Cd127 clone ebiordr5

CD4⁺CD25⁺⁺CD127^−/low putative Tregs, aseptically flow-sorted from peripheral blood lymphocytes (PBL), were expanded using a modification of our previously described protocol (Figure 1) (18 (link)). Briefly, these cells were stimulated with anti-CD3/CD28-coated microbeads (Miltenyi Biotec, Auburn CA, bead: cell ratio of 1:2) on day 0 and cultured in X-Vivo-15 media supplemented as previously described (18 (link)), including 2000 IU/ml of rhIL-2. At days 12 and 24, (20 ) cultures were re-stimulated as on day 0. Treg cultures were pulsed with 100 nM of rapamycin for 48 hours from day 34-36, given our previous results showing that this optimized Treg suppressive activity. (18 (link)). Tregs were then harvested, washed free of rapamycin, magnetic beads removed, and cryopreserved as previously described. (18 (link)) The Treg phenotype was assessed by staining for CD3 (clone SP34-2, BD, San Jose, CA), CD4 (clone SK3, BD), CD25 (clone 4E3, Miltenyi Biotec), CD127 (clone eBioRDR5, eBioscience, San Diego, CA) and FoxP3 (clone PCH101, eBioscience) using the FoxP3 Fix/Perm Buffer Set (BioLegend, San Diego, CA). In some experiments, Tregs were also labeled with an anti-Ki-67 antibody (Clone B56, BD). Data were acquired on an LSR II flow cytometer and analyzed using FlowJo software (Treestar, Ashland, OR). Positively stained cells were identified using appropriate isotype-control antibodies.

Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by Ficoll Hypaque gradient centrifugation. Total CD4⁺ T cells were isolated by negative selection using CD4⁺ T cell isolation kit (StemCell Technologies, Vancouver, BC) and stained for fluorescence-activated cell sorting (FACS) with the following antibodies: anti-CD45RO (clone UCHL1), CD45RA (clone Hl100), CD25 (clone M-A251) (all from BD Biosciences) and CD127 (clone eBioRDR5) from eBioscience (San Diego, CA). The T_reg (CD4⁺CD25^hiCD127^low/neg), memory T cell (CD4⁺CD45RA⁻ CD45RO⁺CD25^low/neg+) and naive T cell (CD4⁺CD45RA⁺CD45RO⁻CD25^lo/neg) populations were sorted on a FACS Aria (BD Biosciences). Unless specified, CD4⁺ T cells used in the experiments were T_reg-depleted CD4⁺ T cells and they were sorted as CD4⁺CD25^pos/loCD127⁺. CD14⁺ cells were isolated by positive selection using EasySep™ Human CD14 Positive Selection Kit (StemCell Technologies).