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. Author manuscript; available in PMC: 2013 Dec 15.
Published in final edited form as: J Immunol. 2012 Nov 14;189(12):5590–5601. doi: 10.4049/jimmunol.1201744

Figure 5. CD8+CTLA-4+ T cells suppress T-cell proliferation in a contact-independent fashion.

Figure 5

PBMC from a patient with CTLA-4− and IL-35-regulated PAP-specific bystander suppressive immune response (ID002) were stimulated for three days with PAP (bottom row), PSA (middle row), or media alone (top row). Cells were sorted by flow cytometry for CD8+CTLA-4+ (black) or CD8+CTLA-4− (grey) T cells, and added-back to the top chamber of a 96 transwell plate at either their natural frequency, or a fold higher or lower (indicated along the x-axis). Autologous PKH26-labeled PBMC were added to the bottom chamber of the transwell plates, and stimulated for seven days with either media alone, anti-CD3/CD28-coated beads along with IgG (solid lines), or anti-CD3/CD28-coated beads along with anti-IL-35 blocking antibodies (dashed lines). Following this incubation, cells were collected from the bottom chamber and measured for the proliferation of naive CD4+ (left panels) and CD8+ (right panels) T cells by PKH26 dilution, and percent suppression was calculated compared to the proliferation of naïve PBMC without any cells added back. Data shown are representative of at least two independent experiments.