The Soluble Form of CTLA-4 from Serum of Patients with Autoimmune Diseases Regulates T-Cell Responses
A soluble form of CTLA-4 is differently found in serum of patients with autoimmune diseases and normal donors. (a) A soluble form of CTLA-4 is found in serum of patients with different autoimmune diseases. The concentration of sCTLA-4 was evaluated by ELISA on sera collected from AIT (), CD (), and PBC patients () and a healthy donor group as a control (). Results are expressed as log of ng/mL. Each sample was diluted 1 : 10 and tested in triplicate. Deviation between triplicates was <10% for any reported value (as previously published [17, 21]). Lines indicate the mean values for each group. The differences among the different groups of patients and the healthy donors were determined by non-parametric Mann-Whitney Rank Sum test (). (b) Data from three independent experiments showing the capability of sCTLA-4 to inhibit anti-CD86 mAb immunoreactivity. The test was performed utilizing a THP-1 cell line expressing CD86 molecules. Three different representative sera from patients with autoimmune disease (21AIT negative, plot 1; 55AIT, plot 2, and 101CD, plot 3, positive for sCTLA-4; and 101CD-deprived sera, plot 4) were preincubated with THP-1 cells prior to stain cells with mAb to CD86. The addition of CTLA-4 Ig fusion protein was utilized as positive control of interference of specific mAb binding to CD86, plot 5. Thinner lines represent controls with a not reacting mAb, thicker lines represent the inhibitory effect of serum sCTLA-4 (and CTLA-4 Ig, plot 5) on anti-CD86 mAb immunoreaction, and shaded curves represent the immunostaining of untreated cells with anti-CD86 mAb. (c) The sera of 15 autoimmune patients with high concentrations of sCTLA-4 were depleted to show that sCTLA-4 is the protein of serum with functional activities (see Figures 3 and 4). Results are expressed as log of ng/mL. Each sample was diluted 1 : 10 and tested in triplicate. The arrow indicates the result of in vitro depletion of sCTLA-4 on 101CD serum (used also for the experiment shown in (b), plot 4).