Generation of CMV-Specific T-Lymphocytes for Adoptive Immunotherapy: A Comparison of Artificial Antigen-Presenting Cells and Autologous Dendritic Cells Pulsed with CMV-pp65 Protein-Spanning Pentadecapeptide Pools.

Adoptive transfer of virus-specific T-cells constitutes a promising approach for the treatment and prevention of cytomegalovirus (CMV) infections complicating allogeneic HSC transplants. Current strategies employing peptide-pulsed donor-derived dendritic cells (DCs) for antigen presentation are effective, but limited by availability of adequate numbers of DCs and the time required for their generation. We established a series of immediately accessible, replenishable, artificial antigen-presenting cells (AAPCs), consisting of mouse 3T3 cells transduced to express human B7.1, LFA-3, ICAM-1, β₂-microglobulin and one HLA class I allele (Papanicolaou et al., Blood 2003). We then compared yields of CMV-peptide-specific T-cells when T-cells from HLA A0201 seropositive donors were sensitized with autologous DCs or HLA A0201 AAPCs loaded with a known HLA A0201-binding immunogenic nonamer of CMV-pp65 (NLVPMVATV) or a pool of pentadecapeptides spanning CMV-pp65, which included the 15-mer No. 123 (AGILARNLVPMVATV) containing this epitope. Peptide loading was performed in the presence or absence of serum in order to distinguish peptide editing mediated by ectopeptidases in the serum or expressed by the different APCs. Results from repeated experiments employing three HLA A0201 CMV-seropositive donors demonstrated that the yields of epitope-specific T-cells following 14–28 day sensitization with AAPCs loaded with the NLVPMVATV nonamer consistently exceeded those generated in response to peptide-loaded autologous DCs, as assessed by enumerating peptide-HLA A0201 tetramer-binding and peptide-specific IFN-γ producing T cells. High and selective yields of NLV-specific T-cells were obtained when the T-cells were sensitized with AAPCs or DCs loaded with the CMV-pp65-spanning pool of 15-mers in the presence or absence of serum suggesting that ectopeptidases expressed on the DCs and AAPCs appropriately clip and edit the 15-mers after binding to HLA A0201. Even higher yields were achieved when the T-cells were sensitized with AAPCs transduced to express the full length CMV-pp65 protein, confirming the potential of the mouse-derived AAPCs to process this protein and load immunogenic epitopes on HLA A0201. T-cells generated in response to peptide-loaded or transduced AAPCs also specifically lysed nonamer-loaded HLA A0201 expressing targets. Taken together, these studies indicate that mouse-derived AAPCs can efficiently process endogenous protein and edit exogenous 15-mer peptides for binding and presentation by the single expressed HLA allele. Use of these AAPCs loaded with overlapping peptides or transduced to express an immunogenic protein may thus provide advantages in terms of immunogenicity, immediate accessibility and broad applicability for rapid production of antigen-specific T-cells for adoptive immunotherapy.