Inclusion of Brefeldin A during dendritic cell isolation allows in vitro detection of cross-presented self-antigens.
The mechanism of cross-presentation enables dendritic cells (DC) to induce immunity against intracellular pathogens and to tolerize autoreactive CD8 T cells. The antigen-presenting cells (APCs) responsible for cross-presentation of self-antigens have been identified as CD8alpha(+) CD11c(+) DC. Isolation of these cells has been notoriously difficult, and the resulting responses of T cell hybridomas were too low to permit further studies. Here, we demonstrate that inclusion of Brefeldin A (BfA), an agent reported to block MHC class I-peptide complex turnover on the cell surface, during DC isolation from transgenic RIP-mOVA mice facilitated activation and proliferation of naïve OVA-specific CD8(+) T cells in vitro. CD8alpha(+) DC were more efficient than CD8alpha(-) CD11c(+). BfA also reversibly preserved expression of costimulatory molecules by DC, as evidenced by their expression of costimulatory markers and by an increased stimulatory capacity of DC matured in vivo by LPS. We conclude that the use of BfA notably improves sensitivity of detection of cross-presented self-antigens.
Benke
Dirk
D
Department of Cardiology, University clinic of the Rheinisch-Westfälische Technische Hochschule, Aachen, Germany. dirk.benke@gmx.net
Krüger
Thilo
T
Lang
Andreas
A
Hamilton-Williams
Emma E
EE
Kurts
Christian
C
D000818:Animals; D001324:Autoantigens / Q000276:immunology*; D020126:Brefeldin A / Q000494:pharmacology*; D039521:CD11c Antigen / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D049109:Cell Proliferation; D045142:Cross-Priming / Q000276:immunology*; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D010047:Ovalbumin / Q000276:immunology
D001324:Autoantigens; D039521:CD11c Antigen; D015395:Histocompatibility Antigens Class I; D020126:Brefeldin A; D010047:Ovalbumin