Abstract Book 2010 - CIMT Annual Meeting

035 Voland | Immune monitoring
Characterisation and functional analysis of T-cell responses in
melanoma patients vaccinated with peptide-loaded
dentritic cells
Steve Voland 1 , Stefanie Gross 1 , Beatrice Schuler-Thurner 1 , Pierre Coulie 2 , and Gerold Schuler 1
1 Dept. Dermatology, University Hospital Erlangen, Erlangen, Germany
2 de Duve Institute and Université catholique de Louvain, Brussels
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T cell responses in melanoma patients vaccinated
with autologous monocyte-derived dendritic cells
(DC) loaded with peptides from different tumorassociated
antigens (TAA) were characterized for
their functional capacity.
To assign a certain functional capacity (cytolytic
activity, cytokine production and degranulation
upon restimulation) to specific T cell clones we
chose a limiting-dilution based approach. Frozen
aliquots of PBMC from 5 melanoma patients who
had received four vaccinations were thawed,
loaded with peptide and seeded in a 96well plate
followed by a 14-day culture. The cells were restimulated
with autologous peptide-loaded PBMC
at day 7. By splitting the cells two times during
the 14-day culture four plates with identical clonal
composition were obtained. Comparative analyses
of each corresponding well were performed with
the following assays:
1. percentage of peptide-specific T cells, determined
by MHC tetramer binding
2. intracellular cytokine production (interferon-γ,
interleukin 2, TNF-α) and degranulation (by CD107a
mobilization) after antigenic stimulation
3. cytolytic activity determined by a standard 51Crrelease
assay
In all 5 patients vaccine-specific CD8+ T cells were
detected after in vitro presensitation with peptide.
Detected responses differed in magnitudes and
overall functional capacity. In most cases a positive
correlation between lytic activity and antigenspecificity
(MHC tetramer positivity) was found.
Furthermore the lytic activity correlated positively
with certain cytokine profiles with a pronounced
IFN-γ and TNF-α proportion and to a lower extend
also with IL-2 and CD107a.
From our data can be concluded that vaccination
with autologous monocyte-derived DCs loaded
with TAA-derived peptides is capapble to induce
antigen-specific CD8+ T cells, with the potential
to produce different immune-stimulatory cytokines
and which show cell mediated cytotoxitcity.
Further investigations of the induced T cells will be
conducted to determine the breadth of the induced
immune response by analysis of the different T cell
clones and their affinities.