1. cHL cell lines induce a suppressive Treg phenotype in cocultured CD4+ T cells.
cHL cell lines induce a suppressive Treg phenotype in cocultured CD4+ T cells. A, Representative examples of induction of CD25hiCD127− Tregs after coculture with cHL (L428, SUP-HD1) or NHL (SUD-HL4) cell lines after 72 hours. P1 indicates a natural Treg population in single T-cell culture; P2 indicates bulk CD4+ T cells after depletion of CD25+ T cells, P3 marks the induced Treg population after cocultivation with L428 cells. B, Summary of coculture experiments. Shown is the mean ± SD. C, qRT-PCR of isolated nTreg (P1) subsets and induced Tregs (P3) relative to the CD25-depleted population P2 as indicated in (A). D, Flow cytometric analysis of Treg cell markers in CD4+ T cells isolated after coculture. E, Flow cytometric analysis of intracellular staining of typical Th1 cytokines after coculture and subsequent stimulation with PMA/ionomycin. Shown is the percentage of positive cells among CD4+ T cells. F, Representative example of a suppression assay using HRS cell-primed T cells and autologous, CD3+ CFSE-labeled RC. Displayed are the conditions of coculture of nonprimed T cells with RC under incomplete stimulation, coculture of nonprimed T cells and RC, coculture of HRS cell-primed T cells and RC, and coculture of a CD25+ subpopulation of HRS cell-primed T cells and RC. Except for the last condition, CD25+ T cells were depleted prior to coculture with HRS cells. CD8+ RC were gated to monitor proliferation during TCR engagement. G, Summary of suppression assay results. All experiments were performed independently at least three times. HRS cell–T-cell cocultures lasted typically for 3 days. *, P < Shown is the mean ± SD.
Frederik Wein et al. Cancer Immunol Res 2017;5:
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