Cytokine production and regulatory activity of CD4+ T cells from patients who received allografts
| . | Activation stimulus (anti-CD3 mAb + TPA) . | Transwell coculture . | |||||
|---|---|---|---|---|---|---|---|
| IL-10, pg/mL−1 . | IL-10, % . | TGF-β1, pg/mL−1 . | TGF-β1, % . | IFN-γ, pg/mL−1 . | IFN-γ, % . | Lower chamber, % (indicator CD4+ T cells) . | |
| Control 1 | 148 | 4.5 | 378 | 3.0 | 5617 | 20 | 20 |
| Control 2 | 280 | 6.3 | 352 | 3.0 | 7700 | 25 | 27 |
| Patient 1 | 82 | 4.3 | 383 | 1.9 | 9518 | 28 | 23 |
| Patient 2 | 61 | 4.0 | 436 | 3.0 | 5815 | 16 | 16 |
| Patient 3 | < 10 | < 1.0 | 70 | < 1.0 | 80 | 3 | 24 |
| . | Activation stimulus (anti-CD3 mAb + TPA) . | Transwell coculture . | |||||
|---|---|---|---|---|---|---|---|
| IL-10, pg/mL−1 . | IL-10, % . | TGF-β1, pg/mL−1 . | TGF-β1, % . | IFN-γ, pg/mL−1 . | IFN-γ, % . | Lower chamber, % (indicator CD4+ T cells) . | |
| Control 1 | 148 | 4.5 | 378 | 3.0 | 5617 | 20 | 20 |
| Control 2 | 280 | 6.3 | 352 | 3.0 | 7700 | 25 | 27 |
| Patient 1 | 82 | 4.3 | 383 | 1.9 | 9518 | 28 | 23 |
| Patient 2 | 61 | 4.0 | 436 | 3.0 | 5815 | 16 | 16 |
| Patient 3 | < 10 | < 1.0 | 70 | < 1.0 | 80 | 3 | 24 |
CD4+ T cells were purified from 2 healthy cytokine-untreated subjects (ctrl) and from 3 patients with leukemia after transplantation with G-CSF–mobilized peripheral blood HSCs from HLA-identical siblings. Patients 1 and 2 showed no clinical signs of GVHD and were on prophylactic cyclosporin A at testing (days 105 and 30 after grafting, respectively). Patient 3 had acute GVHD at testing (day 38 after grafting) and was receiving immunosuppressive therapies, according to standard clinical practice (mycophenolate mofetil and methylprednisolone). To detect cytokine production, purified CD4+ T cells were activated with 0.1 μg/mL anti-CD3 mAbs and 10 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA; both from Sigma Chemical) for 24 hours at 37°C. Culture supernatants were collected and used for cytokine ELISA, whereas cells were used for the flow cytometric evaluation of cytokine production and for Transwell studies, as already detailed. 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 from a healthy control was determined after 7 days by the addition of BrdUrd for the final 24 hours. Data represent median values recorded in 2 experiments performed in duplicate.