Antigen-specific CD8⁺ T lymphocytes are maintained in cultures of anti-CD3– and anti-CD28–stimulated PBLs.

(A) Prevention of CMV-specific CD8⁺ T-cell loss by CD28 costimulation after 12 to 14 days of culture. PBLs (■) from CMV-seropositive donors were stimulated with anti-CD3 (▪) or both anti-CD3 and anti-CD28 (░), and frequency of HLA A* 0201 CMV_pp65 tetramer⁺CD3⁺CD8⁺ cells was assessed as described in “Materials and methods.” Data are expressed as percentage of tetramer⁺ cells in CD3⁺CD8⁺ cells from fresh PBLs. Panels C-E indicate staining with HLA A* 0201 CMV_pp65 tetramer-PE. Human PBLs (panel C) and LV'VFas⁺ T cells (panel D) grown with anti-CD3 and anti-CD28 were stained with PE-coupled HLA A* 0201 CMV_pp65 tetramer, anti-CD3 and anti-CD8 mAbs, or isotype-matched control mAbs (panel B) and evaluated by flow cytometry. Values indicate the percentage of HLA-peptide tetramer⁺cells in CD3⁺CD8⁺ T cells. The results are representative of assays from 6 donors. Panels E-G indicate staining of cell-associated IFN-γ production after antigen stimulation. PBLs (panel F) and LV'VFas⁺ T cells (panel G) from the same donor were incubated for 6 hours with CMV_pp65peptide-pulsed T2 cells or T2 cells alone (panel E) and stained with FITC-coupled anti–IFN-γ mAb, anti-CD3 mAb, and anti-CD8 mAb. Cells were gated to identify CD3⁺CD8⁺ T cells, and assessed for cytokine production. Values indicated the percentage of cells producing the relevant cytokine.