In this study, we analyzed phenotypic changes of islet-resident DCs during the progression of T1D using the MIP-GFP reporter mice in both autoimmune-prone and resistant backgrounds. GFP has a unique tertiary structure that makes it resistant to chemical and physical denaturation (29
). However, under low pH conditions, such as those found in late endosomes and lysosomes of mature DCs, GFP becomes susceptible to proteolysis and quickly loses its ability to fluoresce (32
). These properties of GFP allowed us to visualize and quantitate Ag uptake and processing in live DCs during tissue Ag cross-presentation. Our results demonstrate that intra-islet DCs exhibit a semimature phenotype in steady state and ingest large amounts of tissue Ags, which can remain unprocessed. Upon induction of tissue inflammation, DCs begin to process Ag, acquire a more mature phenotype, and migrate to the PLNs to amplify the ongoing immune response. These results extend the current understanding of the role of DCs in diabetes pathogenesis and suggest that the DCs provide a positive feedback mechanism in amplifying autoimmune responses against islet Ags. This mechanism represents a normal physiological process of an immune response and not a peculiarity of the autoimmune-prone NOD mice because it was similarly observed in the nonautoimmune B6 mice. Thus, the positive feedback loop per se is not sufficient to confer diabetes susceptibility, but it may enhance the pathogenic anti-β cell responses initiated by defects in thymic selection and peripheral regulation in the NOD mice (34
In steady state, tissue-dwelling DCs migrate to the draining LNs where they present self Ags in a process that is thought to contribute to the maintenance of self tolerance (8
). DCs resident in the peripheral tissue are thought to exhibit an immature phenotype with low cell surface expression of MHC and costimulatory molecules and high endocytic capacities, as that observed in Langerhans cells and cultured bone marrow-derived DCs. In contrast, tissue-derived migratory DCs in LNs express higher amounts of MHC and costimulatory molecules on their cell surface, suggesting that only mature DCs gain capacity to enter draining LNs (35
). We found that intra-islet DCs displayed a phenotype distinct from the previously reported immature or mature DCs with high levels of MHC class II and CD86, intermediate levels of CD40, and little CD80 expression. The expressions of MHC class II and CD86 were not further enhanced by inflammatory stimulations, suggesting that they were expressed at the same level as in fully matured DCs. However, their relatively low expression of CD40 and CD80 suggests that they are distinct from fully mature DCs.
The most distinguishing feature of the intra-islet DCs was that they uniformly contained large amounts of unprocessed β cell Ags, as visualized by their bright GFP signals in the MIP-GFP transgenic mice. Previously published electron microscopic analyses have demonstrated that MHC class II Ag-positive cells in islets contained granule-like structures that are recognized by Abs to insulin (6
). This result suggests that intra-islet DCs contain intact Ags taken up from β cells, consistent with our finding with the MIP-GFP reporters. Immature DCs have been shown to maintain Ag in its unprocessed form and begin processing upon receiving a maturation signal, such as LPS (37
). DCs containing unprocessed proteins have also been observed in vivo and are generally found to be present at very low frequencies (38
). The maintenance of large amounts of unprocessed Ags may also be a physiological correlate of maintenance of protein aggregates within specific compartments in DCs (40
). Our observation extends these previous findings to demonstrate that virtually all tissue-dwelling DCs stably maintain unprocessed protein in the absence of inflammatory signals. Because the tertiary structure of GFP and its ability to fluoresce are very sensitive to low pH environment, our results suggest that the endocytosed tissue Ags are maintained in a pH neutral environment, such as early endosomes, and maturation stimuli induced their transition to an acidic compartment for processing and presentation.
