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  3. Medizin - Open Access LMU - Teil 15/22 Podcast
  4. Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70
Background: For optimal T cell activation it is desirable that
dendritic cells (DCs) display peptides within MHC molecules as
signal 1, costimulatory molecules as signal 2 and, in addition,
produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses
towards CD4(+) T helper 1 cells, which then support the development
of CD8(+) cytotoxic T lymphocytes. We therefore developed new
maturation cocktails allowing DCs to produce biologically active
IL-12p70 for large-scale cancer vaccine development. Methods: After
elutriation of leukapheresis products in a closed bag system,
enriched monocytes were cultured with GM-CSF and IL-4 for six days
to generate immature DCs that were then matured with cocktails,
containing cytokines, interferon-gamma, prostaglandin E2, and a
ligand for Toll-like receptor 8, with or without poly (I: C).
Results: Mature DCs expressed appropriate maturation markers and
the lymph node homing chemokine receptor, CCR7. They retained full
maturity after culture for two days without maturation cocktails
and following cryopreservation. TLR ligand stimulation induced DCs
capable of secreting IL-12p70 in primary cultures and after one day
of coculture with CD40L-expressing fibroblasts, mimicking an
encounter with T cells. DCs matured with our new cocktails
containing TLR8 ligand, with or without poly (I: C), induced
alloresponses and stimulated virus-specific T cells after
peptide-pulsing. DCs matured in cocktails containing TLR8 ligand
without poly (I: C) could also be loaded with RNA as a source of
antigen, whereas DCs matured in cocktails containing poly (I: C)
were unable to express proteins following RNA transfer by
electroporation. Conclusion: Our new maturation cocktails allowed
easy DC harvesting, stable maturation and substantial recoveries of
mature DCs after cryopreservation. Our procedure for generating DCs
is easily adaptable for GMP-compliance and yields
IL-12p70-secreting DCs suitable for development of cancer vaccines
using peptides or RNA as sources of immunizing antigens.

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