Anti human pd 1

The expression of PD-1, CD28, PD-L1, and CD86 in NK cells was assayed in the whole peripheral blood samples via flow cytometry within 1 h of blood collection. The cells were stained with monoclonal antibodies and isotype controls according to the manufacturer’s recommendations (anti-human PD-1, PD-L1, and CD86; BD Pharmingen, Franklin Lake, NJ, USA; The other antibodies listed below were obtained from Biolegend, San Diego, CA, USA). The other antibodies listed below were obtained from San Diego, CA, USA. Aliquots of 100 μL of whole blood were incubated with PerCP/cyanine5.5-labeled anti-CD3 (5 μL clone HIT3a), APC/cyanine7-labeled anti-CD56 (5 μL clone HCD56), APC-labeled anti-PD-1 (20 μL clone MIH4), PE.Cy7-labeled anti-CD28 (5 μL clone CD28.2), PerCP/Cy5.5-labeled anti-CD3 (5 μL clone HIT3a), APC/Cy7-labeled anti-CD56 (5 μL clone HCD56), APC-labeled anti-PD-L1 (20 μL clone MIH1), and PE.Cy7-labeled anti-CD86 (20 μL clone 2331 (FUN-1)). The erythrocytes were lysed, and cells evaluated by a researcher blinded to our clinical data. The samples were processed on CytoFLEX (Beckman Coulter Inc. Brea, CA, USA) and analyzed using CytExpert software version 2.0 (Beckman Coulter Inc.). The lymphocytes were gated by forward scatter (FSC) and side scatter (SSC), and NK cells subsets were further identified by CD3⁻ and CD56⁺ staining.

Biopsy samples were fixed in 10 % formalin and paraffin-embedded. Then, the samples were cut into 5-μm-thick sections. Immunohistochemistry was performed according to the appropriate protocols as previously described [14 (link)]. The following antibodies were used as primary antibodies: anti-human CD66b (Anaspec, CA, USA), anti-human CD3 (Gene Tech, Shanghai, China), anti-human PD-L1 and anti-human PD-1 (BD Bioscience, San Jose, USA). A mouse anti-human CD66b monoclonal antibody was used to identify neutrophils (1:50), and rabbit anti-human CD3 monoclonal antibody was used to identify T cells (1:100). A Real Envision Detection Kit (Gene Tech, Shanghai, China) was used for detection. Quantification of immune-cell infiltration was then determined.

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.