Cutting Edge: Merocytic Dendritic Cells Break T Cell Tolerance to b Cell Antigens in Nonobese Diabetic Mouse Diabetes Jonathan D. Katz,*,†,1 Jennifer K. Ondr,*,‡,x,1 Robert J. Opoka,*,† Zacharias Garcia,{ and Edith M. Janssen† In type 1 diabetes, the breach of central and peripheral tolerance results in autoreactive T cells that destroy insulin-producing, pancreatic b cells. In this study, we identify a critical subpopulation of dendritic cells responsible for mediating both the cross-presentation of islet Ags to CD8+ T cells and the direct presentation of b cell Ags to CD4+ T cells. These cells, termed merocytic dendritic cells (mcDCs), are more numerous in the NOD mouse and, when Ag-loaded, rescue CD8+ T cells from peripheral anergy and deletion while stimulating islet-reactive CD4+ T cells. When purified from the pancreatic lymph nodes of overtly diabetic NOD mice, mcDCs break peripheral T cell tolerance to b cells in vivo and induce rapid onset type 1 diabetes in the young NOD mouse. Thus, the mcDC subset appears to represent the longsought APC responsible for breaking peripheral tolerance to b cell Ags in vivo. The Journal of Immunology, 2010, 185: 1999–2003.
T
he autoimmune attack by T cells on insulin-producing pancreatic b cells results in type 1 diabetes (T1D) in both humans and NOD mice (reviewed in Ref. 1). T cells participate in all phases of the disease from the initial insulitis to the selective destruction of b cells by both direct and indirect cytolysis (2, 3). How these rogue T cells escape both central and peripheral tolerance is not fully understood, but both CD4+ and CD8+ T cells are required (4–6). The naive islet-reactive T cells are stimulated by dendritic cells (DCs) that drain from the pancreas and accumulate in the pancreatic lymph nodes (PLNs) after acquiring self-Ag from degranulating and apoptotic b cells (7, 8). Whereas CD4+ T cells are activated by islet Ags presented on class II MHC, CD8+ T cells require cross-presentation on MHC class I by a specialized DC subset (9). The clearance of apoptotic self cells by DCs is generally noninflammatory, even tolerogenic (10). Yet, under certain *Division of Endocrinology, Diabetes Research Center, †Division of Molecular Immunology, and ‡Division of Immunobiology, Cincinnati Children’s Research Foundation; xDepartment of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229; and {La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037 1
J.D.K. and J.K.O. contributed equally to this work.
Received for publication April 30, 2010. Accepted for publication June 18, 2010. This work was supported by Juvenile Diabetes Research Foundation Grant 5-2008944 (to J.D.K. and E.M.J.) and National Institutes of Health Grants R01 DK08179 (to J.D.K.) and R21 AI079545 (to E.M.J.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001398
situations, Ag acquired from apoptotic self cells is proinflammatory, leading to the priming of self-reactive T cells (11); this appears to be the case in T1D-prone animals. However, which DC subset is responsible and how this process unfolds mechanistically are not well understood. Recently, we found that the ablation of conventional DCs (cDCs) in vivo resulted in the loss of CD4+ T cell activation, insulitis, and diabetes (12). This cDC subset expressed CD11c but not CD8a or CD317 and had varied levels of CD11b; we referred to these cells as myeloid DCs but have since found that they contain not only CD11b+ DCs but also a novel subset of CD11c+CD11b2/loCD8a2 DCs that acquired small particulate fragments of Ag from apoptotic cells and, unlike other DC subsets, were capable of processing and presenting Ag in a nontolerogenic manner to both CD4+ and CD8+ T cells (11). These DCs, termed merocytic DCs (mcDCs), stored Ag from apoptotic cells in discrete, punctate vesicles in their cytoplasm, meros (m«ros, particle in Greek). Given that mcDCs resided within the major stimulatory fraction of cDCs and were particularly effective at breaking tolerance to self-Ags, we hypothesized that mcDCs may represent the critical cDC subset necessary for the activation of both b cell-specific CD4+ and CD8+ T cells in vivo.
