Fig 5. PD1⁺CTLA4⁺CD4⁺ T cell-mediated suppression requires cell contact.

To determine mechanisms involved in the suppression mediated by PD1⁺CTLA4⁺CD4⁺ T cells, we conducted suppression assays in transwell systems and in the presence of blocking antibodies for IL10R, TGFβ, CTLA4 and PDL1. The net (stimulated minus spontaneous) proliferation counts for CD4⁺ T cells (ratio 1:0), stimulated with anti-CD3/28, were set as 100%. Proliferation results for other cell culture conditions are expressed as percentage of net proliferation counts of CD4⁺ T cells (ratio 1:0). A) 5 x10⁴ CD4⁺ T cells were stimulated with anti-CD3/28 and cultured with/without an equal number of PD1⁺CTLA4⁺CD4⁺ T cells, separated by a 0.2μm transwell membrane. P = 0.75, using a Wilcoxon matched pairs test (n = 3). B) 2.5 x10⁴ CD4⁺ T cells were stimulated with anti-CD3/28 and cultured with/without an equal number of PD1⁺CTLA4⁺CD4⁺ T cells (abbreviated with PD1⁺) and with/without addition of 10μg/ml blocking antibodies for IL10R, TGFβ, CTLA4 and PDL1 (n = 4). *, overall P = 0.01. Statistical significance was determined using a Friedman test with Dunn´s multiple comparisons test.