Abstract Book 2010 - CIMT Annual Meeting

102 Zimmer | Tumor biology & interaction with the immune system
Impact of endoplasmic reticulum aminopeptidases (ERAP)
on T cell epitope generation in melanoma cells
Christin Zimmer 1 , Silke Lubojanski 2 , Tanja Dannenberg 1 , Xhao Fang 1 , Ulrike Seifert 3 , Dirk
Schadendorf 1 , Annette Paschen 1
1 Department of Dermatology, University Hospital Essen, Essen, Germany
2 Department of Medicine III, University Medical Centre, Mainz, Germany
3 Institute for Biochemistry, University Medicine Charite, Berlin, Germany
Tumor cells presenting antigen epitopes by HLA
class I molecules can be specifically recognized and
effectively killed by autologous cytotoxic CD8+ T
lymphocytes (CTLs). Different proteolytic/peptidolytic
components are involved in the intracellular
generation of antigen epitopes. The cytosolic, multi-catalytic
proteasome cleaves the tumor antigen
into peptide fragments of different length, thereby
defining the C-terminus for the majority of peptides
presented by HLA class I molecules. While some
of the proteasome products are of the correct size
for direct binding to HLA class I molecules others
are N-terminally elongated peptide precursors that
require further trimming by aminopeptidases. In
the endoplasmic reticulum of human cells two
aminopeptidases, ERAP1 and ERAP2, have been
identified. Their function in epitope generation by
precursor trimming has been clearly demonstrated.
Thus, the aim of this study is to determine the influence
of ERAPs on the presentation of specific
tumor antigen epitopes by melanoma cells. At first,
the expression level of ERAP1 and ERAP2 in several
cell lines established from different metastases of
melanoma patients was determined by quantitative
real time PCR and Western Blot analysis. All cell
lines tested expressed ERAP1 and ERAP2 at mRNA
and protein level, albeit at different quantities. To
gain further insight into the role of EARP1 in the
generation of specific CTL epitopes, its expression
was down-regulated either by transient transfection
of melanoma cells with siRNA or stable
transfection with a shRNA expression plasmid. In
either case, knockdown of ERAP1 expression was
verified by RT-PCR and/or Western Blot analysis.
Interestingly, ERAP1 down-regulation increased
the recognition of melanoma cells UKRV-Mel-15
by CTLs specific for an epitope (Melan-A26/27-35)
derived from the melanoma differentiation antigen
Melan-A/MART-1. Conversely, overexpression of
ERAP1 decreased the presentation of the Melan-A/
MART-126/27-35 epitope, pointing to a destructive
role of ERAP1 in the processing of this epitope. In
contrast, down-regulation of ERAP1 in Ma-Mel-86a
cells did not affect the stimulation of autologous
CD8+ T cells directed against a mutated neoantigen
(please see abstract by Lubojanski et al.).
Our results point to a more epitope-specific role of
ERAP1 in antigen presentation: ERAP1 activity can
either contribute to or interfere with the generation
of specific epitopes while others might even be generated
independently.
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