Anti cd45ra

T-lymphocyte purification and their subsequent activation were analyzed by flow cytometry (BD FACSCanto™; Becton Dickinson Immunocytometry Systems, San José, CA, USA). Cells were stained using the following monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): anti-CD4 (CD4⁺ T-lymphocytes), anti-CD25α (activated CD4⁺ T-lymphocytes), anti-CD45RA (naïve CD4⁺ T-lymphocytes), and anti-CD45RO (memory CD4⁺ T-lymphocytes) following the manufacturer’s recommendations (BD Biosciences Pharmingen, San José, CA, USA). Isotype-matched control monoclonal antibodies were used to determine the negative cell populations.

MLNLs (500,000 cells/tube) were stained using mouse anti-rat monoclonal antibodies conjugated to FITC, phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP), allophycocyanin (APC) or APC-cyanine (Cy)7. The antibodies used herein were anti-TCRαβ, anti-CD8α, anti-CD4, anti-TCRγδ and anti-CD45RA (BD Biosciences, San Diego, CA, USA). Cells were mixed with PBS containing 2% FBS and 1% NaN₃ and stained, as previously described [28 (link)]. The data were acquired with a Gallios™ Cytometer (Beckman Coulter, Miami, FL, USA) in the CCiT-UB and assessed by the Flowjo v10 software (Tree Star, Inc., Ashland, OR, USA). Results are expressed as percentages of positive cells in the lymphocyte population, selected according to their forward-scatter characteristics (FSC) and side-scatter characteristics (SSC).

For flow cytometry analysis, lymphocytes (2 × 10⁵) from spleens and MLNs were labeled with mouse anti-rat monoclonal antibodies (mAb) conjugated to FITC, phycoerythrin (PE), peridinin-chlorophyll-a protein (PercP), allophycocyanin (APC), or BD Horizon™ BV421, as in previous studies [21 (link),22 (link)]. In this case, the mAb used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-NKR-P1A, anti-TCRγδ, and anti-CD45RA (BD Biosciences, San Diego, USA). After staining with standard procedures [21 (link)], analyses were performed using a Gallios^TM Cytometer (Beckman Coulter, Miami, FL, USA) at the CCiT-UB. All results were assessed by the FlowJo v.10 software.

Fluorochrome-conjugated isotype controls, anti-CD123, anti-CD3, anti-CD4, anti-CD8, anti-CD33, anti-CD45, anti-CD45RA, anti-CD62L, anti-CCR7, anti-PD-1, anti-TIM3, anti-LAG3, anti-Annexin-V, 7-AAD, DAPI, and anti-mouse CD45 were purchased from BD Biosciences or BioLegend (San Diego, CA). Analysis was performed on at least 20,000 cells per sample using a LSR II flow cytometer (BD Biosciences) and a Gallios Flow Cytometer, and analyzed using Kaluza Analysis Software (Beckman Coulter, Indianapolis, IN).

For the assessment of cytokine secreting populations and transcription factor expression, CD4⁺ T cells were stimulated with PMA (100 ng/ml; Sigma Aldrich) and ionomycin (1 μg/ml; Sigma Aldrich) during 6hrs, and brefeldin A (10 μg/ml; Sigma Aldrich) was added 1hr after the beginning of the incubation. Cells were then stained with anti-CD45RA (BD Bioscience) Abs, where specified, fixed, permeabilized using the FOXP3 Staining Buffer Set (eBioscience) according to the manufacturer’s instructions, stained with Abs specific for IL-2 (BD Bioscience), IL-10 (BD Bioscience), IL-17 (eBioscience), IFN-γ (BD Bioscience), FOXP3 (eBioscience) and Helios (BioLegend), as indicated, and analyzed by flow cytometry (FACSAria II; BD Biosciences).

Heparinized peripheral blood was collected from study participants. The percentages and absolute numbers of CD4⁺ T, CD8⁺ T, CD19⁺ B and CD3^-CD56⁺ NK cells were determined by using TruCOUNT tubes and BD Multitest 6-color TBNK Reagent Kit (BD Biosciences) according to the manufacturer’s instructions. In brief, 50 μl of whole blood was labeled with 6-color TBNK antibody cocktail for 15 min in room temperature. After adding 450 μl of FACS Lysing Solution, samples were analyzed with FACSCanto flow cytometer using FACSCanto clinical software (BD Biosciences).
The following monoclonal antibodies were added to 100 μl of peripheral blood: anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-HLA-DR, anti-CD45RA, and anti-CD45RO (BD Biosciences). Isotype controls with irrelevant specificities were included as negative controls. All of these cell suspensions were incubated for 20 min at room temperature. After lysing red blood cells, the cells were washed and resuspended in 200 μl of PBS. The percentages of CD28⁺CD4⁺ T cells, CD28⁺CD8⁺ T cells, HLA-DR⁺CD3⁺ T cells, HLA-DR⁺CD8⁺ T cells, CD45RA⁺CD4⁺ T cells, CD45RO⁺CD4⁺ T cells, CD4⁺CD25⁺CD127^- Treg cells, CD45RA⁺ Treg cells, and CD45RO⁺ Treg cells were analyzed with FACSCanto flow cytometer.