optimum, 30 °C), pH range pH 6-8 (optimum, pH 7) and in the presence of 0-0.5 % NaCl (optimum, 0 %). Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain K3R-10T belonged to the genus Flavobacterium and shared close similarities with Flavobacterium succinicans LMG 10402T (97.0 %), Flavobacterium chungangense LMG 26729T (96.4 %), Flavobacterium branchiophilum IFO 15030T (96.4 %) and Flavobacteriumpiscis412R-09T (96.3 %), but formed a distinct phylogenetic line of its own in the phylogenetic trees. The polar lipids consisted of phosphatidylethanolamine, an unidentified aminolipid and three unidentified phospholipids. The DNA G+C content was 35.4 mol%, MK-6 was the major isoprenoid quinone, and homospermidine was the predominant polyamine. The predominant cellular fatty acids were iso-C15 : 0 3-OH, iso-C15 : 0, a summed feature comprising C16 : 1ω7c and/or C16 : 1ω6c and iso-C15 : 1 G. The absence of aminophospholipid, acid production from carbohydrates, DNA G+C content and colony morphology differentiated strain K3R-10T from related species of the genus Flavobacterium. Thus, on the basis of phenotypic, chemotaxonomic and phylogenetic features, strain K3R-10T evidently represents a novel species in the genus Flavobacterium, for which the name Flavobacterium keumense sp. nov. is proposed. The type strain is K3R-10T (=JCM 31220T=KCTC 52563T).