Post-G CD4⁺ T cells were activated with HLA-DR-mismatched TCD PBMCs in the presence or absence of 1 μg/mL tacrolimus, 50 μg/mL cyclosporin A, 5 μg/mL human or mouse chimeric anti-TNF antibody infliximab, 1 nM methotrexate, 10⁻⁶ M dexamethasone, or 0.1 μM mycophenolate mofetil. After 7 days of primary culture, supernatants were harvested for cytokine ELISA, and post-G CD4⁺ T cells were plated in secondary Transwell cultures separated from indicator (autologous pre-G) CD4⁺ T cells obtained from the same HSC donor. Both compartments of the Transwell received allogeneic TCD PBMCs as stimulator cells and responder cells at a 1:3 ratio. Proliferation of indicator CD4⁺ T cells in the lower chamber of the Transwell was determined after 7 days by the addition of BrdUrd for the final 24 hours. Data represent median values recorded in 4 independent experiments performed in duplicate. BrdUrd incorporation by pre-G CD4⁺ T cells cocultured in the absence of suppressor post-G CD4⁺ T cells in the Transwell approached 30% in all experiments (data not shown). Pre-G CD4⁺ T cells released comparable cytokine amounts after activation and exerted no measurable suppression of indicator T cells (data not shown).

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