Cd27 apc h7

At indicated times post-infection a proportion of lymphocyte cultures representing ~200,000 cells were pelleted at 400g for 3 minutes into 96-well round bottom plates. Cells were washed once with PBS and resuspended in 200μl PBS containing (0.4ng/ml) fixable viability stain (BD Cat# 564406) and incubated at room temperature for 10 minutes. Cells were pelleted and resuspended in 100μl cold PBS without calcium and magnesium containing 5% FBS, and 0.1% Sodium Azide (FACS Block) and incubated on ice for 15 minutes after which 100μl cold PBS containing 0.5% FBS and 0.1% Sodium Azide (FACS Wash) was added. Cells were pelleted and resuspended in FACS Wash containing B cell phenotype panel as follows for 15 minutes on ice: (volumes indicated were routinely used for up to 0.5(10)^6 cells and were based on titration of the individual antibodies on primary tonsil lymphocyte specimens) Ig Lambda Light Chain-V450 (2μl), CD19-PE (8μl), CD38-PECy7 (3μl, BD Cat# 560667), IgD-PerCP Cy5.5 (2.5μl, BD Cat# 561315), CD138-APC (4μl, BD Cat# 347207), CD27-APC H7 (2.5μl BD Cat# 560222), Ig Kappa Light Chain-Alexa700 (2μl). After incubation, 100μl FACS Wash was added and pelleted lymphocytes were washed with a further 200μl of FACS Wash prior to being resuspended in 200μl FACS Wash for analysis. Data was acquired on a BD LSR2 Flow Cytometer and analyzed using FlowJo software.

Frequencies of activation (HLA-DR, CD38 and CCR5) and maturation (CD27 and CD45R0) markers were determined in fresh, anti-coagulated whole blood at each of the two time points. Blood samples were incubated for 10 minutes with CCR5 PECy7 followed by 30 minutes incubation using the following fluorochrome labeled monoclonal antibodies for cell surface staining (mABs); CD3-Pac Blue (BD), CD4 Per-CP Cy5.5 (eBioscience), CD8 V500 or CD8 Amcyan, CD27 APC-H7, CD45RO APC, HLA-DR FITC and CD38 PE (all from BD). Stained cells were finally fixed with 2% paraformaldehyde prior to acquisition. Acquisition was performed on a FACS CANTO II (BD). Compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Flow cytometry data was analyzed using FlowJo (version 9.5.3; Tree Star Inc.). Depending on the expression of CD27 and CD45RO markers on CD4 and CD8 T cells; T cell subsets were defined as follows: naïve (CD27⁺CD45RO⁻), “central-like” memory (CD27⁺CD45RO⁺), “effector-like” memory (CD27⁻CD45RO⁺) and “terminally differentiated” (CD27⁻CD45RO⁻) CD4 and CD8 T cells. In addition, total memory CD4 T cells were defined as the sum of central memory, effector memory and terminally differentiated CD4 T cells.

Frequencies of activation (HLA-DR, CD38) and maturation (CD27, CD45RO) markers on CD4 and CD8 T cells were determined as previously described [21 (link)] in fresh, anti-coagulated whole blood. CD4^pos T lymphocytes were classified as “naïve” (CD27^posCD45RO^neg), ‘‘central memory-like" (CD27^posCD45RO^pos) and effector memory (CD27^negCD45RO^pos) CD4 T cells. Gating strategies are given in the supplement. Blood samples were incubated for 10 minutes with CCR5 PECy7 (natutec, Frankfurt, Germany) followed by 30 minutes incubation using the following fluorochrome labeled monoclonal antibodies for cell surface staining (mABs): CD3 Pac Blue (Becton Dickinson; New Jersey, USA), CD4 Per-CP Cy5.5 (eBioscience, San Diego USA), CD8 V500 or CD8 Amcyan, CD25 phycoerythrin (PE)-Cy7 (eBioscience), CD27 APC-H7, CD45RO APC, HLA-DR FITC and CD38 PE (all from BD). Intracellular forkhead-box-protein P3 (FoxP3) was detected using FoxP3-Alexa Fluor 647 (ebioscience). Stained cells were then finally fixed with 2% paraformaldehyde prior to acquisition. Acquisition was performed on a FACS CANTO II (BD). Compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Flow cytometry data was analyzed using FlowJo (version 9.5.3; Tree Star Inc.).

The proportion of T cells expressing activation (HLA-DR and CD38) and maturation (CD27 and CD45R0) markers was determined in fresh, anti-coagulated whole blood as previously described [25 (link)]. Briefly, fresh blood samples were incubated for 30 min using the following fluorochrome-labelled monoclonal antibodies (mABs): CD3-Pacific Blue (BD), CD4 Per-CP Cy5.5 (eBioscience), CD8 V500 or CD8 Amcyan, CD27 APC-H7, CD45RO APC, HLA-DR FITC and CD38 PE (all from BD). Acquisition was performed on a FACS CANTO II (BD). Compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Flow cytometry data was analysed using FlowJo (version 9.5.3; Tree Star Inc.).