Adaptive immune responses are initiated by the presentation of peptides from foreign proteins on MHC molecules by DC in the 2º lymphoid sites. We have previously observed that CD8 T cell responses to cytoplasmic and secreted tumor-Ag can be robust but result in overall tolerance induction (35
). In contrast, CD4 T cells remain ignorant throughout disease progression, due to defective MHC-II presentation of tumor-derived proteins on DLN DC. We have now examined the basis for this defective MHC-II presentation in several s.c. cancer models and on two murine backgrounds. We found that tumors are infiltrated by large numbers CD11b+
DC, a large fraction of which have a partially activated phenotype. In comparison to normal dermal DC, these TIDC are very inefficient at taking up soluble proteins, but are comparable in the relative rates of protein degradation and formation of MHC-II-peptide complexes. While TIDC exhibit a partial block in migration to the DLN, these data show that the inability to present sufficient Ag to activate naïve CD4 T cells in the DLN, or stimulate effector CD4 T cells within the tumor appears to result primarily from the limited capacity of TIDC to internalize protein Ag.
Interestingly, the contrasting responses of CD8 and CD4 T cells to the same tumor-secreted protein suggest that MHC-I and MHC-II presentation in the DLN is physically uncoupled and is differentially regulated. In agreement with this notion, we found that while the LN resident CD8α+
DC were efficient at cross-presenting tumor-derived proteins to CD8 T cells, only the activated/migratory CD11b+
DC had any detectable MHC-II-peptide complexes (35
). These findings suggest a model where the DLN receives tumor-derived lymph fluid carrying cell debris along with various tumor proteins and cell membranes, which then gets efficiently taken up by immature CD8α+
DC and selectively directed towards MHC-I processing (43
). At the same time, TIDC are poor at protein uptake and to some degree migration, and are thus unable to function as professional APC in the DLN for initiating CD4 T cell priming. The proposed scenario for anti-tumor responses is similar to one described by Jenkins and colleagues for soluble protein immunization, where Ag presentation to CD4 T cells in the DLN occurs via both resident and migratory DC, with the exception that in the tumor model the draining proteins are cell-associated and hence are targeted to MHC-I presentation and migratory DC are defective at Ag uptake and therefore MHC-II presentation (26
). However, it is entirely plausible that in addition to TIDC defects, resident DLN DC have defective MHC-II presentation machinery and contribute to CD4 T cell ignorance. Elucidating these non-mutually exclusive mechanisms behind the apparent MHC-I/II presentation paradox will require further study.
Microscopic examination revealed that TIDC heavily infiltrated the peripheral and inner parts of the tumor tissues with some indication of infiltration-rich regions. Phenotypic examination of TIDC showed that they were largely indistinguishable from normal interstitial dermal DC, which is in agreement with other data that has described a large prevalence of CD11b+
DC infiltrating various murine tumors (36
). Interestingly, in contrast to the immature peripheral dermal DC, TIDC expressed intermediate to high MHC-II and co-stimulatory levels, and low levels of intracellular IL-12. These results suggest that TIDC undergo a partial maturation program, possibly through experiencing inflammatory cytokines, such as TNFα, at the tumor site (45
). It has been shown that DC maturation decreases phagocytosis and prevents uptake and presentation of proteins on MHC (42
). It is thus possible that the partial activation state of TIDC is at least in part responsible for causing poor Ag uptake and MHC-II presentation. This is consistent with the fact that the immature dermal DC were efficient at Ag uptake, which allowed them to maintain high levels of available intracellular protein after maturation and migration to the DLN. These high intracellular protein levels appeared to directly correlate with the amount of detectable surface MHC-II-peptide complexes in both the periphery and the DLN, suggesting that efficient Ag acquisition is required for optimal MHC-II presentation. We found that TLR ligand co-administration caused an increase in maturation and migration in both dermal DC and TIDC and greatly enhanced Ag presentation in skin DLN. This treatment however failed to alter peripheral Ag uptake in both subsets. While it has been shown that DC undergo a transient increase in Ag phagocytosis post TLR induced maturation in vitro
, our results argue that Ag acquisition in vivo
occurs relatively rapidly and independently of simultaneous TLR signaling (42
). Interestingly, after the initial uptake and degradative phase, dermal DC appeared to store the protein at fairly stable levels, suggesting that the some of the soluble protein can enter the nondegradative intracellular vesicular compartment that has been described to play a role in Ag transfer to B cells (41
). It is also possible that limiting Ag processing for MHC-II presentation in the periphery could be important for reducing tissue inflammation and destruction during the effector CD4 T cell response. It is important to note that the observed DC Ag handling characteristics describe responses to soluble proteins, which are likely to be different from bacterial or viral infections.
