In vitro Treg Cell Differentiation and Polarization

In vitro induced Treg (iT_reg) cells were differentiated as previously described (15 (link)). Briefly, CD4⁺CD25⁻ cells were negatively selected from B6 or Pbx1-d Tg splenocytes using the CD4⁺CD25⁺ Treg isolation kit (Miltenyi Biotech), and then 5 × 10⁵ cells were stimulated with mouse T-activator CD3/CD28 beads (Life Technologies) at the concentration of 1 × 10⁶ beads/mL in the presence of 100 U IL-2, 20 ng/ml TGF-β (Pepro Tech), and 0–5 nM all-trans retinoic acid (RA) for 5 d. T_H1 and T_H17 polarization was performed as previously described (23 ).

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Choi S.C., Hutchinson T.E., Titov A.A., Seay H.R., Li S., Brusko T.M., Croker B.P., Salek-Ardakani S, & Morel L. (2016). The lupus susceptibility gene Pbx1 regulates the balance between follicular helper T cell and regulatory T cell differentiation. Journal of immunology (Baltimore, Md. : 1950), 197(2), 458-469.

Publication 2016

CD4+CD25+ Treg isolation kit CD3/CD28 beads IL-2 TGF-β

All trans retinoic acid Cd25 Cd4 cells Cells Isolation Mouse Pbx1 Tgf β Th17 Treg cells

Corresponding Organization : University of Florida

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Variable analysis

independent variables

Concentration of all-trans retinoic acid (RA) (0-5 nM)

dependent variables

Differentiation of in vitro induced Treg (iT_reg) cells

control variables

Stimulation with mouse T-activator CD3/CD28 beads (1 x 10^6 beads/mL)
Presence of 100 U IL-2
Presence of 20 ng/mL TGF-β

controls

Positive control: CD4+CD25- cells from B6 mice
Negative control: CD4+CD25- cells from Pbx1-d Tg mice

Annotations

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