Cd4 clone 4b12

Briefly, 4 μm tissue slices were prepared from cryopreserved tissues and mounted on glass slides. Immunohistochemistry was performed according to the manufacturer’s instructions using an automated staining facility (Bond Max, Leica, Wetzlar, Germany). The following mouse monoclonal antibodies were utilized: CD3ϵ (clone ab16669, 1:100, Abcam, Cambridge, UK; RRID: AB_443425), CD4 (clone 4B12, 1:150, Leica, Wetzlar, Germany; RRID: AB_563559), CD8 (clone 4B11, 1:100, Leica, Wetzlar, Germany; RRID: AB_442068), CD20 (clone L26, 1:100, Leica, Wetzlar, Germany; RRID: AB_442055), CD163 (clone EPR19518, 1:500, Abcam, Cambridge, UK; RRID: AB_2753196), NKp46 (clone 195314, 1:175, R&D Systems, Minneapolis, MN, USA; RRID: AB_2149153) and FoxP3 (clone 236A/E7, 1:100, Thermo Fisher Scientific, Waltham, MA, USA; RRID: AB_467555).

Sections from tissue blocks of all cases were immunohistochemically stained with STING (anti-TMEM173; clone SP338, dilution 1:150; Abcam, UK). Heat-induced antigen retrieval for STING was performed using a microwave oven and 0.01mol/L of citrate buffer, pH 8.0, for 30 min. Moreover, tumors defined as “excluded” and “inflamed” by hematoxylin and eosin were investigated for the specific composition of different lymphocytes subpopulations, according to the different percentages of expression of the following antibodies: CD3 (clone PS1, dilution 1:200, LEICA), CD20 (clone L26, prediluted, NOVOCASTRA), CD4 (clone 4B12, dilution 1.150, LEICA), CD8 (clone 29S, dilution 1:20, LEICA), and FOXP3 (clone 221D/D3, dilution 1:200, SEROTEC). All samples were processed using a sensitive “Bond Polymer Refine” detection system in an automated bond immunohistochemistry instrument (Leica Biosystems, Germany). Sections incubated without the primary antibody served as a negative control. Cytoplasmic and membranous labeling for the STING was recorded by combining the percentage of positive cells (0–100%) multiplied by staining intensity (0, 1+, 2+, and 3+) to obtain an overall H-score (0-300).

Formalin-fixed biopsies were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate pH 6.0 (for HbcAg), Tris-EDTA pH 9.0 (for CD4 and CD8), pepsin (for CD68), or commercial CC1 buffer Ventana (for PD-1 and PD-L1). Sections were then incubated with antibodies to the following: HBsAg (clone A10F1, 1:100, Cell Marque, Rocklin, CA), HbcAg (1:800, Abcam, Cambridge, UK), CD4 (clone 4B12, 1:200, Leica, Buffalo Grove, IL), CD8 (clone 4B11, 1:1000, Leica, Buffalo Grove, IL), CD68 (clone PG-M1, 1:300, Dako, Santa Clara, CA), PD-1 (clone NAT105, 1:200, Abcam, Cambridge, UK), PD-L1 (clone 28-8, 1:200, Abcam, Cambridge, UK). Expression of the markers was quantified using the Visiopharm software, by dividing the number of strongly positive cells by the total stained area. PD-L1 staining on hepatocytes was scored on a semi-quantitative scale: 1+ 25% positive, 2+ as intermediate, and 3+ diffuse staining.