Abstract
Adoptive T cell immunotherapy has demonstrated clinical efficacy in controlling reactivation of human herpesviruses (e.g., cytomegalovirus, CMV and Epstein-Barr Virus, EBV) that are known etiologic agents of life-threatening illness in immunocompromised populations. Early approaches utilized in the expansion of large numbers of human virus-specific CD4+ and CD8+ T cell often involved laborious culture techniques impractical for widespread clinical use in populations at risk, including stem cell transplant (SCT) recipients. Advances in our understanding of the biology of myeloid dendritic cells (DC) can now facilitate more practical approaches for ex vivo antigen-specific T cell expansion. These advances have included novel approaches to isolate human peripheral blood monocytes (e.g., CD14 selection) and the optimization of cytokine cocktails inducing DC maturation. To define the optimal culture conditions for human antigen-specific T cell expansion, we conducted an iterative series of pairwise CMV-specific T cell expansions, in which important determinants of the expansion process could be directly compared. T cells, derived from apheresis products obtained from CMV-seropositive healthy donors, were expanded under GLP-compliant conditions, following incubation with autologous myeloid DC pulsed with overlapping pentadecapeptide pools spanning the CMV pp65 protein. In each case, we assessed the expanded CMV-specific T cell populations by immunophenotyping of the generated DC, and by phenotypic analysis, cytokine flow cytometry (CFC), ELISPOT analysis and HLA-peptide tetramer staining of the expanded T cells. Specifically, we compared:DC generated from monocytes isolated via plastic adherence to those isolated via positive selection of CD14+ T cells using magnetic beads;DC matured using tumor necrosis factor-α (TNFα) alone or in combination with other cytokines (ITIP, containing TNFα, IL-1β, IL-6 and PGE2);the quality of expansions derived from a starting lymphocyte population selected by non-adherence to plastic vs. the CD14-negative fraction in the monocyte selection process.From this series of experiments, we can conclude that while CD14 selection of monocytes results in a more phenotypically homogeneous population of DC, that this does not improve the quantity or quality of expanded virus-specific T cells. When we compared TNFα to ITIP maturation of DC, we found that while ITIP induced significantly higher CD83 expression on DC, TNFα-matured DC favored the maintenance of CMV-specific CD4+ T cells in culture resulting in ultimately greater numbers and functional proportions of tetramer-stained CD8+ T cells. Finally, we consistently found that the use of non-adherent PBMC resulted in dramatically better quantitative expansions of CMV-specific CD4+ and CD8+ T cells, vs. a lymphocyte population obtained following depletion of monocytes using CD14 positive selection. These results suggest a set of optimal T cell/DC co-culture conditions that lead to the generation of the largest numbers of expanded, functional CD4+ and CD8+ T cells, and are likely to have relevance for human trials wherein adoptive immunotherapy and/or DC immunization is planned.
Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns