Abstract
Background: Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells capable of priming effective T-cell responses. This enhanced ability to prime immune responses, coupled with the recent development of clinical grade techniques for obtaining large numbers of human DCs, make it possible to use them for therapeutic vaccination after antigen loading and maturation. Although the exact mechanisms by which DCs modulate immunity are still under investigation, it is obvious that important parameters for determining DC-based immunotherapy efficacy include their maturation status and ability to elicit primary responses.
Methods: We compared the immune function of DCs generated and matured in culture plates (pla-DCs) with those generated and matured in clinical grade bags (bag-DCs). The maturation cocktail was composed of Poly I:C, IL1β, IFNα, IFNγ, and TNFα. We first analyzed the impact of the culture conditions on the phenotype, secretion of bioactive IL-12 and its kinetics during the maturation process, and the capacity to prime in vitro naïve T-cells against HIV-gp41 protein in seronegative donors. We also investigated immature and mature bag-DCs and pla-DCs, using molecular techniques (EMSA, Western Blot and micro-arrays).
Results: although mature bag-DCs displayed a mature phenotype, they were unable to secrete significant amounts of IL-12 p70 and to initiate primary immune reactions against HIV-gp41, at the opposite of pla-DCs. Furthermore the addition of exogenous IL-12 p70 could not restore their functionality. Molecular analyses performed on immature DCs showed that bag-DCs were already in the beginning of a maturation process, as demonstrated by activation of NFκB, and the Akt pathway. Surprisingly, we found the β-Catenin pathway already activated (known to induce a maturation inefficient for the generation of TH1 response). A micro-array analysis demonstrated that receptors for the maturation cocktail component were under-expressed in immature bag-DCs. When analyzing mature DCs, we found the expression of several genes involved in DCs capacity to induce responses to be downregulated in bag-DCs. Apart from IL-12 p35 and p40 under-expression, other genes crucial for DC function, including chemokines, co-stimulatory and adhesion molecules, were also under-expressed. EMSA and Western blot assays showed that in mature pla-DCs, NfκB and the Akt pathway were normally activated, but not the β-Catenin pathway. Futhermore, in mature DCs, though MAPK p38 was activated in both DCs type, the Mek/Erk pathway was activated only in bag- DCs. It is worth mentioning that the same phenomena occurred when using CD14-positive purified cells, which rules out a possible role of contaminating PBMCs in bags.
Conclusion: we conclude that culture conditions have an important impact on DCs functionality and that results obtained with pla-DCs cannot be directly transferred to bag-DCs. Our results strongly suggest that the differentiation of monocytes-derived DCs in bags lead to the generation of immature cells, which are already engaged in an inefficient type of maturation, together with a decreased expression of genes involved in the response to the maturation cocktail, which in turn results in mature DCs presenting with defects in the expression of genes crucial for the induction of primary TH1-type responses.
HIV gp41