It has been recently shown that i.t. T cell infiltration and accumulation directly correlates with therapeutic efficacy (48
). In our hands, poor Ag uptake and presentation by TIDC not only precluded the initial priming of CD4 T cells in DLN, but also appeared to prevent effector CD4 T cell accumulation at the tumor site and correlated with a lack of tumor regression (35
). Even i.t.administration of CpG, which was observed to partially enhance MHC-II presentation but not Ag uptake, was incapable of rescuing effector CD4 T cell accumulation. Interestingly, we also observed differences between Ag-nonspecific CD8 and CD4 T cell tumor infiltration, with CD8 T cells being much better than CD4 T cells at infiltrating the neoplastic tissues. In agreement, we have previously observed that endogenous CD8 T cells vastly outnumbered CD4 T cells inside the tumor (35
). While we initially attributed these results to reduced initial priming of endogenous CD4 T cells in tumor DLN, it is possible that both priming and ability to infiltrate the tumor is defective in the CD4 T cell population. While defective tumor infiltration by T cells has been previously observed and attributed to a lack of integrin expression on the tumor endothelium, the disparity between CD4 and CD8 T cell infiltration observed in our model suggests that differences in various adhesion molecules exist directly on CD4 and CD8 effector T cells (48
Interestingly, TIDC were efficient at infiltrating and accumulating to great numbers inside the tumor tissue, suggesting that this immune cell subset carries the appropriate adhesion molecules for proper tumor infiltration. On the other hand, TIDC were relatively poor at migrating to the DLN, suggesting that TIDC do not possess the necessary surface molecules to mediate exit or to guide them into the DLN. However, it is also possible that disease-associated tissue microenvironment is simply not permissive for TIDC migration after initial infiltration. Further characterization of integrin and adhesion molecule expression and function on tumor endothelium, effector CD4 and CD8 T cells, and TIDC is necessary to understand the requirements for immune cell trafficking into and out of neoplastic tissues.
While poor TIDC migration likely contributed to CD4 T cell ignorance, it is clear that defective Ag acquisition by TIDC served as the major limiting step in both initiation and facilitation of anti-tumor CD4 T cell responses. This defect can be attributed to several different non-mutually exclusive mechanisms. In addition to partial maturation, improper TIDC differentiation due to a lack of appropriate growth factors in the tumor microenvironment can cause poor Ag uptake. It is also possible that inhibitory factors present in the tumor may cause specific deregulation of TIDC function (50
). While TGFβ and IL-10 have been described to negatively modulate anti-tumor immune responses, we did not observe enhancement of Ag uptake by TIDC or initiation of CD4 T cell priming in DLN after TGFβ antibody blockade or in IL-10 deficient animals, respectively (unpublished observations). However, it is possible that several suppressive factors act to inhibiting TIDC functional competence and/or differentiation, and neutralization of all of them is necessary for restoration of TIDC function (50
). Additionally, nutrient starvation is likely to occur at the tumor site and has been previously shown to induce autophagy and reduce soluble Ag capture, thus specifically decreasing presentation of peripheral proteins and enhancing presentation of self-proteins on MHC-II (52
). Interestingly, we observed that tumor infiltrating macrophages also took up lower levels of Ag as compared to their normal skin counterparts. While clearly not being as efficient at Ag processing for MHC-II presentation as DC, these results suggest that inhibition of Ag uptake by the tumor microenvironment is a global phenomenon that extends to multiple immune cell subsets. Understanding the mechanisms behind defective Ag acquisition as well as immune cell migration should allow for better understanding of clinical data from various immunotherapy trials and more importantly for enhancing design of future therapeutic regimens.