Peripheral blood lymphocytes (PBLs) from 3 recipients ,M8907: d821 pBMT (A), M6007: d930 pBMT (B), and M8314: d261 pBMT (C), in the tolerant (TOL) group were labeled with CFSE and cultured with irradiated donor or third-party PBLs. After 5 days of culture, CD4⁺CSFE^low (proliferating) and CD4⁺CSFE^high (nonproliferating) cells were sorted and added at a varying ratio into the recipient T cells in the presence of CD3/CD28 bead stimulation. (A and B) A dose-dependent suppression of T cell activation was observed by donor-primed proliferating (CSFE^low) cells in both recipients. Although less significant, third-party-primed proliferating cells of a recipient (M8907) also showed a dose-dependent suppression of T cell activation (A). However, such a dose-dependent suppression by the third-party-primed cells was not observed in the other recipient (M6007) (B). Nonproliferating cells primed with either donor or third-party antigens failed to suppress T cell activation. (C) To evaluate whether this suppressive function of the donor-primed CD4⁺ proliferating cells depends on cell-to-cell contact, isolated CD3⁺ cells from a TOL recipient (M8314) stained with CFSE were cultured with CD3/CD28 beads. Donor-primed proliferating CD4⁺ (CSFE^low) cells were added directly (contact) or seeded onto Transwell permeable support cell culture inserts (no contact). Since CD4⁺ cells consisted of both responder CD4⁺ cells and modulator CD4⁺ cells in this experiment, T cell activation was evaluated by CD8⁺ cell proliferation. The ratio of responder to modulator was 1 to 1. Although inhibition of CD8⁺ cell activation was observed by cell-to-cell contact, such inhibition was not observed in the Transwell system, which suggested that direct cell-to-cell contact is required for suppressive function of donor-primed modulator cells. pBMT, post bone marrow transplantation.