Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies

Abstract

Ex-vivo differentiation of regulatory T cells (Tregs) from naïve CD4+ T-cells has been widely used in immunological research. Isolation of a highly pure naïve T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency. Here, we present the results of a comprehensive side-by-side comparison of various magnetic separation strategies and FACS sorting in multiple levels. Additionally, we propose a novel optimized custom made magnetic separation protocol, which not only yields sort level purity and Treg differentiation but also lowers the reagent costs up to 75% compared to the commercially available purification kits. The highly pure naïve CD4+ T-cell population obtained by this versatile method can also be used for differentiation of other T-cell subsets; therefore this protocol may have broad applications in T-cell research.

Regulatory T cells mediate specific suppression by depleting peptide–MHC class II from dendritic cells

“…For iT reg and T activated cell differentiation, naive T cells were isolated and 24 well sterile tissue culture plates (Corning, Corning, NY) were coated anti-CD3ε (145–2C11, Biolegend) and anti-CD28 (37.51, Biolegend) as described 36 . Naive CD4 + T-cells were resuspended in complete RPMI media supplemented with 100 IU/ml recombinant human IL-2 for both iT reg and T activated , with additional 5 ng/ml recombinant human TGF-β for iT reg and 10 μg/ml anti-TGF-β (1D11.16.8, BioXcell, West Lebanon, NH) for T activated cultures.…”

Second signals rescue B cells from activation-induced mitochondrial dysfunction and death

“…Phoenix Eco retroviral packaging cell line (ATCC CRL-3214) was purchased from American Type Culture Collection. B cells and naïve T cells were purified from mouse spleens using negative magnetic selection strategies explained in detail in 49,50 . Cells were maintained in vitro in complete culture media (RPMI 1640 media supplemented with 10% fetal calf serum, 10 mM HEPES pH7.3, 50 U/ml penicillin, 50 μM streptomycin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol) in a humidified 5% CO 2 tissue culture incubator at 37 °C unless otherwise specified.…”

Toll-like receptor 9 antagonizes antibody affinity maturation

“…B cells and naïve CD4 + T cells were isolated from mouse spleen using mouse B cell and naïve T cell isolation kits (Miltenyi Biotech) according to previously published optimized protocols 30 , 31 . NIH3T3 mouse fibroblast cell line (ATCC CRL-1658) was provided by O. Voss.…”