A previous study elegantly demonstrated in the B6.RIP-mOVA model that DCs constitutively migrate from tissues to present tissue- specific Ags in the draining LNs in the absence of overinflammatory stimulations (41
). Once reaching the LNs, migratory DCs are short-lived; thus, they need to be continuously replenished from the tissue. In this study, we observed that virtually all DCs in the islets contained intact tissue Ags, whereas DCs bearing intact tissue Ags were very rare in the draining LNs of the same animal. This result suggests that processing of acquired tissue Ags normally precedes the migration of the DCs to the draining LNs. However, this coordinated process became deregulated under extreme inflammatory conditions, such as those observed in earlier backcrosses of NOD.MIP-GFP mice, in fully backcrossed NOD. MIP-GFP.CD28KO, and fully backcrossed NOD.MIP-GFP receiving a large dose of activated diabetogenic T cells. This deregulation allowed a few of the islet-derived DCs to reach the LN before the complete processing of acquired GFP and other islet Ags. It should be noted that these GFP+
DCs only represented a fraction of islet Ag-bearing DCs in the pancreatic LN because many islet-derived DCs fully process the acquired Ag, thus losing GFP fluoresce before arriving in the pancreatic LN.
A recent study directly examined the steady-state presentation of another islet model Ag, hen egg lysozyme, which was driven by a transgenic insulin promoter (6
). In that system, up to 90% of intra-islet DCs and 50% of PLN DCs expressed processed hen egg lysozyme peptide-MHC complex on the cell surface. Our use of GFP model Ag in this study showed that unprocessed Ags were present in nearly all intra-islet DCs in steady state. These results together suggest that, in the absence of inflammation, intra-islet DCs have a limited capacity to process and present acquired Ag, allowing them to display processed Ags on the cell surface while retaining unprocessed Ag intracellularly. The absence of DCs with intact Ag in the draining LN suggests that a fraction of islet-resident DCs must degrade all acquired tissue Ags before migrating to LNs. This steady-state activity is enhanced by intra-islet inflammation leading to DC maturation, loss of unprocessed Ag in majority of the DCs, and enhanced migration to PLNs.
In contrast to DCs matured by TLR ligands, the mature DCs we observed in inflamed islets did not express inflammatory cytokines; thus, phenotypically they resembled DCs activated by inflammatory cytokines and mechanical disruptions (42
). These previous studies suggest that such alternatively activated DCs are tolerogenic because they fail to elicit IFN-γ production, but promoted IL-10 secretion from T cells. It is important to point out that these studies were conducted with bone marrow-derived or secondary lymphoid tissue-resident DCs that are likely distinct from peripheral tissue-resident DCs in their maturation status and functional capacities (35
). In our models, maturation of islet-resident DCs was clearly associated with T cell activation and tissue destruction. Furthermore, DCs isolated from inflamed islets in NOD mice were sufficient to induce T cell proliferation and secretion of IL-2, IFN-γ, and TNF-α ex vivo (K. Melli and Q. Tang, unpublished result). Thus, these in vivo T cell-matured DCs were immunogenic and not tolerogenic.
Taken together, we have shown that T cell-mediated inflammation in the islets also induced maturation of islet-resident DCs and their LN migration. Thus, islet inflammation results in an increase of mature islet Ag-presenting DCs in the PLNs that may lead to further priming of β cell Ag-reactive T cells. We propose that this represents a positive feedback mechanism to amplify the ongoing autoimmune response. Mature DCs arriving in the PLNs can prime more islet-reactive T cells, thus recruiting a broader repertoire of T cells to the autoimmune response. Additionally, mature DCs that remain in the islets may serve to reactivate newly arrived primed T cells to activate their effector functions and/or to reprime resident T cells to maintain their cytotoxic potential (44
), leading to increased β cell destruction. This model is consistent with a previous report demonstrating an essential role of DCs in sustaining anti-islet immune responses (12
) and provides a mechanistic framework of DC function in this context. Furthermore, results from our study suggest that therapeutic interventions that interrupt this amplification loop may help to prevent further tissue destruction and preserve the remaining islet mass in patients afflicted with autoimmune diabetes.