Here we show that a specific DC population with potent capacity to prime both CD4+ and CD8+ T cells to cell-associated antigens can be exploited to induce protective tumor-specific T cell responses in tumor bearing hosts. mcDCs have a distinct phenotype in the internalization and processing of cell-associated antigens, characterized by antigen storage in compartments with reduced lysosomal degradation resulting in prolonged antigen presentation. mDC-driven activation of T cells to cell-associated antigens endows T cells with a greater capacity for primary expansion, enhanced effector function and increased memory development, even in an otherwise immunosuppressive environment. As a result, treatment of tumor-bearing mice with mcDCs was more effective in the induction of protective tumor-specific CD8+ T cell responses, inhibition of tumor growth and increased survival than was treatment with other types of DC.
DCs are a phenotypically and functionally heterogenous population of leukocytes and, although the intrinsic properties of each DC subset may dictate their functional specificity, their final maturation and functional capacities are also influenced by the tissue environment and the cell-types they interact with (reviewed in (41
)). The mcDCs described in this study account for ~5% of the total splenic DC population and lack the conventional DC classification markers CD8 and CD11b. Recently it has been suggested that the CD11b-CD8- DCs that express cystatin C and are expanded by FLT3L are immediate pre-cursors of CD8α+ DCs, as transfer of CD11b-CD8- DCs results in rapid conversion to CD8α+ DC (10
). The mcDCs described here share many characteristics with CD11b-CD8- DCs in that they express cysteine protease inhibitor cystatin C and are expanded by FLT3L (data not shown). However, our data indicates that all characteristic features of mcDCs, including type I IFN production after phagocytosis of cell-associated materials, and CD4+ and CD8+ T cell priming to cell-associated antigens, neither required nor affected CD8α expression on mcDCs. Moreover, transfer of mcDCs on a CD45.1 background into a CD45.2 host, showed that only a small population of mcDCs expressed CD8α around 3 d after transfer (supplemental figure 2
). Given that CD8α+ DCs do not produce type I IFNs after uptake of cell-associated material and show relatively poor capacity to prime CD4+ T cells to cell-associated antigens, the mcDC population could be regarded as a functionally distinct population.
Several mechanisms may contribute to the effective CD8+ T cell priming capacity of the mcDC, including the distinct internalization, processing and presentation of antigen, the production of type I IFNs by the mcDC upon uptake of cell-associated material, and increased induction of CD4+ T help.
Pulse-chase studies showed that mcDCs were capable of activating T cells to cell-associated antigens over a longer time-span than CD8α+ DCs. Importantly, when DCs were pulsed with the high affinity CD8+ T cell OVA epitope SIINFEKL, both mcDCs and CD8α+ DCs showed comparable priming, which was significantly less sustained over time than the priming by mcDCs presenting cell-associated antigens. Similarly, transfer of peptide-pulsed mcDCs and CD8α+ DCs into naïve mice resulted in comparable CD8+ T cell responses that did not protect mice from subsequent tumor challenge (data not shown). Together with the observation that mcDCs “store” cell-associated antigen in a compartment with relatively little lysosomal degradation, these data suggest that mcDCs may use these compartments as a source to continuously supply MHC ligands for presentation. This is in line with the observations of Van Montfoort, et al., who showed the formation of long-term storage compartments in DCs after receptor-mediated endocytosis of IgG-OVA(35
). These compartments were lysosome-like organelles, distinct from MHC class II compartment and the recently described early endosomal loading compartments, and functioned as antigen depots which enabled the DCs to maintain high MHC-peptide levels on their surface over time.
Recent studies indicated that cross-presenting CD8α+ DCs limit endophagocytic proteolysis through low expression and low recruitment of proteolytic enzymes to phagosomes, limited acidification by the V-ATPase and active alkalinization of the endosomal and phagosomal lumen by the NAPDH oxidase NOX2(29
). In CD8α+ DCs, RAC2-mediated assembly of the NOX2 complex to phagocomes resulted in the production of reactive oxygen species (ROS), which causes alkalinization of the phagosome and prevents antigen degradation(37
). mcDCs and CD8α+ DCs have comparable ROS production in the resting and activated states (data not shown). In addition, DNA arrays showed that mcDCs express mRNA for all genes of the NAPDH complex at similar or even higher levels than and CD8α+ DCs (data not shown), suggesting that mcDC scould possible exploit a comparable mechanism as CD8α+ DCs to delay antigen degradation and enhance their antigen presenting capacity.
