Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured in
GM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are the
most potent APCs known. These DCs have many features of primary DCs, including the
expression of molecules that enhance antigen capture and selective receptors that guide
DCs to and from several sites in the body, where they elicit the T cell mediated immune
response.
For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC)
with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials of
different cancers.
However, the efficacy of DCs in the development of immunocompetence is critically
influenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the time
of exposure of the tumor antigens used for loading in the presentation phase.
The aim of the present study was to create instruments to acquire more information about
DC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbased
vaccine.
In particular, two different tumor antigen were studied: the monoclonal immunoglobulin
(IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanoma
tissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate the
kinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetric
and fluorescence microscopy analysis, respectively.
iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours after
loading. By the cytofluorimetric analysis it was observed that the monoclonal antibody was
completely captured after 2 hours from pulsing, and was decreased into mDC in 5 hours
after maturation stimulus.
To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, then
were monitored in the 2h to 22 hours interval time after loading.
Then, to reveal difference between increasing lysate concentration, iDC were loaded with
20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h from
pulsing.
By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8
hours loading was completed reaching a plateau phase. For 200 and 400μg the mean
fluorescence of cells increased until 13h from pulsing.
The lysate localization into iDC was evaluated with conventional and confocal
fluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensive
and diffuse fluorescence was observed within the cytoplasmic compartment. Moreover,
after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shaded
area with a typical polarized aspect. In addition, small fluorescent spots clearly appeared
with an increment in the number and fluorescence intensity.
The nature of these spot-like formations and cloudy area is now being investigated
detecting the colocalization of the fluorescence lysate and specific markers for lysosomes,
autophagosomes, endoplasmic reticulum and MHCII positive vesicles.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:692 |
| Date | 06 June 2008 |
| Creators | Ancarani, Valentina <1978> |
| Contributors | Neyroz, Paolo |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
Page generated in 0.0023 seconds