Purified CD4
+ T cells (CD4 T cells beads, Miltenyi or
CD4 Dyna Beads, Invitrogen) were labeled with CFSE or CellTrace Volet (CTV) (Thermofisher) according to manufacturer’s protocol and cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (FBS, GIBCO Sigma), 100 U/mL
penicillin, 100 μg/mL
streptomycin and 2 mM
L-glutamine (all from GIBCO) together with DC and irradiated feeder cells (40 Gy) in 2:1:1 ratio for 72 or 96h. Polyclonal CD4
+ T cells were stimulated with
αCD3 (2C11) and
αCD28 (37.51) (BioXcell); OT-II cells with OVA
323-339 (ISQAVHAAHAEINEAGR) peptide (Pepscan) and Trp1 cells with Trp1
106-130 (SGHNCGTCRPGWRGAACNQKILTVR) peptide (Pepscan) at concentration indicated in the
Figure 2. Cells were additionally supplemented with
IL-2, IL-15 or
IL-7 (Peprotech) at a concentration indicated in the
Figure 2. Mouse CD4
+ T cells were cultured with αCD25 (PC61, BioXcell) and α
IL-2 (
JES6-1A12, BioXcell), added to the culture 24h post stimulation with αCD3 and αCD28 in a concentration of 5 μg/ml.
Śledzińska A., Vila de Mucha M., Bergerhoff K., Hotblack A., Demane D.F., Ghorani E., Akarca A.U., Marzolini M.A., Solomon I., Vargas F.A., Pule M., Ono M., Seddon B., Kassiotis G., Ariyan C.E., Korn T., Marafioti T., Lord G.M., Stauss H., Jenner R.G., Peggs K.S, & Quezada S.A. (2020). Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4+ T Cells. Immunity, 52(1), 151-166.e6.