Ex vivo regulatory functions of 9 bovine cell populations in a co-culture assay. As a read out system (grey bars) proliferation of 35 000 CD4⁺CD25^- Tresp cells (population R1 in Fig. 1, indicated as R along the Y-axis), isolated from peripheral blood (cow A as a representative example) combined with 70 000 irradiated CD4^-CD25^- APC (population R5 in Fig. 1, indicated as A along the Y-axis) is shown. In white bars control results of co-culturing 30 000 (indicated as Reg 1 along the Y-axis), 60 000 (indicated as Reg 2 along the Y-axis) or 90 000 (indicated as Reg 3 along the Y-axis) potential regulatory cells (a) CD4⁺CD25^high, (b) CD4⁺CD25^low, (c) CD8⁺, (d) NKp46⁺, (e) WC1⁺, (f) W15B (WC1⁺ T cell line), (g) WC1.1⁺, (h) WC1.2⁺ and (i) CD14⁺ monocytes combined with 70 000 irradiated CD4^-CD25^- APC are shown. In black bars results are shown of co-cultures with the 9 potential regulatory cells (30 000–90 000 cells indicated as Reg 1-3 along the Y-axis) in combination with CD4⁺CD25^- Tresp (R) and irradiated CD4^-CD25^- APC (A). The data are representative experiments and are presented as dose dependent (30 000, 60 000 and 90 000) potential Treg proliferation or ratio dependent (potential Treg: Tresp = 0.9:1, 1.7:1 and 2.6:1, R1-R3:R) as the mean of proliferation on day 5 (+ 1 SD) in cpm. The stimulus used was plate-bound anti-bovine CD3 at a sub maximal concentration of 3 μg/mL. (p values reflect comparison of R1 + R (2.6:1) + A versus R + A, ** p ≤ 0.01).