Anti il 2 clone mq1 17h12

To determine by flow cytometry the phenotype and cytokine profiles of vaccine-specific CD4+ and CD8+ T-cells 12 days after in vitro stimulation (IVS), patient PBMCs were re-challenged with individual peptides overnight at 37 °C, 5%CO2 in presence of Brefeldin A (51-2301KZ, BD Bioscience). The next day, the cells were washed with PBS and stained for 20 min on ice with anti-CD3 (clone SK7; # 344840, Biolegend), anti-CD8 (clone RPA-T8; #301042, Biolegend), anti-CD4 (clone RPA-T4; #558116, BD Biosciences), and a viability dye Zombie UV (#77474, Biolegend). After fixation and permeabilization for 20 min at 4 °C (BD Bioscience Cytofix/Cytoperm Kit), anti-CD154 (CD40-L, clone 24–31; #310826, Biolegend), anti-Granzyme B (clone GB11; #GRB17, Invitrogen), anti-Perforin (clone B-D48; #353310, Biolegend), anti-IL-2 (clone MQ1-17H12; #559334, BD Biosciences), anti-TNF-α (clone MAb11; #557647, BD Biosciences), and anti-IFN-γ (clone B27; #554702, BD Biosciences). All samples were acquired on a 5-laser BD FORTESSA instrument equipped with the FACS DiVa software. Analyses were performed with FlowJo v10.5 (FLOWJ,.LLC, Ashland, OR, USA) and SPICE 6 software.

Antigen-specific cellular immune responses were assessed in multiparameter ICS assays as described previously^{57 (link),58 (link)}. Briefly, thawed cryopreserved PBMCs were co-cultured for 6 h with autologous herpes virus papio-immortalized B-LCL cells that had been infected with a recombinant vaccinia virus expressing SIV Gag/pol. They were incubated with brefeldin A (BD) for the final 5 h of stimulation. Then immunostaining was performed using a CytofixCytoperm kit (BD) and the following monoclonal antibodies: anti-CD3 (clone SP34-2, Alexa700; BD), anti-CD4 (clone L200, APC-H7; BD), anti-CD8 (clone DK25, APC; Dako), anti-IFN-γ (clone 4 S.BS, PE; BD), anti-TNF-α (clone MAM11 PE-Cy7), and anti-IL-2 (clone MQ-1-17H12). A fixable-dead-cells stain kit (Invitrogen) was used to exclude dead cells from the analysis. Samples were fixed with 1% freshly prepared paraformaldehyde for at least 1 h and then analyzed in a FACSCanto II flow cytometer (BD). Data were analyzed using FACSDiVa (BD) software.