Cd3 clone sp34 2

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.

Antibodies used in this study are as follows: CD3 (clone SP-34–2; BD Biosciences), CXCR5 (MU5UBEE; eBioscience), GagCM9 tetramer (NIH tetramer core), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; clone EH12.2H7; BioLegend), CD8 (SK1; BD Bioscience), Ki67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), GrzB (GB11; BD Biosciences), perforin (Pf-344; Mabtech), FoxP3 (206D; BioLegend), HLA-DR (L243; BioLegend), IFNλ (B27; BD Biosciences), TNFα (MAb11; BD Biosciences), and IL-2 (MQ1- 17H12; BD Biosciences).

Cryopreserved PBMCs from BCG-vaccinated NHP were thawed in RPMI 50% FCS and plated at a concentration of 3×10⁶ cells/well in a 24 well plate for 2 to 4 hours for monocyte plastic adhesion. Non-adherent cells were collected and stained with 1:800 dilution of viability dye (Thermofisher) for 10 minutes at 4°C. Cells were washed once with FACS buffer (PBS 0.1% BSA) and divided to be stained with 1:50 dilution of different tetramers for 15 minutes at 37°C. HLA-E tetramers were produced and peptide loading was confirmed by mass spectrometry as described previously (Ruibal et al. submitted) (18 (link)). Antibodies for CD3 (clone SP34–2, BD Biosciences) and CD8 (clone RPA-T8, BD Biosciences) staining were added and incubated for 30 minutes at 4°C. Cells were washed once as before, fixed with 1% paraformaldehyde and immediately acquired on a FACSLyric (BD Biosciences). All data were analyzed using FlowJo software v10.7.1 (BD Biosciences).

Mononuclear cells from liver and spleen were stimulated with 1 ug of total NS3 antigens (pool of NS3 (aa 941 to 1250; 30 peptides), NS3/4a (aa 1241 to 1615; 37 peptides) at 37°C for 6 hours. After stimulation, the cells were stained with antibodies against CD3 (clone SP34-2, BD Biosciences), CD4 (clone L-200, BD Biosciences) and CD8 (clone LT8, AbCam) followed by intracellular staining for IFN-γ (clone B27, BD Biosciences) similar to the flow cytometry staining protocol described above. Unstimulated cells and cells stimulated with PMA (0.05 ug/reaction) and Ionomycin (1 ug/reaction) served as negative and positive controls, respectively.