Cd45ra pe cy7

Thawed PBMC were washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol and 0.02 mg/ml DNAse.
CD2⁺ cells were isolated through immunomagnetic separation technique (Miltenyi Biotec, Bergish Gladbach, Germany). Purified CD2⁺ cells were washed with PBS and stained 20’ at room temperature using LIVE DEAD Red (ThermoFisher) viability marker. Then, cells were washed and stained for 20’ at room temperature with the following mAbs: anti-CD4 FITC, -CCR7 PE and -CD45RA PE-Cy7 (Biolegend, San Diego, CA, USA).
CM CD4⁺ T cells were identified and sorted with a purity more than 98% by using a Biorad eS3 Sorter (Bio-Rad, Hercules, California, USA), operating inside a specifically designed Benchtop Biocontainment Enclosure (bioBUBBLE, Fort Collins, Colorado, USA). The gating strategy and purity are shown in Supplementary Fig. 9.

The procedure of flow cytometric analysis of CMV- and IAV-specific CD8⁺ T cells prior to and post in vitro stimulation was described [40 (link)]. Briefly, freshly PMBCs were stained with fluorescently labeled dextramers or tetramers specific to CMV-pp65 (NLVPMVATV) and IAV-M1 (GILGFVFTL) (Immudex, Copenhagen, Denmark and NIAID tetramer core) first at room temperature for 20 min; followed by staining of cell surface markers including antibody against CD3, CD8, CD62L, CD45RA, CD95, CD27, CD28 CD70, CD127, and CD69 at 4°C for 30 min. Cells were washed again with FACS buffer (Hanks solution with 0.3% Sodium Azide), and then fixed immediately with 3% formaldehyde and 1% FBS in FACS buffer. Fixed cells were further stained with intracellular markers (perforin and granzyme B) at 4°C for 30 min. Stained cells were collected by BD_Symphony and were analyzed using FlowJo version 7.6.5 software.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.

PBMCs thawed on day one were washed twice in PBS + 1% BSA (PBS/BSA) and resuspended in 100µL PBS/BSA containing previously titrated combinations or singles of fluorescently conjugated antibodies against human 1 µg/mL CD3 Pacific Blue (Clone UCHT1; BD Bioscience), 1 µg/mL CD4 FITC (Clone OKT4; BioLegend), 0.2 µg/mL CD28 PE (Clone CD28.2; BioLegend), 1.5 µg/mL CCR7 APC (Clone G043H7; BioLegend), 0.5 µg/mL CD45RO APC-Cy7 (Clone UCHL1; BioLegend), 0.5 µg/mL CD45RA PE-CY7 (Clone HI100; BioLegend), 0.1 µg/mL CD56 PE (Clone HCD56; BioLegend), 1.5 µg/mL CD19 APC-Cy7 (Clone HIB19; BioLegend), and 5µL CD16 FITC (Clone B73.1; BD Biosciences). Cells were analyzed separately for T cells (CD3, CD4, CD28, CD45RA, CD45RO, and CCR7), NK cells (CD3, CD56, and CD16), and B cells (CD3 and CD19). Cells were incubated with antibodies for 30 min on ice in the dark before washing once in PBS/BSA and fixed with 1% formalin (Sigma Aldrich) for 20 min at RT in the dark. Cells were then washed and resuspended in 300 µL PBS/BSA and immediately analyzed on a BD FACS Canto II equipped with 3 lasers at the Duke Cancer Institute Flow Cytometry Core. All analyses were completed after acquisition using FCS Express v6 (DeNovo Software, CA).

