Immortalized HLA Class I Negative Daudi B Cells Coated with HLA/Peptide Monomers Promote Efficient In Vitro Expansion of Antigen Specific CD8+ T Cells.

Monocyte derived dendritic cells (DCs) have historically been used to stimulate antigen specific T cells in vitro for both experimental and clinical applications. However, this is a time consuming procedure that is restricted by the limited number of DC precursors within peripheral blood. We developed an alternative source of antigen presenting cells (APCs) exploiting the immortalized HLA class I negative human B cell line Daudi. A fusion protein comprising the variable region of an anti CD20 antibody linked to streptavidin was used to target recombinant biotinylated HLA class I peptide complexes to Daudi cells via the cell surface antigen CD20. These alternative APCs were used to induce in vitro proliferation of memory T cells specific for Cytomegalovirus (CMV). CD8+ T cells from an HLA-A*0201 positive, CMV positive individual were stimulated with irradiated Daudi cells coated with HLA-A*0201 monomers presenting a peptide epitope from the CMV coat protein pp65. Staining with fluorescent-labelled HLA class I tetramers showed a 40-fold expansion of pp65 peptide specific CD8+ T cells after 14 days of culture that was comparable to expansion produced by stimulating with peptide antigen presented by DCs. The alternative APCs were also used to induced proliferation of allo-restricted peptide specific CD8+ T cells from an allo-naive individual yielding a population in which 78% bound tetramers representing the HLA-A*0201/peptide combination used for stimulation. Functional specificity of the expanded CD8+ T cells was demonstrated using HLA class I monomer coated Daudi cells as targets in cytolytic killing assays and to stimulate cytokine production that was measured by intracellular staining. Our results show these easy to prepare novel APCs induce efficient in vitro expansion of functional peptide specific CD8+ T cells.