Fig. 4

Effect of circulating EVs on immune-cell infiltration and killing into LND1 spheroids. Overview of LND1 spheroid co-cultures with PBMCs of a responder (RES), a non-responder (NRES) or healthy donors (HEALTHY) which were or not preincubated with circulating EVs from the same responder or non-responder. Pictures by fluorescence microscopy were taken after 2 (A) and 24 h co-culture (B). C PBMCs obtained from healthy donors, a responder and a non-responder were preincubated and not with RES EVs or NRES EVs and seeded with LND1 spheroids adding 20μg/ml nivolumab. After 96 h, the viability of tumor cells was evaluated as described in M&M section. D FCM characterization of PBMCs obtained from healthy donors, a responder and a non-responder:CD3⁺CD8⁺ T cells, CD3⁺CD8⁺IFNg⁺ activated T cells, CD3⁺CD4⁺CD25⁺FOXP3⁺ Treg cells, CD14⁺CD68⁺CD80⁺ M1 macrophages, CD14⁺CD68⁺CD206⁺ M2 macrophages, CD11b⁺HLA-DR⁺CD303⁺ pDCs, CD11b⁺HLA-DR⁺CD303⁺CD83⁺ activated pDCs, CD11b⁺HLA-DR⁺ cDCs, CD15⁺CD14⁻HLA-DR⁻ PMN-MDSC, CD15⁻CD14⁺HLA-DR⁻ M-MDSC