Cd8 pe

CD4-PE, CD25-FITC, CD127-APC, FITC-lineage-cocktail (CD3, CD14, CD16, CD19, CD20, CD56), HLA-DR-PerCP, CD123-PE, CD11c-APC, CD3-PE-Cy5, CD4-FITC, CD8-PE, mouse isotype controls, and lysis solution were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Whole-blood aliquots (60 µl), at T3, were withdrawn from Nil (negative control) and mitogen (positive control) tubes and from the three Ag tubes (20 µl each, mixed together) of the QuantiFERON SARS-CoV-2 kit (Qiagen, Hilden, Germany) before centrifugation, and stained with the following combination of anti-human fluorescent monoclonal antibodies: CD3BV786, CD4FITC, CD8PE, CD137APC, CD69BV711 (Becton Dickinson, San Jose, CA, USA), CD154(CD40L)APC-VIO770, and CD134(OX-40)PE-VIO770 (Miltenyi Biotec, Auburn, CA, USA). Pharm Lyse solution (Becton Dickinson, San Jose, CA, USA) was used to remove red blood cells. Then T cells were analyzed by using a 16-color FACS Celesta SORP flow cytometer (Becton Dickinson, San Jose, CA, USA) with the same instrument setting. At least 10⁴ cells were analyzed using the Kaluza Version 2.1.1 software (Beckman Coulter, CA, USA). Cells were gated on the forward scatter/side scatter cell gate and then on the CD3+CD4+ gate for the quantification of CD40L+CD69+ and CD137+OX-40+ SARS-CoV-2-specific CD4 T cells, and on the CD3+CD8+ gate for the quantification of CD137+CD69+ SARS-CoV-2-specific CD8 T cells.

T, B, and NK cell counts were determined in months + 1, + 2, + 3, and + 6 post-transplantation. Lymphocyte subsets in peripheral blood were quantified using Trucount Tubes (Becton Dickinson, Albertslund, Denmark) together with the following panel of conjugated monoclonal antibodies and analyzed on a FC500 flow cytometer (Beckman Coulter, Copenhagen, Denmark): CD3-PerCP, CD3-FITC, CD4-FITC, CD8-PE, CD45-PerCP, CD16/56-PE, CD20-FITC, and CD19-PE (Becton Dickinson). CD3⁺ T cells, CD3⁺CD4⁺ T cells, and CD3⁺CD8⁺ T cells were determined. NK cells were differentiated by CD3⁻CD45⁺CD16⁺CD56⁺ phenotype. The following B cell phenotypes were distinguished: total B cells (CD45⁺CD19⁺), mature B cells (CD45⁺CD19⁺CD20⁺), and immature B cells (CD45⁺CD19⁺CD20⁻). Data of these immune cell populations have been published previously in a different context [46 (link)].

Fifty microlitres of whole blood was collected and anticoagulated using heparin and stained for CD3-AF700, CD4-PeCy7 and CD8-PE (all Becton Dickinson, USA) and erythrocytes were lysed using Utilyse reagents (Agilent Technologies Inc, USA) and Flow Count Fluorosphere (Beckman Coulter, USA) beads were added to enable cell population quantification. Samples were analysed on a Beckman Coulter Cytomics FC500 flow cytometer (Beckman Coulter, USA) and data was analysed using CXP analysis V 2.0, 2.1 and 2.2 (Beckman Coulter, USA) Lymphocytes and monocytes were gated using forward and side scatter characteristics. Pre-infection, 9 CCM, 12 RM and 9 MCM had data collected using whole blood flow cytometry. Post-infection, 7 CCM, 9 RM and 4 MCM had data generated using this method, and was carried out at ACDP3 level of containment.

BALF cells were resuspended in FACS buffer (PBS 1% FCS). Lungs were perfused with ice-cold PBS to remove excess blood before single cell suspensions were obtained using collagenase. Briefly, whole lungs were digested with RPMI containing collagenase D (1 mg/mL) and DNase I (Roche, Mannheim, Germany) and cells were washed and recovered by centrifugation. Erythrocytes were lysed by incubation with RBC lysis buffer. To avoid non-specific binding of Abs to FcRγ, FACS Buffer containing anti-mouse CD16/32 mAb (Mouse BD Fc Block) (2.4G2, BD) was added to all primary stains. Cells were labeled with fluorophore-conjugated antibodies at pre-optimized dilutions to CD3-FITC, CD4-PE, CD8-PE, CD49b-PE (NK/NK T marker) and CD69-FITC (all from Becton Dickinson) for 1 h at 4°C and then washed twice in FACS buffer and resuspended in a final volume of 0.5 ml of FACS buffer. Data was acquired on a BD FACSCalibur flow cytometer (Becton Dickinson) and typically up to 10⁵ viable cell events were collected for analysis. A strict gating strategy was used to determine different immune cell populations as follows: single cell gate (FSC-H vs FSC-A), live cells (propidium iodide exclusion), granularity/size cell gate (FSC-A vs SSC-A) and specific surface marker gates. Flowjo software (version 7.2.4, Tree Star, OR) was used to generate plots for data analysis.