Materials and Methods Mice All mice were purchased from The Jackson Laboratory (Bar Harbor, ME), except BDC2.5/NOD.Rag12/2 (13) and OT-I (CD45.1) TCR transgenic, B6.PL (CD90.1), rat insulin promoter (RIP)-membrane-bound OVA (mOVA) transgenic B6 (14), and mOVA/B6.Kb2/2 (15) mice, which were bred in our existing specific pathogen-free barrier colony. All experiments were performed under approved Institutional Animal Care and Use Committee guidelines.
Abs All Abs were purchased from Biolegend (San Diego, CA) except anti-murine plasmacytoid DC Ag-1 (PDCA-1), which was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Address correspondence and reprint requests to Dr. Jonathan D. Katz, Cincinnati Children’s Research Foundation, 3333 Burnet Avenue, Room S5.405, MLC 7021, Cincinnati, OH 45229. E-mail address:
[email protected] The online version of this article contains supplemental material. Abbreviations used in this paper: cDC, conventional dendritic cell; DC, dendritic cell; FLT3L, FLT3 ligand; mcDC, merocytic dendritic cell; mOVA, membrane-bound OVA; PDCA, plasmacytoid dendritic cell Ag; PLN, pancreatic lymph node; RIP, rat insulin promoter; T1D, type 1 diabetes; WT, wild-type. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
2000
CUTTING EDGE: mcDCs IN T1D IN THE NOD MOUSE
DC purification and transfer DCs were expanded in vivo using the B16-FLT3 ligand (FLT3L) model (described in Ref. 16). cDCs were purified from spleen and lymph nodes, by MACS, followed by cell sorting (to 93–97% purity). Cell transfers were performed as described (12). For isolation of DCs from recent-onset diabetic mice, the PLNs were harvested from ∼40 NOD mice with a blood glucose $ 300 mg/dl cells, and the cells were stained with anti-CD11c, anti-CD11b, anti-CD8a, and a mixture of FITC-conjugated mAb to B220, CD19, TCR-b, CD3, NKR-P1C, and CD49b to exclude plasmacytoid DCs, NK DCs, IFNproducing killer DCs, B and T cells, NK cells, and NKT cells. CD11c+ cells were sorted into either CD11b2 (mcDCs) or a pool of CD11b+ DCs and CD8a+ DCs and transferred (i.v., 4–5 3 105) into 28-d-old NOD mice as above.
Islet cell Ag B6 islets were isolated using the technique of Lacy and colleagues (17) for rat islets, dispersed using trypsin, irradiated (30 Gy) to induce apoptosis, and labeled with CFSE, then mixed at a ratio of 20 cDCs to 1 islet cell overnight. Dead cells were removed using a dead cell removal kit (Miltenyi Biotec).
T cell proliferation Ag-loaded DCs were used to stimulate sort-purified naive T cells (CD62L+ T cells from BDC2.5/Rag12/2 mice). T cell proliferation assays were performed in vitro using [3H]thymidine deoxyribose incorporation (ICN Biomedicals, Irvine, CA) as described previously (12), whereas in vivo proliferation assays were performed using CFSE decay as described previously (18).
Type 1 IFN assay Detection of IFN from DCs incubated with irradiated splenocytes was performed using a bioassay as described previously (19).
Imaging flow cytometry analysis The uptake of CFSE-labeled irradiated islet cells was assessed using an ImageStream analyzer (Amnis, Seattle, WA). Internalization of CFSE particles from irradiated islet cells was determined by masking DC cytoplasm, followed by a subtracting of the nucleus (DRAQ5+; Biostatus, Leicestershire, U.K.), and then applying a spot mask on CFSE particles.
Insulitis and diabetes Insulitis and diabetes was assessed as described previously (20).
Statistics Statistical analysis was performed using the GNU/Linux application, Qtiplot (version 0.9.7).
