Figure 5.

MultiStem-mediated suppression of T-cell cytotoxicity is contact-dependent. (A): Purified CD8⁺ cytotoxic T lymphocytes (CTLs) were stimulated with irradiated allogeneic Epstein-Barr virus-positive B cells (stimulator:responder [S:R] ratio of 1:10) with or without irradiated third-party MultiStem (ratio of 1:2) and with or without interleukin-2 (IL-2) for 7 days. Data are expressed as mean ± SEM percentage of SR of P815 targets (effector:target ratio of 10:1) and pooled from four experiments with three CTL and two MultiStem donors (donors 2 and 4). (B): Standard experimental setup was used (left). Right: After a 3-day resting period, CTLs from the primary mixed-lymphocyte culture, cultured with or without MultiStem, were restimulated during 4 days with the same alloantigens in the absence of MultiStem. Results are expressed as mean ± SEM percentage of SR against P815 targets of three experiments with two CTL and two MultiStem donors (donors 2 and 4). (C): Results of anti-CD3-redirected cytotoxicity of B cell-stimulated CD3⁺ T cells (S:R ratio of 1:20) against P815 targets with or without MultiStem, in direct contact or separated by a transwell system, during the 7-day activation phase. Data are pooled from six experiments with five T-cell and two MultiStem donors (donors 2 and 5). (D): Flow-cytometric analysis of MultiStem, pretreated or not with interferon-γ, for the PD-1 receptor and its ligands programmed death ligands 1 and 2 and for the ligand of the Fas pathway. Average mean fluorescence intensity values ± SD of three experiments (MultiStem donors 2, 4, and 5) are shown. The background signal of unstained cells is included. Statistical significance was calculated with the paired t test. ∗, p < .05. Abbreviations: FasL, Fas ligand; IFN-γ, interferon-γ; IL-2, interleukin 2; ns, not significant; PD-1, programmed death-1; SR, specific ⁵¹Cr-release; w/, with; w/o, without.