Anti human cd8

CD8 T, CD4 T and B cells were purified from PBMCs with EasySep Human positive or negative selection (STEMCELL Technologies, 17953, 17852 and 17954) according to the manufacturerʼs instructions. The cell purities were checked for over 95% with anti-human CD8 (BioLegend, 344721), anti-human CD4 (BioLegend, 317408), anti-human CD19 (BioLegend, 392504) and anti-human CD20 (BioLegend, 302326), respectively. Total RNA was isolated from CD8 T, CD4 T and B cells of three randomly selected young and old donors individually at baseline (before vaccination) and 7 days after the second vaccination dose by using TRIzol reagent (Invitrogen) (Supplementary Table 1). RNA purity was checked by the NanoPhotomerer spectrophotometer (IMPLEN), and integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). Then, cDNA libraries were constructed using 0.1 µg of RNA per sample with the NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer’s recommendations; the libraries were sequenced on an Illumina NovaSeq platform; and 250-bp paired-end reads were generated.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood donated by 5 healthy male and 5 healthy females between 26 and 46 years of age. CD3 T cells were isolated from PBMCs using a human Pan T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. CD8 T cells were isolated from PBMCs using CD8 T isolation kits (Miltenyi Biotec). T cells separated for allogeneic–antigen stimulation were labeled with 0.5 μM CFSE for 5 min at room temperature and washed four times with xenofree media or DPBS. T cell-depleted PBMCs (Td-PBMCs) were isolated from PBMCs using a CD3 T-positive isolation kit (Miltenyi Biotec). For allogeneic–antigen stimulation, separated Td-PBMCs were irradiated at 10 Gy (IBL-437C, CIS Bio International, Gif-sur-Yvette, France) for their role as antigen presentation cells of B cells, as the function of B cells was significantly reduced at 20 Gy [54 (link),55 (link)]. To examine the production of memory T cells, T cells were stained with anti-human CD3, anti-human CD4, anti-human CD8, anti-human CD45RA and anti-human CD62 L (BioLegend) antibodies, as well as with the respective isotype antibodies corresponding to these target antibodies. The phenotypic changes in T cells after allogeneic–antigen stimulation was analyzed by flow cytometry (BD Biosciences).

For cell lines, cells were firstly harvested, centrifuged, and washed in PBS. The cell pellets were resuspended in 100 μL PBS + 1% BSA and stained with fluorescent dye-conjugated antibodies. All antibodies were obtained from Biolegend: anti-human Fas (Cat#305608), anti-mouse/human CD11b (Cat#101206), anti-human HLA-DR (Cat#307609), anti-human CD8 (Cat#344706), anti-mouse Fas (Cat#152607), anti-mouse Gr1 (Cat#108426), anti-mouse CD8 (Cat#100732), and anti-mouse CD45 (Cat#147712). Stained cells were analyzed by flow cytometry. Data files were analyzed using FlowJo.V10 software.

Peripheral blood mononuclear cells (PBMC) were extracted from 4ml of venous blood obtained from HCC patients for flow cytometry analysis. We used the anti-human CD8 (Biolegend) marker to identify CD8⁺T cells. Notch1⁺CD8⁺T cells were identified using the anti-human Notch1 (BD) antibody. Additionally, we assessed the expression of co-inhibitory molecules on the cell surface using anti-human TIGIT (BD), anti-human TIM-3 (BD), anti-human CD39 (Biolegend), and anti-human PD-1 (BD) antibodies. Furthermore, we evaluated the killing function of the cells by detecting the expression of anti-human Perforin (Biolegend) and anti-human GranzymeB (BD) antibodies.

Portions of primary tumor and metastatic tissues were digested with collagenase IV/DNase (0.2 mg/mL and 10 μg/mL) at 37 °C for 30 min. Cells were put through 40-μm filters to obtain single-cell suspensions and stained as previously described [21 (link)]. Cell surfaces were stained with anti-mouse CD4, anti-mouse CD206, anti-mouse CD11b, anti-mouse F4/80, anti-mouse Gr1.1, anti-mouse CD3, anti-mouse CD45, anti-mouse CD8a, anti-mouse CD28, anti-mouse NKG2D, anti-human CD45, anti-human CD8, anti-human CD33, and anti-human HLA-DR antibodies (BioLegend, San Diego, CA) labeled with fluorescein isothiocyanate, PerCP/Cy5.5, phycoerythrin, or Brilliant Violet 421. Flow cytometry was performed using an Attune flow cytometer (Life Technologies), and the data were analyzed with FlowJo software.