Blocking CD28 or CTLA-4 counter-receptors in combination with B7-1 or B7-2. PBMCs (1.5 × 10⁵/well) from five healthy subjects and five patients with type 1 diabetes were stimulated with 10 μg/ml GAD65 in the presence or absence of each one of the blocking Ab’s or with different combinations of Ab’s. Ten replicates were established for each condition and harvested after 10–12 days of culture, and counts per minute per well were determined. Each column represents the mean percentage of inhibition of proliferation to GAD65 in the presence of single blocking Ab’s or combinations of them in five patients with recent onset of type 1 diabetes and five normal control subjects. As shown previously in Figures 2 and 3, the blockade of CD28, B7-1, or B7-2 caused strong inhibition of proliferation in normal subjects. (a and b) Blocking both B7-1 and B7-2 molecules at the same time induced a more significant decrease of the response than blocking just one of the molecules alone in both patients and controls. While anti-CD28 in combination with anti–B7-2 had almost no effect on proliferation or cytokine secretion (data not shown), anti-CD28 in combination with anti–B7-1 induced significant decreases of responses to GAD65. In contrast, coblockade with anti–CTLA-4 and anti–B7-1 enhanced T cell proliferation, while anti–CTLA-4 in combination with anti–B7-2 suppressed GAD-specific response. While anti-CD28 induced significantly more suppression of GAD responses in the normal subjects, the effects of combining the B7-1, B7-2, CD28, and CTLA-4 F(ab′) mAb’s were not different as compared with patients with diabetes.