Generation of Influenza HA-Specific Lymphocytes from Autologous Umbilical Cord Blood-Derived Dendritic Cells.

INTRODUCTION: Umbilical cord blood transplantation (CBT) is being performed with increasing frequency for patients lacking HLA-matched donors. CBT is associated with delayed immune reconstitution and high rates of viral infections. Particularly problematic is the reactivation of latent viral infections such as CMV, EBV, and VZV, as is overt infection from common agents like adenovirus and influenza. To address these issues, we proposed to generate influenza-specific CB T-cells ex vivo for potential cell therapy applications, allowing for effective therapeutic interventions until such time that immune reconstitution has occurred.

METHODS: Dendritic cells (DC) were generated from both the adherent and the CD14⁺ CB fractions cultured in AIM-V medium supplemented with human serum, GM-CSF, and IL-4. Antigen loading was performed by incubation with recombinant, purified A/New Caledonia HA antigen. Maturation was performed with the ITIP inflammatory cocktail (IL-1β, TNF-α, IL-6, and PGE₂). Autologous CB lymphocytes were co-cultured with mature DC at a ratio of 10:1 for 4 days in IL-12 after which they were expanded in a cytokine cocktail of IL-2, IL-7, IL-12, and IL-15. Lymphocytes were restimulated similarly every 7-10 days. Antigen specificity was demonstrated by IFN-γ ELISpot.

RESULTS: Mature DC expressed the appropriate markers including CD80, CD83, CD86, CD209, and HLA-DR. Some DC expressed CD14, others CD11c, an indication of a mixed interstitial/Langerhans myeloid phenotype. We demonstrated a 4–10 fold enhancement of IFN-γ ELISpots when primed lymphocytes were incubated with HA-loaded DC in comparison to autologous unloaded DC (p<0.003) or loaded allogeneic DC (p<0.0001). The CD8 content of the HA-specific lines was typically greater than 50% after two stimulations. At the time of phenotyping CD3⁺ cells were primarily CD62L^posCD197(CCR7)^neg and CD127(IL-7Rα)^neg, an effector phenotype; however, 6–22% of cells expressed CD127 (effector or central memory), and up to 6% of cells were CD62L^posCD197^pos (central memory).

CONCLUSIONS: The results demonstrate that, despite the generally naïve nature of CB lymphocytes, influenza-specific responses can be generated ex vivo, and could potentially be used clinically in CBT patients who develop infectious complications prior to immune reconstitution.