Results and Discussion 2/lo Elevated levels of CD11b
DCs in NOD mice
mcDCs reside within the CD11b2/lo subset of cDCs. Thus, to determine whether NOD mice had altered numbers of mcDCs, we compared the frequency of mcDCs in the spleen and PLNs of age-matched NOD, C57BL/6 (B6), and BALB/c mice. As shown in Fig. 1A and 1B, the frequency and absolute numbers of mcDCs were initially similar among NOD, B6, and BALB/c mice but increased in NOD mice as they aged; this is consistent with the overall rise in DCs seen by others in the spleen and blood of aging NOD mice (21). The expansion of mcDCs coincided with the appearance of insulitis (reviewed in Refs. 2, 22), and it is important to note that the mcDCs differed both phenotypically and functionally from the abnormal or altered DC subsets described by others (21, 23, 24) in the NOD mice and were found in the nonactivated DC fraction in all mice strains tested (including nonobese resistant; data not shown). Enhanced type 1 IFN production by NOD mcDCs
One hallmark of mcDCs is their production of type 1 IFNs upon an encounter with irradiated/dying cells that is vital for the priming of both CD4+ and CD8+ T cells (11). We assessed IFN production by mcDCs from both the PLNs and spleen
of NOD mice and found that compared with BALB/c mice, NOD mcDCs produced elevated levels of IFN upon coculture with irradiated splenocytes (Fig. 1C). These data suggested not only that NOD mice show elevated levels of mcDCs with age, but also show elevated production of proinflammatory IFNs. These data agree with recent findings suggesting that type 1 IFNs are vital initiators of T1D in NOD mice (25). Expansion of mcDC in vivo by FLT3L
Although the existence of mcDCs in NOD mice was considerable, their overall numbers per mouse, like that of other DC subsets, remain low. Thus, to undertake meaningful mechanistic studies, we took advantage of the ability of FLT3L to expand these cells in vivo. This was done using the B16-FLT3L model (described in Ref. 16). Briefly, the FLT3L-expressing tumor B16-FLT3L was injected s.c. (4–5 3 107) on the back of NOD mice and produced biologically active FLT3L that drove the in vivo expansion of several DC subsets, including mcDCs by 30–35-fold within 7–10 d. To assess whether FLT3L altered the phenotype of mcDCs, FLT3L-driven and wild-type (WT) NOD DC subsets were compared. FLT3L did not alter the expression of costimulatory ligands or MHC class I and II on mcDCs or any other DC subset (Supplemental Fig. 1). Moreover, in vivo-expanded mcDCs acquired and presented Ag similarly to WT NOD mcDCs (see below). Thus, unless otherwise indicated, subsequent experiments were performed using in vivo FLT3L-expanded mcDCs. Acquisition of cell fragments from irradiated islet cells by NOD mcDCs
mcDCs are functionally unique. Unlike their other cDC counterparts, mcDCs acquire Ag by merocytosis, or nibbling small fragments of Ag from dead or dying cells, rather than by macropinocytosis, hence their original description as nibbling DCs (11). This includes cellular Ag from cells that die by either gamma or UV irradiations and CD95 cross-linking (11). Moreover, the antigenic fragments acquired by merocytosis are retained by mcDCs as visually distinct punctate cytoplasmic vesicles. Thus, to establish whether the elevated numbers of CD11b2/loCD8a2PDCA-12 DCs observed in NOD mice were in fact functional mcDCs, we performed Ag acquisition studies with NOD DCs using CFSE-labeled irradiated islet cells as targets. In this study, CD11b+ DCs and mcDCs were isolated by magnetic beads from NOD splenocytes and then mixed with CFSE-labeled irradiated islet cells overnight. Postculture, the DCs were purified away from dead cells, labeled with mAb to CD11b, CD11c, and the nuclear dye DRAQ-5, and analyzed using an imaging flow cytometry. (Overall DC survival postculture can be found in Supplemental Fig. 2.) Representative single and composite images for each subset incubated with or without irradiated b cells are shown in Fig. 2A. Note that the CD11b+ DCs incubated with irradiated, CFSE-labeled islet cells exhibited diffuse CFSE staining within the cytoplasm, typical of phagocytosis and catabolism of ingested cells, whereas the corresponding mcDCs contained cells with distinct punctate vesicles containing CFSE. This is more easily seen in the composite image galleries shown in Fig. 2B. In this study, several of the CD11b+ DCs have not only widely distributed CFSE staining but also cytoplasm-localized apoptotic bodies (DRAQ-5+), consistent with macropinocytosis. In contrast, the mcDCs exhibit multiple merocytic vesicles of moderate to high intensity for CFSE.
