Evasion of the immune system is currently regarded as the seventh hallmark of cancer progression, with some of the more recently discovered strategies affecting DC differentiation and maturation (44
). Considering that the steady-state tumor microenvironment does not contain strong stimuli for DC maturation, it becomes heavily dependant on CD4 T cell help, ablation of which would lead to tolerant anti-tumor CD8 T cell responses (44
). It is therefore not surprising that the progressing tumor would evolve mechanisms to inhibit this last safeguard against CD8 T cell tolerance induction.
By examining TAg-specific naïve CD4 and CD8 T cell responses to two tumor cell lines we found that the CD8 T cells rapidly responded to a subcutaneous challenge by recognizing cross-presented cytoplasmic and secreted TAg, but were tolerogenic in nature and did not alter tumor growth. We have previously shown that this is in part due to low levels of signal three cytokines available for CD8 T cells during priming in DLN (22
). In contrast to the CD8 responses, OT-II TAg-specfic CD4 T cells remained completely ignorant and naïve throughout tumor progression. Even though OT-II CD4 T cells have been reported to have poor responsiveness to low levels of MHC-II-peptide complexes (33
), we found that they responded equivalently or better than OT-I CD8 T cells to immunization with soluble OVA into various solid and subcutaneous sites, but their response was dramatically decreased to i.t. OVA administration. Interestingly, OT-II responses remained intact in tumor-bearing animals if OVA was administered at a distal from the tumor site. These results indicated that T cell response differences were due to a localized tumor-specific mechanism for selective inhibition of MHC-II-restricted TAg presentation, and not due to T cell intrinsic differences, a mutation of tumor OVA protein, or DC cross-dressing themselves with tumor membrane-derived MHC-I complexes (42
Examination of polyclonal endogenous T cells indicated that they respond similarly to transgenic T cells, with CD8 T cells infiltrating the tumor site in greater magnitude and specificity than CD4 T cells. We additionally found that Treg CD4 T cells are enriched in the tumor with greater than 50% of the population sensing antigen. We hypothesize that the Treg population senses self-antigen MHC class-II-peptide complexes, that are derived from the intracellular autophagy-dependent pathway and not from external phagocytosis/pinocytosis dependent pathways of tumor-antigen processing. It has been recently shown that nutrient starvation, most likely found at the tumor site, causes an increase in presentation of intracellular self-proteins on MHC class-II due to increased autophagy. This enhanced presentation of self by DC could thereby be responsible for the observed defect in presentation of soluble OVA or EaGFP on MHC-II, and increased presentation to the self-specific Treg population (38
). Interestingly, TIDC did not have sufficient MHC-II-peptide complexes to cause priming of OT-II T cells directly ex vivo
. This indicates that while presentation of self-Ag by TIDC can occur, exogenous proteins are strongly inhibited from being presented on MHC-II.
We found that the tumor DLN CD8α+ DC subset was responsible for cross-presenting TAg for CD8 T cell priming, with neither the CD8α+ nor the CD8α- DC subset being able to cause CD4 T cell activation. Interestingly, CD8 T cell responses to both cytoplasmic and secreted tumor proteins were fundamentally identical, indicating that cross-presentation of different TAgs by DLN DC is a relatively efficient process. Moreover, we were able to characterize the CD8α+ DC subset in tumor DLN to be functionally immature, explaining the tolerogenic nature of the CD8 T cell response. Since MHC-II presentation by the CD8α+ DC subset is not as efficient as cross-presentation, its possible that CD4 T cells are not primed by this subset due to limiting TAg levels and overall low MHC-II levels (18
). By utilizing soluble EαGFP protein, we were able to directly visualize MHC-II presentation inhibition in tumor DLN in comparison with tumor-free controls. We found both a decreased frequency of migratory DC carrying the fluorescent protein, as well as decreased levels of intracellular protein and surface MHC-II-peptide complex on DC in tumor DLN. Interestingly, the migratory DC displayed functionally high levels of total MHC-II, potentially displaying self-antigens and evoking Treg responses, as witnessed at the tumor site. Put together, selective inhibition of the migratory DC MHC-II presentation machinery coupled with CD8α+ DC limited MHC-II presentation capacity could lead to a blockade in anti-tumor CD4 T cell responses. However, it is also possible that MHC-II presentation machinery in both migratory and resident LN DC is affected by the tumor microenvironment.
