Characterization of Cultured TIL Phenotypes

The cultured TIL phenotypes after culture were characterized using flow cytometry with anti-CD3 (Cat#: 555339, 1.5 μL/10⁶ cells), anti-CD4 (Cat#: 557871, 2 μL/10⁶ cells), anti-CD8 (Cat#: 563823, 2 μL/10⁶ cells), anti-CD56 (Cat#: 56275, 3 μL/10⁶ cells), and anti-PD1 (Cat#: 561272, 5 μL/10⁶ cells) for 30 minutes on ice in the dark [35 (link), 37 (link)]. Thereafter, the cells were washed once with PBS and resuspended in 400 μL PBS. 7AAD was used to distinguish live cells and dead cells, and the cells were run on a BD Fortessa (BD Biosciences). Fluorescence minus one (FMO) was used as the negative control. Moreover, FlowJo software was used to analyze the data generated by flow cytometry. FoxP3 staining was conducted using an intracellular staining protocol from BD Biosciences as follows: anti-CD3 and anti-CD4 were stained for 30 minutes on ice in the dark; TILs were washed, fixed, and permeabilized following protocols for BD Fix Buffer I (Cat#: 557870, BD Biosciences, USA) and Perm Buffer III (Cat#: 558050, BD Biosciences, USA). The cells were washed thrice with Perm Buffer III and incubated with anti-FoxP3 (Cat#: 560460, 5 μL/10⁶ cells) for 30 minutes on ice in the dark. The cells were run on a BD Fortessa (BD Biosciences). Fluorescence minus one (FMO) was used as the negative control. FlowJo software was used to analyze the data generated by flow cytometry.