Anti cd3 v450

Cryopreserved PBMNCs were thawed, incubated with an Fc receptor blocking reagent (ChromPure Mouse IgG, Jackson ImmunoResearch Suffolk, UK) to inhibit non-specific binding before incubated with the following, pre-titrated antibodies (all from BD Biosciences, San Jose, CA): anti-CD3 V450, anti-CD45RO FITC, anti-PD-1 (CD279) PE, and anti-CD8 AF700. Flow cytometry was acquired by a LSRFortessa (BD Biosciences, San Diego, CA) and data analyzed using FlowJo version 10.2 (Tree Star Inc., Ashland, OR). Compensation was performed using BD CompBeads (BD Biosciences). Applying gating strategies based on unstained controls and for PD-1 the fluorescence minus one (FMO), the expression of PD-1 was measured on CD3⁺/CD45RO⁺/CD8⁺ singlets and given by the median fluorescense intensity (MFI) (Supplementary Figure 4).

The ex vivo Ag presentation assays were performed using a protocol adapted from Langlet et al. OVA-specific CD8⁺ T and CD4⁺ T cells were isolated from OTI and OTII transgenic mice, respectively, and labeled with 10 µM CFSE. 10⁵ OT-I T cells or 10⁵ OT-II T cells were subsequently co-cultured for 48 hours with respectively 10⁴ sorted Ly6C^hi monocytes, MHCII^hi DCs or MHCII^int DCs obtained from the DLN of mice previously (24 h) immunized with 20 µl of an OVA (100 µg/ml)/CpG (100 µg/ml) mixture. OT-I and OT-II proliferation was determined by the loss of CFSE fluorescence by flow cytometry. Sorts were performed using a FACSAria sorter (BD Biosciences).
In vivo antigen presentation assays were performed through adoptive transfer of 2 × 10⁵ CFSE labeled OT-I or OT-II cells to respectively WT C57BL/6 J mice, CCR2^−/− mice, CCR7^−/− mice and Flt3L^−/− mice 48 hours prior to OVA/CpG immunization. Four days post immunization, DLN were dissected, stained with anti-CD3-V450, anti-CD4-PerCP, anti-CD8-PerCP, anti-Vα2 TCR-PE, anti-CD19-APC-Cy7 (all BD Biosciences), PE-labeled SIINFEKL specific dextramers (Immudex) and measured on a LSRII flow cytometer (BD Biosciences).

PBMCs (5 × 10⁵cells/well) from active TB patients were thawed and rested 6 h before labeling with Carboxyfluorescein succinimidyl ester (CFSE) according to manufacturer’s procedure. In the COX-i treated samples, indomethacin was added 2 h prior to stimulation with ESAT-6 and Ag85. The samples were incubated for 6 days and then washed and stained for surface markers and viability staining. Fluorochromes used in the 6 days proliferation assay; anti-CD3-V450, anti-CD4- APC H7, anti-CD45RA -BV 605, anti-HLA DR- APC, anti-CD25-PE, anti-CD127- PeCy7 and 7AAD- PerCP (all from BD Bioscience) and CellTrace™ CFSE Cell Proliferation (Life technologies).

Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation through Ficoll-Hypaque of blood samples obtained at the time of allograft biopsy, and stored at—80°C until analyzed. After thawing, 0.5–1 X 10⁶ PBMC were incubated with various antibody combinations for 30 min at 4°C. The fluorochrome-conjugated monoclonal antibodies used were anti-CD19 V500, anti-CD3 V450, anti-CD56 PE, anti-CD14 PE-Cy7, anti-CD45 APC, anti-CD38 PE-Cy7 from BD Biosciences, anti-CD24 APC from Miltenyi Biotec and anti-IgD FITC and anti-CD27 PE from Beckman Coulter. Samples were analyzed with Canto II cytometer (BD Biosciences) and the data were processed using FlowJo software (Tree Star, Ashland, USA).

Resting CD4⁺ T-cells were labeled with the proliferation dye eFluor670 and cultured alone or with one of seven sorted syngeneic APC subpopulations at a ratio of 10:1 for 24 h in IL-2 (2U/mL, Roche Diagnostics, Basel, Switzerland) supplemented RF10 media. APC included monocyte subpopulations (CD14⁺CD16⁻ and CD14^loCD16⁺), DC subpopulations (pDC, CD1c⁺, CD141⁺ and SLAN⁺), and B-cells. Co-cultures were then infected with NL(AD8)ΔnefEGFP for 2 h, after which time excess virus was washed away and cells were cultured for an additional 5 days. In order to compare APC stimulatory capacity between APC-T-cell co-cultures, at day 3 post-infection, cells were stained with anti-CD3-V450 (BD Biosciences) to differentiate between T-cell and APC, and the proportion of proliferated (eFluor670^lo) CD4⁺ T-cells were determined. Day 3 was used because this is when productive infection reached is maximum and remained high until day 5 (unpublished data). Additional APC-T-cell ratio’s were not used due to low APC yields. At day 5 post-infection, productive infection was determined by EGFP expression and non-proliferating, non-productively infected (eFluor670^hi EGFP⁻) CD4⁺ T-cells were sorted using a FACSAria.