Figure 2.

Enforced IL-10 Expression in Alloantigen-Specific CD4⁺ T Cells Promotes Their Conversion into Tr1-like Cells with the Ability to Kill Myeloid Cells

(A) Protocol to generate allo-CD4^IL-10 cells. Enriched alloantigen-specific CD4⁺ T cells were transduced with LV-IL-10 or LV-GFP at an MOI of 20 during a secondary stimulation with the same allo-mDCs used for priming (see also Materials and Methods). (B) Allo-CD4^IL-10 and allo-CD4^ΔNGFR cells equally expanded in vitro. Purified allo-CD4^IL-10 and allo-CD4^ΔNGFR cells were expanded with feeder mixture (see Materials and Methods), and numbers of recovered cells at the indicated time points are presented. Mean ± SD of n = 6 independent experiments are presented. (C) The proliferative responses of allo-CD4^IL-10 and allo-CD4^ΔNGFR cells. Allo-CD4^IL-10 and allo-CD4^ΔNGFR cells were stimulated with allogeneic (allo-mDCs) or 3^rd party mDCs (ratio 10:1), and proliferation was evaluated by [³H]thymidine incorporation on days 3 and 5, respectively. Mean ± SEM of n = 19 for allogeneic stimulation and n = 16 for 3^rd-party stimulation tested in more than five independent experiments. All samples were tested in duplicate-triplicate. p, Wilcoxon matched-pairs signed rank test. (D and E) Cytokine production of allo-CD4^IL-10 and allo-CD4^ΔNGFR cells. (D and E) Allo-CD4^IL-10 or allo-CD4^ΔNGFR cells were stimulated with allo-mDCs or a third-party mDC (ratio 10:1), and IFN-γ (D) and IL-10 (E) production were determined in culture supernatants 48 hr after activation by ELISA. Mean ± SEM of n = 12–26 donors tested in five independent experiments. All samples were tested in duplicate-triplicate. p, Wilcoxon matched-pairs signed rank test. (F) Suppressive activity of allo-CD4^IL-10 cells. Autologous allo-specific CD4⁺ T cell lines (responders) were labeled with eFluor dye and stimulated with allo-mDCs (ratio 10:1) alone or in the presence of allo-CD4^IL-10 or allo-CD4^ΔNGFR cells at a 1:1 ratio. After 3 days of culture the suppressive ability was determined by CFSE/eFluor dilution of CD4⁺ΔNGFR⁻ cells. One representative donor (left panel) and mean ± SEM of n = 6 for allogeneic stimulation and 3^rd-party stimulation tested in three independent experiments (right panel) are shown. The suppression mediated by allo-CD4^IL-10 cells or allo-CD4^ΔNGFR cells was calculated as follows: ([proliferation of responders in the presence of transduced cells/proliferation of responders alone] × 100). p, Wilcoxon matched-pairs signed rank test. (G) Allo-CD4^IL-10 cells killed myeloid cell lines. CD4^IL-10 and CD4^ΔNGFR cells were co-cultured with ALL-CM, U937, and K562 cell lines at 1:1 ratio for 3 days. Residual leukemic cell lines (CD45^lowCD3⁻) were counted by FACS. Cytolysis mediated by CD4^IL-10 cells was measured as elimination index (see Materials and Methods) for each target cell. Analysis was performed in two independent experiments. Dots represent the elimination index of CD4^IL-10 cells generated from different healthy donors, and lines represent mean values of the elimination index. Gray area indicates the threshold of cytotoxicity.