T bet 4b10

For Western analysis, whole-cell protein lysates were obtained from effector CD8⁺ T cells at 4 time points (0, 3, 5, and 8 days post-stimulation) with lysis buffer Proprep (INTRON Biotechnology) by suspending 106 cells in 10 μl buffer and incubating on ice for 30 minutes in the presence of Halt Protease & Phosphatase inhibitors cocktail (Thermo Scientific). Cell lysates were resolved by 10% Bis-Tris SDS PAGE Gel, transferred onto PVDF membrane (Merck), blocked with 5% skim milk in TBST buffer containing 0.1% Tween-20, and probed with the following mAbs: T-bet (4B10, Santa Cruz), Eomes (Y-20, Santa Cruz), Foxp1 (polyclonal, Abcam), and β-actin (C4, Santa Cruz). Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of β-actin (1:5000) detected in each lane.

Formalin-fixed, paraffin-embedded (FFPE) tumor tissues obtained from each patient before the vaccination were analyzed. Serial 2-μm thick sections were cut and processed for IHC. Primary antibodies used: CD3-Policlonal (A045201–2, 1:400), CD8 (C8/144B, 1:20), CD68-KP1 (M081401–2, 1:3000) and CD68-PGM-1 (M087601–2, 1:50), all purchased from Dako; CD163-10D6 (NCL-CD163, 1:200), GZMB (11F1, 1:80) (Novocastra, Leica Biosystems); Tbet (4B10, 1:80) (Santa Cruz); MHC-I-EMR8–5 (Ab70328, 1:4000) (Abcam); NY-ESO-1-E978 (35–6200, 1:200) (Thermofisher); PD1-NAT105 (3137, 1:50) (Biocare) and PDL1-RBT (BSB2654, 1:50) (Bio SB). All these primary antibodies were processed using the Autostainer Link 48 Dako System.
Immune infiltrating cells were quantified by counting the number of immune reactive cells at 400X magnification. The three areas with the most intratumor inflammatory cells were selected. Results are reported as mean value of immune reactive cells/mm².