Phenotypic and Functional Studies of Dendritic Cell Vaccines.

摘要

Recent investigations have confirmed that circulating monocytes are recruited to sites of infection, where they differentiate into immature dendritic cells (iDCs) in situ and contribute to the generation of an immune response. Monocyte-derived DCs (moDCs; hereafter referred to as 'DCs') are responsive to oxygen concentration, and as a consequence of their role in antigen acquisition and presentation, are exposed to pathology-induced low oxygen environments throughout the course of their functional lifespan. We studied the effect of oxygen concentration on the phenotypic and functional characteristics of monocyte-derived DCs throughout the DC generation and maturation phases.;Results The data from studies described herein demonstrates that contrary to our prediction, generating DCs in a hypoxic environment attenuates their immunostimulatory potential. After optimizing the culture conditions for our assays and defining the limits of hypoxia and hyperoxia, several parameters of DC functionality were investigated. It was first established that when cultured under hypoxic conditions, DCs were less immunostimulatory in MLR experiments than when they were generated and matured at 20% or 40% O 2. It is predicted that phenotypic characteristics of hypoxia-generated DCs contributed to this observation. In our phenotypic studies of AOE-generated DCs, this hypothesis was proved to be true. Not only were hypoxia-generated DCs less viable than DCs cultured at 20% or 40% O2, but they exhibited distinct differences in their immunophenotypic profile that could explain their attenuated ability to stimulate allogeneic T cells in MLR. These differences were imparted on DCs during the differentiation phase, and not during the maturation phase of DC generation. Furthermore, hypoxia drove DCs to produce a different profile of immunomodulatory cytokines than DCs that were generated and matured at 20% or 40% O2. In this regard, hypoxia-generated DCs produced lower levels of chemotactic factors for leukocytes, and instead produced a cytokine milieu that promoted the survival and differentiation of myeloid cells.;Additionally, it was demonstrated that while hypoxia-generated DCs participated in the uptake of particulate pathogen faster than DCs generated at 20% or 40% O2, that ultimately, a smaller percentage of hypoxia-generated DCs were competent in phagocytosis than their counterparts at 20% or 40% O 2. In DCs that were targeted with fluorescently-labeled fusion proteins (FP; B11 or 3G9), the average rate at which AX647-FP was degraded in mDC that had been incubated at 5% O2 was significantly different from the average rate at which AX647-FP was degraded in mDCs that were incubated at 20% or 40% O2. It is predicted that hypoxia affects the manner in which antigen is degraded in B11-FP or 3G9-FP targeted DCs. In almost every measurement that was made, the effect of hyperoxia on DCs was negligible, as compared with DCs that were generated at atmospheric oxygen concentrations; despite the fact that a 1.8-fold increase was measured in the [O2] in CM incubated at 40% atmospheric oxygen (14.7% O2 in CM), versus CM incubated at 20% atmospheric O2 (8.3% O2 in CM).;The observation of prolonged antigen persistence (.48 hours) of AX647 3G9 FP in AOE-generated DCs was unexpected, regardless of their maturational status. Conversely, based on previous data from DCs generated at 20% O 2, this characteristic of AX647 B11-pulsed AOE-generated DCs was confirmed in this study. Targeting antigen to either CD205 or CD206 via 3G9 or B11 FP respectively, resulted in the prolonged retention of targeted antigen within discrete endosomal punctae; which based on hypotheses from antecedent reports in the literature, may contribute to enhanced cross presentation in FP-pulsed DCs. Functional studies of cross presentation will need to be performed in order to prove our hypothesis. Interestingly, mDCs cultured in hypoxia degraded targeted antigen at an accelerated rate, as compared with DCs that were matured at 20% or 40% O2, which may be due to modified cellular metabolism or perturbed endosomal trafficking at low oxygen concentrations. The localization of internalized FP that is targeted to DCs is further studied in Chapter 2 of this disquisition. (Abstract shortened by UMI.)