Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+) T lymphocytes.
BACKGROUND
In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells).


METHODS
We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.


RESULTS
Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.


CONCLUSIONS
We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.
von Euw
Erika M
EM
Fundación Instituto Leloir, Patricias Argentinas 435 (1405), Buenos Aires, Argentina. evoneuw@leloir.org.ar
Barrio
María M
MM
Furman
David
D
Bianchini
Michele
M
Levy
Estrella M
EM
Yee
Cassian
C
Li
Yongqing
Y
Wainstok
Rosa
R
Mordoh
José
J
D000951:Antigens, Neoplasm / Q000276:immunology*; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology* / Q000528:radiation effects; D002454:Cell Differentiation / Q000187:drug effects / Q000528:radiation effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000528:radiation effects; D054415:Chemokine CCL19 / Q000494:pharmacology; D018920:Coculture Techniques; D045142:Cross-Priming / Q000187:drug effects / Q000276:immunology* / Q000528:radiation effects; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D000939:Epitopes / Q000276:immunology; D005720:Gamma Rays; D006801:Humans; D016753:Interleukin-10 / Q000276:immunology; D018664:Interleukin-12 / Q000276:immunology; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology / Q000473:pathology* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000276:immunology*; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D009336:Necrosis; D009363:Neoplasm Proteins / Q000276:immunology*; D010587:Phagocytosis / Q000187:drug effects / Q000276:immunology / Q000528:radiation effects; D054400:Receptors, CCR7 / Q000276:immunology; D013997:Time Factors; D058951:gp100 Melanoma Antigen
D000951:Antigens, Neoplasm; C515370:CCR7 protein, human; D054415:Chemokine CCL19; D000939:Epitopes; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D054400:Receptors, CCR7; D058951:gp100 Melanoma Antigen; D016753:Interleukin-10; D018664:Interleukin-12