Anti cd3 bv510 hit3a

The following anti-human fluorochrome-conjugated antibodies were used: Alexa Fluor 700-conjugated antibody to human CD3 (UCHT1); anti-IL-7Rα-BV421 (A019D5), anti-CD4-PE-Cy5 (OKT4), anti-CD45RA-APC-Cy7 (HI100), anti-Fas-BV650 (DX2), anti-CCR7-BV785 (G043H7), anti-CD31-Alexa Fluor 488 (WM59), anti-TCR γ/δ-Percp-Cy5.5 (B1), anti-CD25-BV650 (BD96; BioLegend), anti-CD19-BV711 (SJ25C1), anti-CD14-BV711 (MφP9), anti-CD3-BV510 (HIT3a), anti-CD8-Alexa Fluor 700 (RPA-T8; all from BD Biosciences); anti-CD19-PE-eFluor610 (HIB19); anti-CD14-PE-eFluor610 (61D3), and anti-CD56-APC (CMSSB; Thermo Fisher). Simultaneously, dead cells were stained using a Live/Dead Fixable Red Dead Cell Stain Kit (Thermo Fisher). The cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (Thermo Fisher), and then intracellular molecules were stained (4°C, 20 minutes) using anti-Bcl-2-Alexa Fluor 488 (100; BioLegend), anti-Ki-67-PE-Cy7 (20Raj1), and anti-Foxp3-PE (236A/E7; Thermo Fisher). Detailed information about the multicolor flow cytometry panel used in this study is shown in supplemental Table 4.

The naive and memory subsets of CD8⁺ and CD4⁺ T cells were isolated from cryopreserved cells using Moflo Astrios EQ (Beckman Coulter), and the dead cells and surface markers were stained with anti-CD4-PE (RPA-T4), anti-CD8-Alexa Fluor 700 (RPA-T8), anti-CD3-BV510 (HIT3a; all from BD Biosciences), anti-CD45RA-APC (HI100; Thermo Fisher), and anti-CCR7-Percp-Cy5.5 (G043H7, BioLegend). We extracted RNA from isolated cells using an RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. CDR3 regions of the TCR chain were amplified using RNA as a template, with primer sets carrying a specific barcode (iRepertoire). The cDNA amplicons were sequenced using HiSeq 4000 (Illumina), as previously described.²⁴ (link) iRepertoire analyzed CDR3 sequences²⁴ (link) and provided raw data regarding variable (V), diversity (D), and joining (J) segment usage and CDR3 length. We measured TCR repertoire diversity using the following indices: $\text{Inverse}\text{Simpson}\text{index}(1/\mathrm{\lambda })=1/{\sum }_{i=1}^R{p}_i^2$ ${\text{Shannon}\text{index}(\mathrm{H}}^{\prime })=-p_i{\sum }_{i=1}^R\ln p_i$ $\text{Normalized}\text{Shannon}\text{index}=\frac{Shannonindex\left(H´\right)}{\log \left(thenumberoftotalproductiventsequences\right)}$ $\mathrm{U}/\mathrm{T}\text{index}=\frac{\text{the}\text{number}\text{of}\text{productive}\text{unique}\text{nt}\text{sequences}}{\text{the}\text{number}\text{of}\text{total}\text{productive}\text{nt}\text{sequences}}$

Cryopreserved PBMCs were stained with cell trace violet (CTV; Thermo Fisher) (20 minutes, RT), and then stimulated with 1 μg/mL epitope peptide (NLVPMVATV; JPT Peptide Technologies) for 6 days at 37°C in 500 μL of 10% fetal bovine serum/RPMI 1640 medium (WELGENE). Cells were labeled with HLA-A∗0201-restricted CMVpp65 pentamer (HLA-A2∗0201: CMVpp56_495, ProImmune). Next, surface molecule staining, dead cell staining, fixation, permeabilization, and intracellular molecule staining were performed using the following antibodies: anti-CD3-BV510 (HIT3a), anti-CD8-Percp-Cy5.5 (SK1; BD Biosciences), anti-CD19-PE-eFluor610 (HIB19), anti-CD14-PE-eFluor610 (61D3), anti-Ki-67-PE-Cy7 (20Raj1; Thermo Fisher), anti-CD45RA-APC-Cy7 (HI100), and anti-CCR7-BV785 (G043H7; BioLegend), and a Foxp3 Staining Buffer Set. Data were collected using an LSR II instrument.