Isotype matched igg

Flow cytometry was performed as described (40 (link)). Mf were resuspended with FACS buffer (PBS supplemented with 0.2% BSA, 0.01% NaN₃) and stained with PE-conjugated mouse anti-human mAbs against CD14, CD80, CCR7, and TREM-1 (clone 193015, obtained from BioLegend, Milano, Italy), CD68 (obtained from Dako, Milano), CD206 (obtained from Miltenyi Biotec), CD86, HLA-DR, CD36 (BD-Pharmingen, Milano, Italy), and isotype-matched IgG (obtained from BioLegend) for 30 min at 4°C, after preincubation with rabbit IgG (obtained from Sigma) to block non-specific sites. Four-color flow cytometric analysis was carried out to analyze SFMCs using the following Abs: anti-CD68-FITC (obtained from Dako), anti-CD80-PE/Cy7, anti-CD206-APC, and anti-TREM-1-PE Abs (obtained from Biolegend). Fluorescence was quantitated on a FACSCalibur flow cytometer equipped with CellQuest software (BD-Biosciences). Cells were gated according to their light-scatter properties to exclude cell debris.

To assess CRLF2 expression on the surface of T-ALL blasts the following combination of antibodies was used: CRLF2PE (Clone 1B4, Biolegend, London, UK) [24 (link)] or isotype matched IgG (Biolegend), CD45PerCP (BD Biosciences, Franklin Lakes, NJ, USA) and CD7ECD (Beckman Coulter, Brea, California, USA). Leukemic blasts were gated as CD45 intermediate/CD7+. The T-ALL cell lines were stained only with the CRLF2PE or the isotype antibody.

Human T-lymphocytes were isolated from healthy donors using RosetteSep Human T-cell Enrichment Cocktail (STEMCELL Technologies). Lymphocytes were cultured in a cell culture plate coated with anti-human CD3 antibody (OKT3; eBiosciences, San Diego, CA, USA) and human CD28 antibody (BioLegend, San Diego, CA, USA). The proliferation of lymphocytes was examined by the BrdU incorporation assay (Cell Proliferation ELISA kit, Roche, Basel, Switzerland). Anti-PD-1 antibody (clone EH12.2H7) and isotype-matched IgG were obtained from BioLegend.