While the majority of studies agree that adoptively transferred CD8 T cells undergo tolerance and do not control tumor growth, there are some examples in the literature of naïve CD8 T cells controlling tumor progression without immunization. Recently Boissonnas and colleagues have shown that 1×107
transferred naïve OT-I CD8 T cells undergo efficient activation and can control E.G7 tumor growth (47
). However, such a high transfer number has been shown to cause a CD4 T cell help independent activation of CD8 T cells in both non-tumor and tumor models (23
). Additionally, several reports show that naïve CD4 T cells respond to a cognate tumor challenge and help CD8 T cell responses without immunization (23
). However, these same studies also show clear dependence of the effective anti-tumor responses on the high transfer number of CD4 and CD8 T cells. In our hands, adoptive transfer of 1×106
transgenic T cells does not result in CD8 T cell clonal expansion and control of tumor growth, indicating that the transfer numbers are insufficient for effective CD8 T cell activation to occur independently of CD4 T cell help. This is additionally supported by the fact that the low precursor frequency tumor-specific endogenous T cells appear to mirror the transgenic T cell response.
In addition, certain leukemia and prostate tumor models show relatively robust tolerogenic CD4 T cell activation to TAg (50
). One difference between the systems is that CD4 T cell responses described in our model represent responses to tumor-specific and not tumor-associated antigens, as described in the prostate model (50
). More importantly, it is likely that in liquid tumors, antigen levels in the blood and lymph are higher than if they were derived from a solid tumor, thereby having greater access to resident spleen and lymph node DCs. In agreement with this, transgenic and endogenous CD4 T cells undergo a tolerogenic response to the E.G7 thymoma when it is injected i.p. and grows in ascities fluid (52
). This tolerance can be reversed through CTLA-4 blockade, which allows for CD4 T cell help-dependent control of the tumor by CD8 T cells. This further supports the notion that provision of CD4 T cell help critically depends on CD4 T cell activation by antigen presenting cells. It is also important to note that these particular studies did not examine both the CD8 and CD4 T cell response kinetics, which introduces the possibility that unsynchronized CD8 and CD4 T cell responses may occur (50
). Thereby CD4 T cells could respond much later than CD8 T cells and not be able to provide help during priming, causing tolerance in CD8 T cell responses. Moreover, the majority of the aforementioned studies utilize either fairly immunogenic tumor cell lines, or poorly characterized spontaneous tumor models where immunogenicity is difficult to control (23
). Thus, while clearly not universal to all tumor models, our experimental system describes the behavior of low T cell precursor frequencies in response to certain poorly immunogenic, solid tumors.
Overall, these results point to a novel mechanism of tumor immune evasion, where the tumor microenvironment causes aberrant DC functionality by selectively restricting MHC-II presentation by activated interstitial DC that migrate from the tumor site to the draining lymph nodes, while leaving cross-presentation by immature, lymph node resident CD8α+ DC virtually intact. The decrease in the ability of DC to present TAg to CD4 T cells thereby prevents them from providing help for effective CD8 T cell priming. However, it is possible that even if CD4 T cells were activated, CD4 T cell help would not be sufficient to rescue CD8 T cell tolerance induction, as more than one dominant inhibitory mechanism might be in place in tumor-bearing animals. Nevertheless, lack of MHC class-II presentation on DC does represent the earliest block in the helper pathway and hence is at least in part responsible for tolerance induction in CD8 T cells.
While the mechanisms of impairment of DC presentation capacity are currently unresolved, the tumor microenvironment is known to harbor a milieu of suppressive cytokines derived from tumor cells themselves, or produced by infiltrating Treg CD4 T cells or M2 macrophages. Possible mediators include but are not limited to, VEGF, IL-6, M-CSF, TGFβ, IL-10, COX2, PGE2, and gangliosides, and multiple factors may be involved (44
). Additionally, nutrient deprivation, which is most likely found at the tumor site, has been shown to alter MHC-II presentation machinery (46
). Functionally, a number of steps in DC MHC-II processing and presentation could be altered to cause the observed phenotype, including: DC pinocytosis of extracellular proteins at the tumor site, lysosome acidification, cathepsin activity, H2M and H2-O function, DC migration to DLN, and others (34
). It will be important to understand the basis for the selective inhibition of DC MHC-II presentation demonstrated here, and determine whether this phenotype is observed in human cancer patients and, if so, its association with clinical outcome. In addition, a means of reversing the MHC-II defect would have obvious potential for improving T cell based anti-tumor therapies.