Factors Affecting the Efficiency of CD8+ T Cell Cross-Priming with Exogenous Antigens

Abstract

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, “cross-priming” can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor’s blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual’s APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

Dominance of Virus-Specific CD8 T Cells in Human Primary Cytomegalovirus Infection

“…The use of exogenously derived antigens may in general be less powerful to stimulate CD8 compared with CD4 T cells and thus rather lead to an underestimation of specific CD8 T cell frequencies. Interestingly, to overcome this limitation, sets of overlapping peptides spanning immunodominant proteins instead of soluble proteins have recently been applied to increase efficiency in stimulating CD8 T cell responses (32)(33)(34). Although the here determined CMVspecific CD8 T cell frequencies might be underestimated to some extent, the data further emphasize the particular dominance of virus-specific CD8 T cells during primary infection.…”

“…Although the use of whole blood precludes the retrospective analysis of frozen samples, stimulation of whole blood as compared with isolated peripheral blood mononuclear cells not only leads to the detection of higher frequencies but also allows for a more reliable detection of specific CD8 T cells (unpublished observations). This fact may be due to the opsonizing effect of CMV-specific antibodies present in whole blood in stimulating antigen uptake, because detectable CD8 T cell responses were shown to be diminished upon depletion of immunoglobulins in vitro (32). The use of exogenously derived antigens may in general be less powerful to stimulate CD8 compared with CD4 T cells and thus rather lead to an underestimation of specific CD8 T cell frequencies.…”

Inability to Induce Tolerance Through Direct Antigen Presentation

“…Although CD8π T cells have been shown to recognize exogenous peptides presented in the context of MHC CI in a process called 'cross-priming' (40)(41)(42)(43), it is generally agreed that antigen presentation to CD8π T cells is predominantly via the direct pathway. Thus, an alternative interpretation of these results is that direct MHC/peptide recognition is less costimulation dependent irrespective of whether CD4π or CD8π T cells are mediating the graft rejection.…”

Multiparameter Intracellular Cytokine Staining

“…For whole proteins requiring intracellular processing, a pre-incubation of 2 h prior to adding brefeldin A and/or monensin is recommended (12). CD8 responses to whole protein antigens can sometimes be detected and are increased with longer incubation in antigen alone, but not in all donors (13). 10 Automating incubation times: A programmable heat block, incubator, or water bath can be used for time activation, cooling the samples to 4-18°C at the end of a specified period at 37°C, and holding them for later processing.…”

“…Store at 4°C in the dark until ready for data acquisition, which should be performed within 24 h. Optional: resuspend pellets with 150 μL of 1% paraformaldehyde in PBS or BD Stabilizing Fixative (see Note 16). 13 Freezing of activated samples: Samples can be frozen at −80°C directly in FACS lysing solution (12,14). This allows for samples to be sent to another laboratory for processing, or for longitudinal samples to be accumulated for batch processing.…”