Identification of CRA as an immunosuppressive compound. (A) Two-way allo-MLR microcultures were tested in the presence of vehicle alone (0.1% DMSO; left panel) or test compounds (2 μM; right panel). Each dot represents the actual level of ³H-thymidine uptake observed in each coculture, and the mean value observed in the vehicle treatment group is shown with a bold line. Twenty compounds reducing T-cell proliferation below the cut-off level (the mean − 2 SD in the vehicle-treated group) were considered as positive in the first screening. The arrow indicates the impact of CRA. (B) The chemical structure of CRA. (C) T cells purified from C57BL/6 mice were cultured with CRA at the indicated concentrations in the presence (●) or absence of allogeneic BM-DCs (○). (D) CD4⁺ T cells purified from the DO11.10 TCR transgenic mice and BM-DCs were cocultured with the indicated concentrations of CRA in the presence of OVA_323-339 peptide (●) or vehicle alone (○). Data shown are ³H-thymidine uptake on day 4 (mean ± SD, n = 3). (E) Mononuclear cell fractions isolated from unrelated donors were cocultured (circles) or cultured independently (triangles) in the presence of CRA in the indicated concentrations. Data shown are the means ± SD (n = 3) of ³H-thymidine uptake on day 5 (open symbols) and day 7 (closed symbols). Asterisks indicate statistically significant (**P < .01) differences compared with the vehicle-treated control samples.