Work

Generation of HLA-Restricted T-Lymphocyte Responses Against Recombinant Influenzavirus Hemagglutinin A/New Caledonia by an In Vitro Umbilical Cord Blood Model of Dendritic Cell Vaccination

Year: 2009

Type: article

Abstract: Introduction: Influenza infection following treatment with chemotherapy and/or concomitant radiation therapy remains a significant cause of morbidity among immuncompromised patients in the hospital setting. Further, such patients tend to seroconvert inefficiently and with suboptimal titers following prophylactic vaccination with either the standard or recombinant influenza vaccines. Toward the goal of addressing this issue, we have developed an in vitro model of dendritic cell (DC) immunotherapy utilizing DCs generated from umbilical cord blood (UCB). Methods: UCB monocytes were harvested by adherence, and UCB DCs were generated by incubation in GM-CSF and IL-4. Immature DCs were loaded with purified rHA protein [A/New Caledonia (H1N1)], matured with an inflammatory cytokine cocktail, and used to stimulate autologous T-lymphocytes. T-lymphocyte priming and development were supported by supplementation with IL-12, IL-2, IL-7, and IL-15. DC and T-cells were analyzed by flow cytometry. Antigen specific responses were documented by IFN-γ ELISpot, 51Cr lysis, and tetramer staining. Recombinant hemagglutinin HLA-restricted peptides were synthesized by Sigma-Genosys (The Woodlands, Texas). MHC class II DR15-restricted tetramer was synthesized by Beckman-Coulter (Fullerton, CA). Results: DCs were primarily of myeloid Langerhans phenotype (CD11c+, CD14-) and expressed high levels of CD209, HLA-DR and costimulatory molecules CD80, CD86, and CD83. Primed and expanded T-cells were predominantly CD62L+CCR7-, indicative of effector status; however, there were also small but distinct populations CD127+ and CCR7+ memory cells. Upon recall with HA-loaded autologous DC, a 4 to 10-fold increase in the number of IFN-γ producing T-lymphocytes was observed in comparison to T-cells stimulated with autologous unloaded DCs or allogeneic DCs loaded with irrelevant antigens. Antigen-specific T-cell functionality was determined by 51Cr lysis assay. Using a peptide library of predicted HA binding epitopes, we mapped an HA-specific, DR15-restricted CD4 T-cell epitope (GNLIAPWYAFALSRG) and documented tetramer-positive CD4+ cells in a DR15+ background. Conclusions: The model demonstrates that HA-specific, HLA-restricted immune responses may be generated from UCB lymphocytes and suggests that dendritic cell immunotherapy for the prevention of influenza might be feasible.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2008.12.274