Anti human cd4 sk3

Live immune cells from peripheral blood, spleen, and BM were determined by staining with live/dead fixable blue dead cell stain kit (Life Technologies) for 30 min prior to cell-specific marker labeling. Cells were labeled with anti-human CD34 (clone 581; BD Biosciences), anti-human CD3 (UCHT1; Biolegend), anti-human CD56 (MEM-188, Biolegend), anti-human CD14 (63D3; Biolegend), anti-human CD19 (SJ25C1; BD Biosciences), anti-human CD117 (104D2; Biolegend), anti-human CD38 (HB-7; Biolegend), anti-human CD33 (WM53; BD Biosciences), mouse CD45.1 (A20; BD Biosciences), anti-human CD8 (SK1; Biolegend), anti-human CD4 (SK3; BD Biosciences), and anti-human CD45 (HI30; Biolegend) monoclonal antibodies for 30 min at room temperature. After incubation, cells were washed and resuspended in FACs buffer containing phosphate buffered saline (PBS), 0.2% bovine serum albumin (GE Healthcare Life Sciences), and 0.05% sodium azide (Merck) for flow cytometry data acquisition. Data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analysis was performed using FlowJo software (version 10; Tree Star Inc). Absolute count of cells in peripheral blood was determined using CountBright™ Absolute Counting Beads (Thermo Fisher Scientific).

Liver cancer cell line PLC/PRF/5 (1 × 10⁶ cells) was injected subcutaneously into the flank of 18- and 60-week-old humanized mice, and tumor growth was monitored weekly by caliper measurement to derive the volume using the formula $\frac{\pi }{6}\left(l,e,n,g,t,h,\times ,{\mathit{width}}^2\right).$ Four weeks post engraftment, tumors were harvested and dissociated using the human Tumor Dissociation Kit (Miltenyi Biotec) with the gentleMACS™ Octo Dissociator (Miltenyi Biotec). Cell suspension was layered over 35% Percoll® (GE Healthcare) and centrifuged to purify the tumor infiltrating lymphocytes (TILs) for downstream flow cytometry analysis. Antibodies used include anti-human CD4 (SK3, BD Biosciences), anti-human KLRG1 (14C2A07, Biolegend), anti-human CD3 (HIT3a, BD Biosciences), anti-human CD45RA (HI100, Biolegend), anti-human CTLA-4 (BN13, Biolegend), anti-human CD45 (H130, BD Biosciences), anti-human PD1 (EH12.2H7, Biolegend), anti-human LAG3 (11C3C65, Biolegend), anti-human CD27 (O323, Biolegend), anti-human CD8 (SK1, BD Biosciences), anti-human TIGIT (MBSA43, ebioscience), and anti-human TIM3 (F38-2E2, Biolegend). Data was acquired on a LSR II flow cytometer (BD Biosciences) and analyzed using the FlowJo software v10 (FlowJo LLC). Exclusion criteria include humane endpoints as stated in the IACUC protocol, such as maximum tumor size, weight loss and animal constitution.