Isolation and characterization of mHag-specific CD4⁺ T-cell clones. (A) Activated (HLA-DR⁺) CD4⁺ T cells were single cell sorted from bone marrow cells obtained from the patient 5 weeks after DLI by flow cytometry. Indicated are the percentages of bone marrow cells expressing CD4 (3%) and CD4⁺ cells expressing HLA-DR (28%). A total number of 106 CD4⁺ T-cell clones were growing out of 1100 single sorted cells. (B) Sixteen CD4⁺ T-cell clones were directed against unknown mHags based on differential recognition of patient (pat) and donor (don) Epstein-Barr virus transformed B-cell lines (EBV-LCLs). Mean release of IFN-γ (ng/mL) in 50-μL supernatants of duplicate wells is shown. (C) All CD4⁺ T-cell clones recognizing unknown mHags and releasing > 250 pg/mL IFN-γ upon incubation with patient EBV-LCLs were tested for specific lysis of patient and donor EBV-LCLs in a 10-hour ⁵¹Cr release assay. Mean specific lysis of triplicate wells at effector-target ratios of 10:1 is shown. (D) CD4⁺ T-cell clones were tested for cytokine release by multicytokine ELISA. Release of IL-2, IL-5, IL-13, and IFN-γ in 50-μL single supernatants is shown (ng/mL). CD4⁺ T-cell clones did not produce IL-4, IL-6, IL-10, IL-12, IL-17A, TNF-α, granulocyte CSF, and transforming growth factor β1 (data not shown).