1a) Using these boundaries and the level of CD127 expression by

1a). Using these boundaries and the level of CD127 expression by CD4+ lymphocytes, CD4+ CD25inter CD127low/− and CD4+ CD25high CD127low/− Treg cells and CD4+ CD25− CD127−/+ and CD4+ CD25+ CD127+ effector T cells were identified and isolated (Fig. 1b), with the prevalence of Treg cells expressed as a percentage of the total CD4+ population (mean ± SEM). Foxp3 expression on the two Treg cell populations (CD4+ CD25inter CD127low/− and CD4+ CD25high CD127low/−) was assessed following fixation and permeabilization of

the cells, as directed (Human Foxp3 Buffer Set; BD Biosciences), before incubation with a mouse anti-human Foxp3-Alexa Fluor 488 antibody (clone 259D/C7; BD CH5424802 concentration Biosciences) or its corresponding isotype control (BD Biosciences) for 30 min protected from light. The labelled cells were washed, re-suspended and the same gating strategy as detailed above was applied during the acquisition of the samples. The suppressive activity of isolated Treg cells on the proliferation of autologous effector T cells was determined by a co-culture carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. Effector T-cell populations (CD4+ CD25− CD127−/+ or CD4+ CD25+ CD127+) were incubated with 5 μm of CFSE (Sigma, Poole, UK) for 10 min EMD 1214063 solubility dmso at 37°C. The labelling

was quenched by the addition of 2·5 ml of ice cold culture medium [X-VIVO 20 medium (Lonza, Slough, UK) supplemented with 5% volume/volume heat-inactivated AB serum (Invitrogen) and penicillin/streptomycin (final concentration:

0·1 U/ml and 0·1 mg/ml, respectively; PAA)] before the cell suspension was incubated on ice for 5 min. Following three washes with pre-warmed medium the labelled effector T cells were co-cultured with Treg cells (CD4+ CD25inter CD127low/− and CD4+ CD25high CD127low/−) in 200 μl of culture medium at various ratios (Treg : effector; 0 : 1, 1 : 1, 1 : 2, 1 : 5 and 1 : 10). Depending on the number of Treg cells available; the 1 : 1 ratio was always prepared. Where possible http://www.selleck.co.jp/products/Adrucil(Fluorouracil).html the CFSE assay was run with 5 × 104 effector cells cultured in each well of a 96-well round-bottomed plate, however, when insufficient cells were isolated the number of effector cells plated was successfully scaled down to 1 × 104/well. Lymphocyte stimulation was provided by Human T-Activator CD3/CD28 Dynabeads (Invitrogen) at a cell : bead ratio of 1 : 3 and 100 U/ml recombinant human IL-2 (AbD Serotec, Kidlington, UK). Following 4 days of co-culture, the cells were harvested and the proliferation of the CFSE-labelled effector T cells was determined using flow cytometry.

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