Cd4 clone sk3

A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10.7 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g. nontransduced T cells or B7-H3-negative cell lines) served as gating controls. CAR detection was performed using F(ab’)₂ fragment-specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), and CD45RO (clone UCHL1, BD Biosciences). LM7 and the negative control leukemia cell line BV173 were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). Cells were additionally stained with DAPI (BD Biosciences) to gate for live cells.

T-cell lines were analyzed after stimulation with “WT CD4⁺ pool” or “Omicron CD4⁺ pool” (1 μM/peptide) for 6 h. During the last 5 h, a mixture of Brefeldin A and Monensin (Biolegend, San Diego, CA, USA) was added. Cells were stained for antihuman CD3 (clone HIT3A; BioLegend), CD4 (clone SK3), and CD8 (clone RPAT8; both BD Biosciences, Franklin Lakes, NY, USA). After fixation and permeabilization, using FoxP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA, Thermo Fisher Scientific, Waltham, MA, USA), cells were stained intracellularly for antihuman, CD154 (clone TRAP1; BD Bioscience), and cytokines: IFN-ɣ (clone 4S.B3; BD Bioscience), IL-2 (clone MQ1–17H12; Thermofisher), or TNF-α (clone Mab11; Thermofisher). Cells were acquired on a FACS Symphony A3 analyzer (BD) and analyzed using FlowJo (V10, Tree Star, Ashland, OR, USA). On average, 40,000 events were acquired; however, in the T-cell lines obtained from the convalescent subjects, the number of events was often somewhat lower (i.e., 8000 events).

PBMCs were isolated from peripheral blood through ficoll and were stored in liquid nitrogen until use. Monoclonal antibody staining of PBMCs was performed as described earlier [9 (link)]. Briefly, cells were incubated for 15 min at room temperature with either the first panel including human antibodies: CCR10 (clone 314305; R&D), CD45RO (clone UCHL1; BD Biosciences), CCR6 (clone 11A9; BD Biosciences), CCR4 (clone 1G1; BD Biosciences), CD3 (clone UCHT1; BD Biosciences), cutaneous leukocyte antigen (CLA, clone HECA-452; BD Biosciences), CD4 (clone RPA-T4; BD Biosciences), and CXCR3 (clone G025H7; Sony), CD25 (clone bc96; Sony). CLA was added to the panel to visualize skin-homing T cells. Or cells were incubated with the second panel including CD3 (clone UCHT1; BD Biosciences), CD4 (clone SK3; BD Biosciences), CD25 (clone bc96; Sony), CD45RO (clone UCHL1; eBioscience), CD127 (clone hIL-7R-M21;BD Biosciences), and CLA (clone HECA-452; BD Biosciences). Subsequently, dead cells were excluded by incubation with Fixable Viability Dye eFluor506 (eBioscience) for 15 min at 4°C. All incubation steps were performed in the dark. Samples were measured on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed manually using FlowJo v10.7 software (Tree Star Inc. Ashland, OR). Additional file 1A and B depict the manual gating strategy of the two panels.

For analysis of intracellular cytokines, treated PBMCs were incubated with brefeldin A (Sigma Aldrich; 5 μg/ml) for the last 3 to 4 h of treatment. Afterwards, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and subsequently stained for 40 min with fluorescent antibodies detecting TNF-α (clone MAb11, BioLegend), CD14 (clone RMO52, Beckmann Coulter), and CD4 (clone SK3, BD Biosciences).
To assess the purity of isolated memory T-helper cells, cells were stained with fluorescent antibodies detecting the following cell surface markers as described previously [23 (link), 24 (link)]: CD45RA (clone HI100), CD45RO (clone UCHL1), CD3 (clone SK7), CD4 (clone SK3), CD56 (clone NCAM16.2) (all from BD Biosciences) as well as CD16 (clone 3G8), CD14 (clone RMO52), and CD19 (clone J4.119) (all from Beckmann Coulter).
For analysis of PBMC proliferation, total cell numbers after treatment were assessed by flow cytometry.
For analysis of PBMC apoptosis, treated cells were stained with fluorescent annexin V antibodies and propidium iodide using the Annexin V apoptosis detection kit APC (eBioscience) according to the manufacturer’s instructions. Afterwards, the proportion of apoptotic cells (annexin V⁺ cells) were evaluated.
All data acquisitions and analyses were carried out using a FACSCalibur device and CellQuest software (BD Biosciences).