Generation of influenza recombinant haemagglutinin-specific lymphocytes from cordblood-derived dendritic cells: immunotherapy model from an unprimed source
Abstract number: P765
Safdar A., Decker W., Shpall E.
Background: Cordblood-derived (CB) is an important source of mostly unprimed stem cells. We sought to generate CB-derived dendritic cells (DC) immunotherapy against influenzavirus.
Methods: Recombinant hemagglutinin (rHA) was expressed in insect (SF9) cells by recombinant baculovirus. The HA gene of influenza A New Caledonia/20/99 (H1N1) influenza virus was independently cloned into the plasmid baculovirus expression vector pPSC12. Generation of Immature DCs. Umbilical cord blood units discarded from the MDACC CB bank were used after IRB approval. DC Loading and Maturation. After six days of culture in GM-CSF and IL-4, immature dendritic cells were loaded with rHA protein. Immature dendritic cells were suspended at a concentration of 4×107 cells/ml, mixed with rHA and incubated for 10 minutes on ice in an electroporation cuvette. T-cell Priming and Re-Stimulation. Upon maturation of loaded DCs, they were incubated at a ratio of 1:10 with autologous non-adherent PBMCs (typically 50% CD3+), plus IL-12. ELISpot Assay. Typically, 104 to 105 lymphocytes from each culture were mixed at a ratio of 10:1 with thawed dendritic cells, plated in triplicate on anti-IFN-gamma coated ELISpot plates (BD Biosciences) and cultured overnight (1218 hours). ELISpots for with putative HA peptide epitopes were performed similarly using 1×1052×105 lymphocytes incubated overnight in 3.75 mg/ml peptide.
Results: rHA-specific lymphocytes demonstrate identifiable HLA-restriction. As demonstrated by Figure, HA-primed lymphocytes (HLA DRbeta1*1503) from a different CB demonstrated a nine-fold increase in statistically large spots when restimulated with the DR15262 epitope (p < 0.00002). These data suggest that 1 in 1,900 of the HA-specific T-cells were DR15262 restricted in a highly-specific fashion. Total ELISpot numbers (p < 0.0002) and total IFN-gamma release (p < 0.00004) between lymphocytes restimulated with DR15262 and the control peptide were statistically distinct as well. Incubation of peptide DR15262 in conjunction with HA-primed/DR15- lymphocytes or in conjunction with adenoviral hexon-primed (irrelevant) lymphocytes did not demonstrate significant numbers of IFN-g spots.
Conclusions: The results demonstrate that, despite the generally naive of CB lymphocytes, influenza HA-specific responses can be generated ex vivo and could be potentially be used to enhance immune reconstitution following allogeneic stem cell transplantation.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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