Cd27 apc

Patients' blood was collected in EDTA tubes (BD Biosciences) at 6 time points: pre-, 15, 30, 90 min and 24 h post reperfusion as well as 3 months after the acute event. Whole blood samples were used for quantification of absolute counts and percentage distribution of lymphocyte subpopulations by multicolor flow cytometry. Absolute counts of granulocytes, monocytes, CD4⁺ and CD8⁺ T lymphocytes were measured using the BD MultiTEST cocktail (anti-CD3-FITC, CD8-PE, CD45-PerCP, CD4-APC) and BD Trucount tubes (BD Biosciences). NK and NKT-like cells as well as naïve (CCR7⁺CD45RA⁺), central memory (CCR7⁺CD45RA⁻), effector memory (CCR7⁻CD45RA⁻) and terminally differentiated effector memory (CCR7⁻CD45RA⁺) subpopulations of CD4⁺ and CD8⁺ T lymphocytes were identified with the following monoclonal antibodies: anti-CD3-FITC, CD4-V500, CD8-APC-H7, CD16-PE, CD27-APC, CCR7(CD197)-PE-Cy7 (BD Biosciences), CD45RA-Pacific Blue (Invitrogen) and CD56-PerCP-eFluor710 (eBioscience, San Diego, CA, USA). Following incubation with these antibodies, samples were lysed, washed and immediately analyzed on a flow cytometer.

Flow cytometry of PBMCs from one healthy donor with five times of vaccinations were conducted following methods described previously^{47 (link)}. All collections were conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Ethics Committee of Huashan Hospital (2021-749). This participant provided written informed consent. Briefly, PBMCs were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Invitrogen) at ambient temperature for 20 min, followed by washing with RPMI-1640 complete medium and incubation with 10 μg/mL SARS-CoV-2 Omicron BA.1- and MERS-CoV-S trimers with His tag at 4 °C for 1 h. Afterward, the cells were washed again and incubated with a cocktail of flow cytometry antibodies, containing CD3 APC-H7 (BD Biosciences), CD19 BV421 (BD Biosciences), CD27 APC (BD Biosciences), and anti-His PE (Biolegend), at 4 °C for 1 h. Stained cells were then washed, resuspended in RPMI-1640 complete medium and sorted for S trimer-specific memory B cells (CD3⁻CD19⁺CD27⁺S trimer⁺ live single lymphocytes). The sorted cells were loaded into the BD Rhapsody single-cell analysis platform, which could effectively capture and separate single cells. The BCR library preparation and quality control were performed according to the manufacturer’s protocol and sequenced on the NovaSeq PE150 platform (Illumina).

In separate experiments, CFSE-labeled PBMCs were resuspended in a culture medium and incubated with saturating concentrations of dye-conjugated mAbs for 30 min at 4°C. Naïve (CD20⁺CD19⁺IgD⁺CD27⁻), non-switched (CD20⁺CD19⁺IgD⁺CD27⁺), and memory (CD20⁺CD19⁺IgD⁻CD27⁺) B-cell populations and non–B-cell populations (CD19⁻) were isolated by FACS with a FACSAria II (BD Biosciences) dependent on the experiment. In addition, from the non–B-cell fraction, total CD3⁺ T cells (CD3⁺CD19⁻) were isolated for specific experiments. The following mAbs were used for isolation: CD3 PE (347247; BD Biosciences), CD19 APC-R700 (564977; BD Biosciences), CD27 APC (337169; BD Biosciences), and IgD PE (555779; BD Biosciences). Different combinations, at a fixed number of cells (25,000 B cells with 125,000 non-B cells) or 100,000 T cells, were then cultured in 96-well U-bottom plates for 6 d. During culture, the cells were stimulated with CpG or αCD40 + IL-21 with or without concentrations of daratumumab as described above. T cells were stimulated with anti-CD3 (αCD3) (clone 1xE; Sanquin) and 10 μg/ml anti-CD28 (αCD28) (clone 15E8; Sanquin) with or without daratumumab. After 6 d, cells were analyzed by flow cytometry as described above. Supernatants were collected for further analysis.