In vivo models have shown that tissue-restricted antigen may be captured by bone marrow–derived cells and cross-presented for the tolerization of CD8+ T cells. Although these studies have shown peripheral tolerization of CD8+ T cells, the mechanism of antigen transfer and the nature of the antigen-presenting cell (APC) remain undefined. We report here the establishment of an in vitro system for the study of cross-tolerance and show that dendritic cells (DCs) phagocytose apoptotic cells and tolerize antigen-specific CD8+ T cells when cognate CD4+ T helper cells are absent. Using this system, we directly tested the “two-signal” hypothesis for the regulation of priming versus tolerance. We found that the same CD83+ myeloid-derived DCs were required for both cross-priming and cross-tolerance. These data suggested that the current model for peripheral T cell tolerance, “signal 1 in the absence of signal 2”, requires refinement: the critical checkpoint is not DC maturation, but instead the presence of a third signal, which is active at the DC–CD4+ T cell interface.
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We thank N. Blachère, N. Bhardwaj and S. Amigorena for helpful discussions and critical review of the manuscript. Supported by the NIH-NCI and the NCC (M. L. A.), The Susan G. Komen Breast Cancer Foundation (R. B. D.) and the Burroughs-Wellcome Fund (R. B. D.).
Mature DCs exposed to CD40L do not further up-regulate DC maturation markers. To evaluate the phenotype of macrophages and DCs at different stages of differentiation, cells were surface labeled with lineage markers (CD14 and CD83) as well as with markers that correlate with the state of DC maturation (HLA-DR and CD86). Additionally we analyzed cells for candidate molecules that may distinguish the phenotype of "tolerizing" versus "activating" DCs (CD40, RANK and OX40). While we confirmed the previously reported phenotypic differences between macrophages, immature DCs and mature DCs, we did not identify phenotypic differences between the mature DCs +/- CD40L treatment. (GIF 38 kb)
Mature DCs +/- CD40L express similar amounts of MHC class I. To evaluate surface MHC class I expression at different stages of DC differentiation, cells were surface labeled with anti-HLA-A,B,C. While we confirmed the previously reported two-fold increase in MHC class I expression during DC maturation, we did not identify up-regulation of MHC class I upon CD40L treatment. Immature DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4 as described. Treatment of immature DCs with TNF-α and PGE-2 for 36 h. resulted in the generation of mature DCs. An additional 40-h incubation with CD40L yielded the CD40L-mature DC cultures. Cells treated with IgG control antibody were used to establish FACS settings (black lines). (GIF 14 kb)
Schematic representation of two models for distinguishing antigen cross-priming from cross-tolerance. We propose two models for how DCs might trigger the distinct immunological outcomes of antigen cross-presentation. (GIF 20 kb)
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Albert, M., Jegathesan, M. & Darnell, R. Dendritic cell maturation is required for the cross-tolerization of CD8+ T cells. Nat Immunol 2, 1010–1017 (2001) doi:10.1038/ni722
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