The following antibodies were used:

Antibody mix 1: CD3 PerCP Cy5.5 (BD552852), CD4 V450 (BD560345), CD8 V500 (BD560774), CD197 Alexa Flour 647 (BD557734), CD45RA PE Cy7 (BD560675), CD183 Alexa 488 (BioLegend 353710), and CD196 PE (BioLegend 353410)

Antibody mix 2: CD4 FITC (BioLegend 357406), CD25 PE (BD555432), CD194 BV421 (BioLegend 395414), CD127 Alexa Flour 647 (BD558588), CD45 V500 (BD560779), CD45 RO PeCy7 (BD337168), CD3 PerCP Cy5.5, and HLA-DR APC H7/Cy7 (BD561358)

Antibodies specific for TCF1 (#2203 S) and pERK (T202/Y204, #4377 S) from Cell Signaling Technology, and DUSP6 (ab76310) from Abcam, were used at a dilution of 1 in 1000 for immunoblotting or 1 in 50 for ChIP-PCR. Antibodies specific for β-actin were from Santa Cruz Biotechnology (sc-47778 HRP) and used at 1 in 10000. Fluorochrome-conjugated antibodies were from BD Biosciences including CD4-APC (555349), CD69- PerCP-Cy5.5 (560738) and CD45RA-PE-Cy™7 and BioLegend including CD62L-PE (304806), CD25-APC (302610) and human IL-2 Alexa-Fluor-488 (500314). All antibodies for flow cytometry were used at 1 in 50. PIM1 inhibitor (526521), GSK3β inhibitors, BIO (2’Z,3’E)-6-Bromoindirubin-3ʹ-oxime (361550) and SB216763 (S3442), were from Sigma.

All flow cytometry data were acquired on BD LSRII flow cytometer, and results were analyzed using FlowJo software (v.X.0.7, Tree Star, Ashland, OR). Compensation was carried out using single-color controls of either cells or beads (OneComp eBeads, eBioscience). Appropriate isotype controls, fluorescence minus one (FMO), or NTD cells were used to validate gating. All samples were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies) or Fixable Viability Dye eFluor 780 (eBioscience) before antibody staining. The following fluorochrome-conjugated mouse anti-human antibodies were used: CD3 PerCP-Cy5.5 (BioLegend, clone UCHT1), Vδ2 PE (BioLegend, clone B6), Vδ2 fluorescein isothiocyanate (FITC) (Miltenyi, clone 123R3), Vδ1 APC-Vio770 (Miltenyi, clone REA173), Vδ1 PE (Miltenyi, clone REA173), anti-TCR γδ PE-Vio770 (Miltenyi, clone 11F2), anti-TCR αβ Brilliant Violet (BV) 421 or PE-Cy7 (both BioLegend, clone IP26), QBend10 APC (R&D Systems, clone 4H11), CD27 BV711 (BioLegend, clone O323), CD45RA PE-Cy7 (BioLegend, clone H100), PD1 FITC (BioLegend, clone EH12.2H7), TIM3 BV605 (BioLegend, clone F38-2E2), and TCR Vβ12 FITC (Abcam, clone S511).

0.8–1 × 10⁶ MNCs were stained with antibodies for 30 min at 4° C in PBS with 0.1% bovine serum albumin (BSA, Applichem, Darmstadt, Germany), followed by live/dead staining with fixable viability dye eFluor-780 (eBioscience, Frankfurt, Germany) in PBS for additional 30 min at 4° C. Cells were resuspended in 200 µL PBS + 0.1% BSA for flow cytometry. The following antibodies were used: CD3-FITC (clone BW264/56, Miltenyi Biotec, Bergisch–Gladbach, Germany); CD8-V500 (clone RPA-T8), CD45RO-FITC (clone UCHL1), CD62L-V450 (clone DREG-56), (both BD Bioscience, Heidelberg, Germany); CD45RA-PE-Cy7 (clone HI10 Biolegend, Koblenz, Germany); CCR7-PE (clone 150503, BioTechne, Wiesbaden, Germany); PD-1-APC (clone eBioJ105),TIM-3-PerCP-eFluor710 (clone F38-2E2, both eBioscience). Cells were analyzed on a FACSCanto II using the Diva software 6.0 (BD Bioscience).