Figure 4. CD8+CTLA-4+ T cells suppress T-cell proliferation in an IL-35-dependent fashion.

PBMC from a patient with CTLA-4- and IL-35-regulated PAP-specific bystander suppressive immune response (ID014) were stimulated for three days with PAP, PSA, or media alone. Cells were sorted by flow cytometry for CD8+CTLA-4+ or CD8+CTLA-4− T cells, labeled with CFSE, and added-back to autologous PKH26-labeled PBMC at either their natural frequency, or a fold higher or lower. Co-cultures were stimulated for seven days with either media alone, anti-CD3/CD28-coated beads along with IgG, or anti-CD3/CD28-coated beads along with anti-IL-35 blocking antibodies. Following this incubation, the proliferation of CFSE- CD4+ and CD8+ T cells was measured by PKH26 dilution, and percent suppression was calculated compared to the proliferation of unstimulated PBMC without any cells added back. Panel A shows the proliferation of CD4+ (left panels) and CD8+ (right panels) to which were added increasing frequencies of PAP-stimulated CD8+CTLA-4+ or CD8+CTLA-4− sorted T cells (indicated next to y-axis). Co-cultures were also treated with either a control IgG (top panels) or IL-35 blocking antibodies (lower panels – indicated to far left of x-axis). Panel B, graphical representation of suppression assays conducted using sorted CD8+CTLA-4+ (black) or CD8+CTLA-4− (grey) T cells that were stimulated with either PAP (bottom row), PSA (middle row), or media alone (top row). Co-cultures were treated with anti-CD3/CD28-coated beads along with IgG (solid lines), or IL-35 blocking antibodies (dashed lines). Following this incubation, the proliferation of CFSE- CD4+ (left panels) and CD8+ (right panels) T cells was measured by PKH26 dilution. Data shown are representative of at least two independent experiments from the same patient.