In this study, we demonstrate that the conditions of DC maturation differentially influence the expression of APM components and the ability to cross-present tumor-derived antigens to HLA class I-restricted CTL. Although several APM components were modulated in their expression, we found that only selected APM components, TAP1 and TAP2, were significantly correlated with cross-presentation to T cells, being particularly high in the cells matured with the DC-1 cytokine combination (). A number of other factors, besides expression of APM components TAP1 and TAP2, could be responsible for this capacity, including surface costimulatory markers, adhesion molecules, and stimulatory cytokines. However we observed no significant differences in expression of CD80, CD83, CD84, CD40 or CCR7 between the different mDC. Additionally, the results observed in CTL recognition of tumor fed DC differed with those observed with DC pulsed with exogenous peptide. Furthermore, MAGE-3 specific CTL recognition was not affected by an IL-12 blocking mAb, demonstrating that differences in cross-presentation are more likely a consequence of a different intracellular phenotype, owing to differences in APM component expression rather than differences in cell surface markers or secretion of IL-12 or other cytokines. Furthermore, whereas we and others16,17
have previously demonstrated that the ability of DC to produce IL-12 is important for their ability to induce TA-specific functional CTL responses from resting T cells,14
the current findings indicate that that this cytokine is not essential for the ability of DC to cross-present antigen to already primed, functional CTL.
Because of the strong correlation of TAP1 and TAP2 expression with DC stimulatory capacity, we studied the importance of selective APM component up-regulation in mDC, through maturation with IFN-γ alone. However, despite the marked up-regulation of TAP1/TAP2 by IFN-γ similar to that seen using the most stimulatory DC-1 cytokine combination () DC treated with IFN-γ alone had poor stimulatory capacity and cross-presentation of a MAGE-3-derived peptide was not robust in IFN-γ matured cells, suggesting that other factors are necessary to achieve the full DC function desired for cancer vaccines, including antigen uptake and surface costimulation. This result also implies that TAP1/2 provide only some of the properties of DC necessary for optimal activity to generate and maintain CTL responses. Similarly, it has been demonstrated that IFN-γ used in conjunction with other maturation stimuli (such as lippopolysaccharide), promotes CCR7-driven migration, IL-12p and IL-27 secretion, and decrease IL-10 production, but is unable to achieve these effects when used alone.18
Thus, our and other's findings, suggests that further studies should be conducted to investigate the value of incorporating IFN-γ into DC maturation and T cell stimulation, and Th1 instruction, in vitro and in vivo.
Clinical vaccine trials incorporating TAP1 and TAP2 expression measurements may allow us to directly address the efficacy of this approach. Thus, we conclude that a complex maturation combination is necessary to fully mature DC, and that TAP1/2 expression alone is not sufficient to generate the most stimulatory DC for cancer vaccines. Irrespective of the mechanism underlying optimal cross-presentation, our results also suggest that monitoring of DC preparations for TAP1 and TAP2 expression may represent useful biomarkers to permit standardization of phenotypic and functional attributes for improved cancer vaccines.
For the first time, we also show the role of APM component expression is strongly related to the ability of DC to cross-present TA to naive and primed CTL in vitro. These findings are consistent with the observations of others, where transfection of selected APM components result in higher levels of cross-presentation and cross-priming in mice.19,20
If these results are borne out by further studies, including our identification of a putative marker to standardize DC, significant utility for optimizing and monitoring vaccine trials might be obtained. The potential to characterize DC by their intracellular, antigen processing phenotype according to APM component expression would enable consistent generation of DC most likely to generate antigen-specific immune responses in vaccines loaded with the tumor-related particulate material, rather than peptides. In addition, future studies may address whether APM components are involved in the processing of long peptides used in some recent vaccine regimens.21
The effect of DC maturation protocols may also facilitate access of exogenous TA into the HLA class I antigen-processing pathway, as this process begins with cytoplasmic degradation and is generally TAP1/2 dependent.22
We have shown that cross-presentation is strongly influenced by DC maturation phenotype, leading to alterations in TAP1/2 expression. It is possible that further studies will elucidate the effect of DC maturation pathways on facilitating access of antigen taken up exogenously into the cytoplasmic compartment, where interaction with these APM components can occur efficiently.
The most striking aspect of our finding was that DC, which express very similar levels of surface phenotypic markers of maturation, differed significantly in their ability to cross-present exogenous antigen, Thus, it is possible that measuring TAP1 and TAP2 would be sufficient to indicate appropriately matured DC, as a direct measure of intracellular processing and presenting function for DC-based immunotherapy. We believe that these APM markers should be incorporated routinely into DC-based vaccines and in vitro studies which require comparable and full DC maturation between experiments. Additional studies should also validate these findings in vivo, by incorporating measurement of APM component expression into ongoing DC-based vaccine trials using tumor-derived antigenic material. Our data suggest that appropriate in vivo manipulation of mDC can yield cells capable of both efficient antigen processing as well as surface HLA-TA peptide specific stimulation of antigen-specific T cells.