D7u8c

Two-micron tissue sections underwent single-marker immunostaining for Programmed Cell-death ligands 1 (PD-L1) and 2 (PD-L2), using antibody clones E1L3N (Cell Signalling Cat. Nr. 13684, dilution 1:100) and D7U8C (Cell Signalling Cat. Nr. 82723, dilution 1:100), respectively, on a Leica Bond RX stainer (Leica, Buffalo, IL, USA). Multiplex immunostaining for CD4 (Spring Biosciences, Pleasanton, CA, USA clone SP35), CD8 (clone SP239), FOXP3 (BD Biosciences, San Jose, CA, USA, clone 346/E7) and PD-1 (Novus Biologicals, Centennial, CO, USA, clone NAT 105/E3) followed a pre-optimised protocol [7 (link)]. Individual counts of CD4+, CD8+, CD4+/FOXP3+ and CD8+/PD-1+ co-immunopositive cells were performed in tissue photomicrographs, assessed at 450× magnification across intra-tumoural (IT) and peri-tumoural (PT) areas and reported as cellular density per mm² of tissue. PD-L1/2 expression in tumour cells was presented using a semi-quantitative score (H-score, range 0–300), derived from multiplying the percentage of positive cells (1% cut-off) by chromogenic intensity (ranked from 0 to 3). Immunopositivity was scored categorically, using a 1% cut-off value, as routinely employed in clinical trials of ICI.

Tested were: PD1 (NAT105, Ventana Medical System, Tucson, USA), CD8 (SP57, Ventana Medical System, Tucson, USA), PD-L2 (D7U8C, Cell Signaling Technology, Danvers, MA, USA).
Sections were deparaffinized, rehydrated and heated for antigen retrieval, 20 min in high buffer (DAKO) (PD-L1 SP142, PD-L1 E1L3N, and PD-L2), and 64 min CC1 (PD1 and CD8). Immunohistochemistry was performed on Dako Link autostain (PD-L1 and PD-L2) with envision flex system or on Ventana Benchmark (PD1 and CD8) with Optiview revelation system. Slides were incubated with primary antibodies 1 h at a 1/100 dilution (PD-L1, SP-142), 1 h at a 1/500 dilution (PD-L1, E1L3N), 1 h at 1/100 dilution (PD-L2), 32 min (prediluted PD1) and 20 min (prediluted CD8).