Specific expansion of cytotoxic T cells by stimulation with long-term CD40-stimulated B cells.

<p>(A) Frequency of antigen-specific CD8+ T cells in four T cell lines after 33 days of repeated stimulation with peptide-loaded autologous long-term CD40 B cells. B cells had been cultured for 693 days at the beginning of the T cell stimulation. Either of four HLA-A2-restricted antigenic peptides derived from viral or melanoma antigens (abbreviated NLV, GLC, CLG, and ELA) was used for stimulation. The frequency of specific T cells was assessed by staining with the corresponding specific HLA/peptide multimer reagent. For control stainings, an HLA/peptide multimer containing an irrelevant peptide was used for staining (lower row): ELA for NLV-stimulated cultures and NLV for all other cultures. (B) Expansion of antigen-specific T cells after stimulation with peptide-loaded long-term CD40 B cells. Total cell numbers (top) in the T cell cultures were determined by microscopic counting. The proportion of antigen-specific CD8+ T cells, represented in linear (middle) and logarithmic scale (bottom), was determined by specific HLA/peptide multimer staining at various times of T cell culture. (C) The cytotoxic reactivity of two of these T cell cultures against cells endogenously presenting the target antigen was assessed in a calcein release assay. Top: T cells expanded with the peptide NLV (from CMV pp65) were tested against HLA-A2-matched mini-LCLs endogenously expressing pp65 or not, autologous CD40 B cells loaded with the target peptide NLV or the control peptide GLC, or K562 cells to test for natural killer-like reactivity. Bottom: T cells expanded with the peptide ELA from MelanA were tested against HLA-A2-matched melanoma cell lines either expressing MelanA (Mel624) or not (A375) that had either been additionally loaded with the ELA peptide or not. Further targets included autologous CD40 B cells loaded with the target peptide ELA or the control peptide NLV, and K562 cells.