MHC-antigen density and length of T cell stimulation significantly affect clonal burst size, acquisition of effector function, and development of memory(42
). The sustained antigen presentation by mcDCs therefore could result in a longer, and therefore more robust activation on a per cell level. In addition, maintenance of sufficient MHC-peptide complexes on the mcDCs would provide the mcDC with a greater timeframe to encounter and activate T cells.
A second adjuvant mechanism in the enhanced priming capacity of mcDCs could be assigned to their production of type I IFNs after uptake of cell-associated materials. Our transfer data shows that type I IFNs act in both an autocrine and paracrine fashion, as transfer of ifnar−/−
mcDCs into WT recipients, and WT mcDCs into ifnar−/−
recipients, significantly reduced antigen-specific CD8+ T cell responses. Type I IFNs have been shown to mature DCs and enhance the processing and presentation of antigenic peptides by increasing proteasome components involved in the generation of peptides and components that target the peptides for interaction with MHC class I and class II molecules(47
). In addition, type I IFNs have been shown to enhance MHC expression and soluble and membrane-associated costimulatory molecules that affect T cell priming (48
). Besides direct effects on DCs, type I IFNs can act directly on T cells and enhance their proliferation and accumulation by inhibition of apoptosis(50
). Additional paracrine effects on bystander cells may result in the induction of cytokines and chemokines that would provide a favorable milieu for T cell priming(49
The ligand that induces type I IFNs in mcDC after uptake of cell-associated materials is still unknown, but recent research indicates that apoptotic cell-derived nucleotide structures that escape lysosomal degradation can induce type I IFNs(53
). Yoshida, et al., showed that mice deficient in DNase II, the lysosomal enzyme that digests the chromosomal DNA of apoptotic cells and expelled nuclei, die in feto
due to anemia caused by type I IFN production(54
). Mice deficient in both the DNase II
genes, however, were born relatively healthy. In light of the fact that mcDCs show prolonged antigen retention and delayed lysosomal degradation of cell-associated materials, it is not unlikely that nucleotide structures can escape from such retained material. Our previous work showed that this type I IFN induction was not dependent on TLR signaling (13
) which suggests a possible role for cytosoli nucleotide sensors.
The capacity of the mcDC to prime strong CD4+ T cell responses to cell-associated antigens might also be instrumental in the induction of the potent anti-tumor effect. We and others have shown that CD4+ T cell help during priming of CD8+ T cells is required for optimal CD8+ T cell activation, primary expansion, acquisition of effector function, and the development of memory(32
). Increasing CD4+ T cell help through transfer of (transgenic) CD4+ T cells or pre-immunization of mice has been shown to enhance the induction of CD8+ T cell responses(57
). In addition, ample studies indicate that CD4+ T cell help plays a supporting role in the maintenance, reactivation, and expansion of existing memory cells(59
DCs provide an attractive mechanism for therapeutic manipulation of the immune system in the priming of protective anti-tumor responses. However, the complexity of the DC system underscores the necessity for its rational manipulation in order to achieve protective and therapeutic immunity. Although current therapies with ex-vivo generated DCs have been proven feasible, there are still many obstacles to overcome to improve clinical trial success and offset the cost and complexity of customized cell therapy. Here we show that DC therapy using DCs that, after uptake of irradiated tumor cells, exhibit prolonged antigen retention, sustained antigen presentation, production of pro-inflammatory type I IFNs, and the capacity to prime both CD4+ and CD8+ T cells, is efficient in the induction of protective anti-tumor responses in vivo in tumor bearing hosts. Targeting DCs with these characteristics in humans, or manipulation of other DCs that would induce similar behavior, could be instrumental in the design of effective therapeutic and preventive DC-based cancer vaccines.