Figure 2.

NY-ESO-1-specific proliferation, cytokine expression and CD107a expression of T cells with no alloreactive immune responses. (A) CD4⁺ and CD8⁺ T cells show a specific proliferation in response to NY-ESO-1. T cells of the final in vitro expanded T-cell products from 4 healthy donors were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated with NY-ESO-1 or control actin peptide (ACTS) overlapping peptide pool-pulsed dendritic cells (DCs), respectively. After 6 d cells were re-stimulated for 6 h with the same antigens and analyzed by flow cytometry. Bars represent NY-ESO-1-specific proliferation and background proliferation (ACTS) for each donor ; representative CFSE staining combined with IFNγ analysis of T cells from donor 4. (B) T cells show a T_H1 driven CD4 response to NY-ESO-1. CFSE-stained CD4⁺ T cells were analyzed for production of the cytokines IFNγ, TNFα and IL-10 by intracellular staining and flow cytometry (n = 4 donors). Results are mean +/− SD; representative staining of T cells from donor 1. (C) CD4⁺ T cells show cytolytic responses to NY-ESO-1. T-cell products of donors 1 and 4 were analyzed in 4 independent experiments for CD107a expression after 6 h of re-stimulation with NY-ESO-1 or ACTS overlapping peptide pools pulsed DCs. Bars show mean results of 4 experiments +/− SD and the results of different DC:T-cells ratios during re-stimulation are shown; representative staining of T cells from donor 4. (D) For final analysis of T-cell lines, T cells from donor 1, 7 and 4 were re-stimulated for 6 h with overlapping peptide (NY-ESO-1, ACTS) pool-pulsed dendritic cells (DCs) and analyzed by immunostaining and multispectral fluorescence cytometry. Bars represent mean values of double positive cells gated on CD4⁺/IFNγ⁺ T cells. T-cell subpopulations were defined as naïve T cells (CD27⁺/CD28⁺, CD62L⁺/CD45RO⁻), central memory T cells (CD62L⁺/CD45RO⁺, CD45RA⁻/CCR7⁺) and effector memory T cells (CD62L/CD45RO⁺, CD45RA⁻/CCR7⁻ and CD27⁻CD28⁺).