A Panel of Artificial Antigen-Presenting Cells (AAPC) Expressing Prevalent HLA Alleles Permits Generation of Cytotoxic T Cells Specific for Both Subdominant and Dominant Viral Epitopes for Adoptive Therapy.

Adoptive transfer of antigen specific T cells can be effective in treating viral infections complicating allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, in practice, generation of T cells is often limited by insufficient supply of autologous antigen presenting cells; therapeutic activity in HLA disparate patients may also be impaired if the immuno- dominant T cells generated are restricted by HLA alleles not shared by the host. AAPCs have theoretical advantages for T cell therapies in terms of sustained supply and capacity to selectively stimulate T cells restricted by HLA alleles shared by donor and host. However, to date, only AAPC systems expressing HLA A*0201 have been characterized. Accordingly, we established a panel of AAPC consisting of NIH 3T3 mouse fibroblast cells, each transduced to express β2- microglobulin and a prevalent HLA class-I allele, specifically HLA A*0201, A*0301, A*2402, B*0702, B*0801 or C*0401, as well as the human co-stimulatory molecules B7.1, LFA-3 and ICAM-1. Novel promotor sequences were introduced to secure stable high expression of the allele on the AAPCs. Sensitization of T cells from seropositive donors with AAPCs expressing each of these alleles (4-8 donors/allele), either loaded with overlapping 15-mer peptides spanning the CMVpp65 sequence or transduced to express the CMV pp65 protein, resulted in 12-35 fold expansions of CD8 + T cells exhibiting CMV pp65 epitope-specific, HLA restricted activity, as quantitated by peptide -HLA tetramer binding, epitope specific production of interferon gamma, and cytotoxic activity against peptide loaded or CMV infected targets. Although both peptide pool loaded and transduced AAPCs induce CMV pp65 epitope specific T cells, yields were higher when transduced AAPCs were employed. In studies of T-cells from 5 donors when sensitized with either peptide pool loaded autologous dendritic cells (DC) or HLA sharing AAPCs, sensitization with DC selectively induced T-cells specific for 1-2 immunodominant CMV pp65 epitopes. In contrast, while sensitization with a panel of peptide loaded or transduced AAPCs expressing shared HLA alleles elicited responses to the same dominant epitopes, we could also regularly generate comparable cytotoxic T cell responses to subdominant epitopes which were either not produced or only present at low frequencies in T cells sensitized with autologous DC. Thus, this panel of AAPCs stably expressing a series of HLA alleles which, in aggregate, are detected in 70% of the patients referred for HSCT, can be employed for rapid generation of CMV-pp65 specific T cells of desired HLA restriction for adoptive therapy.