Anti cd4

Th17 cells were analyzed by CD4 and IL-17 staining. Briefly, single cells were fixed with 4% formaldehyde and further permeabilized by methanol. The cells were then incubated with anti-IL-17 (1:200, Abcam) and anti-CD4 (1:100, R and D Systems) antibodies, followed by incubation with APC or FITC labeled secondary antibody (Invitrogen). The samples were analyzed using a Beckman Coulter flow cytometer. The raw FACS data were analyzed with the FlowJo software.

Immunofluorescence staining utilized tyramide signal amplification staining performed using OPAL Reagents (Akoya Biosciences, Inc, Marlborough, MA). Briefly, tumor tissues were formalin fixed and embedded in paraffin blocks and cut into 10 μm sections at the Cedars-Sinai Biobank and Research Pathology Core. Tissue sections were deparaffinized and rehydrated, then antigen retrieval was performed in Tris-EDTA or citrate buffer. Primary antibodies anti-CD8 (ebioscience, 4SM15), anti-CD4 (R&D, GK1.5), anti-CD90 (Sino Biological, Cat #50461-T44), anti-CD73 (Sino Biological, Cat # 50231-T56), anti-GranzymeB (ebioscience, 16G6), anti-CD90.1 (BioLegend, OX-7), anti-CD45.1 (Invitrogen, A20) diluted to 1:200 were incubated overnight at 4°C. Secondary antibodies conjugated to HRP-polymers (Abcam, Cambridge, UK) were incubated for 15 min and then washed before slides were incubated with OPAL fluorophores (OPAL 520, OPAL, 650, OPAL 570) diluted 1:200 for 10 min at RT. Slides were washed in PBST before performing subsequent rounds of antigen retrieval and staining. Tissues were mounted with ProLong Gold Antifade Mountant with DNA Stain DAPI (Invitrogen). Images were acquired on ECHO Revolution upright microscope equipped with sCMOS Mono camera.