Page 203 - Haematologica Vol. 110 - January 2025

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Figure 2. Classic Hodgkin lymphoma cells induce GPNMB expression and release from macrophages as well as their polarization to M2 phenotype. (A) Flow cytometry for glyco- protein non-metastatic B (GPNMB) expression on M1 or M2 macrophages following their culture in L1236 conditioned media (+CM) or their direct co-culture with L1236 cells at different macrophage:HL cell ratios for 24 hours (h). Macrophages generated from at least 9 different individuals were tested per condition. Anti-GPNMB-PE antibody (HOST5DS) and CD68/PE-Texas Red (Thermo Fisher, eBio- science, Waltham, MA, USA) were used. (B) Flow cytometry for CD163+CD206+ M2 marker expression on M1 or M2 macrophages follow- ing their culture in L1236 CM or by their co-cul- ture with L1236 cells as in (A). CD163/APC and CD206/PE-Cy7 (Thermo Fisher) antibodies were used. Macrophages generated from at least 9 different individuals were tested per condition. (C) Enzyme-linked immunosorbent assay measurement of GPNMB release by M1 and M2 macrophages (ELISA) exposed to L1236 CM or directly co-cultured with L1236 cells for 24 h. GPNMB Duoset ELISA kit (R&D Sys- tems, Minneapolis, MN, USA) was used. Shown are the results of 3 separate donors. Means (solid bars) for all experiments were compared by Student’s t test. The results for additional 2 classic Hodgkin lymphoma (cHL)-derived cell lines are shown in the Online Supplemen- tary Figure S2A-C. EBV-ve: Epstein-Barr vi- rus-negative.
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