Figure 4.

CM from spheroids induces T cells suppression that can be prevented by CXCR4 inhibition. (A) FACS analysis for Treg population within T lymphocytes, from N=8 healthy volunteers. T lymphocytes were stimulated with anti CD3/CD28 beads and incubated for 72 h with CM from adherent or spheroids cell lines, untreated or treated with peptide R. Data are the fold-change in % T reg population compared to proper control medium (RPMI 10% for adherent cells and Stem cells medium- SCM- for spheroids). Data are the mean value ± SD. N=2 independent experiments were performed for each tested NSCLC cell lines (A549/H3122/H1299/Sw900). (B) MTT assay measuring the proliferation of healthy volunteer T cells, unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies, after exposure for 72 h to CM from A549 and H3122 spheroids or control RPMI or SCM medium for 72 h. Data are the mean value ± SD of N=4 independent experiment for each cell line. (C) CSFE assay measuring proliferation of healthy volunteers T cells, stimulated with anti-CD3 and anti-CD28 microbeads, after exposure for 72 h to CM from adherent or spheroids, treated or not with peptide R. Data are the fold-change in % of proliferating cells compared to proper control medium (RPMI 10% for adherent cells and SCM for spheroid). Data are the mean value ± SE of each NSCLC cell line (A549/H3122/H1299/SW900) tested in triplicate experiment. (D) FACS analysis for CD8+ T cytotoxic cells expressing IFNγ in N=8 healthy volunteers incubated for 72 h with CM from adherent or spheroids cell lines, untreated or treated with peptide R. Data are the fold-change in % CD8 T cytotoxic population compared to proper control medium (RPMI 10% for adherent cells and Stem cells medium- SCM- for spheroids). Data are the mean value ± SE of N=2 independent experiments were performed for each tested NSCLC cell lines.