The Journal of Immunology
2001
FIGURE 1. Dynamic rise in frequency of mcDC in NOD mice. A, Six-week-old NOD and B6 mice were compared using flow cytometry. Total CD11c+ DC (top panels) were gated for plasmacytoid DCs (PDCA1+B220+) and cDC populations (middle panels), and then the cDCs (PDCA-12B2202) further subdivided into CD11b+ DCs, CD8a+ DCs, and mcDCs (bottom panels). B, NOD mcDCs increase with age in spleen compared with controls, shown as mean 6 SD of three to five mice per time point. Similar results were seen in PLNs (not shown). C, Increased IFNs produced by activated mcDCs from NOD mice with age as measured from PLNs and spleen DCs after overnight stimulation with irradiated cells; three mice per time point. *p , 0.01; ‡p , 0.001.
To quantify the frequency and intensity of the Ag particles, we applied a spot mask filter described in Supplemental Fig. 3. The mcDC fraction had more cells with multiple CFSEcontaining cytoplasmic vesicles compared with CD11b+ DCs (Fig. 2C), and the mean particles per cell for mcDCs was statistically elevated compared with CD11b+ DCs (Fig. 2D). The CFSE particle intensity was likewise greater in mcDCs than in CD11b+ DCs, reflecting the tight punctate staining seen in the mcDC subset (Fig. 2E). Thus, the imaging flow cytometry revealed that the CD11b2/lo DCs defined in Fig. 1 were one in the same with the functionally defined mcDC subset (Fig. 2).
Efficient presentation of islet cell Ags to naive CD4+ T cells by mcDCs
To establish if NOD mcDCs were more efficient at presenting self-Ag than CD11b+ DCs on a per-cell basis, we undertook Ag presentation studies using mcDCs and CD11b+ DCs as APCs and naive CD4+ BDC2.5 T cells as responders. The mcDCs and CD11b+ DCs were prepared as above and mixed with irradiated islet cells. After 16 h, the mcDC and CD11b+ DC subsets were sort-purified and used as APCs in a 3-d T cell proliferation assay. The Ag-exposed mcDCs were substantively better APCs than their CD11b+ DC counterparts (9.2 6 1.2-fold; Fig. 2F). The CD11b+ DCs were, however, equally effective at presenting exogenous synthetic
FIGURE 2. mcDCs have small, high-intensity particles of irradiated islet cells and present islet Ags efficiently to CD4+ T cells. A, Representative cell morphology of CD11b+ DCs and mcDCs fed irradiated, CFSE-labeled islet cells in vitro imaged using an imaging flow cytometer. The composite image at far right is an overlay of the CD11c, CFSE, and DRAQ-5 (nuclear) images (original magnification 340). B, Representative composite images from CD11b+DCs and mcDCs fed irradiated CFSE-labeled islet cells. Yellow scale bar, 10 mm. C, Quantitative analysis of particles per cell for mcDCs and CD11b+ DCs from NOD mice. The mcDC subset had a higher mean particle score (D) and higher fluorescence intensity (E). Representative result from seven independent experiments. F, Efficient presentation of islet cell Ags to naive CD4+ T cells by mcDCs as compared with CD11b+ DCs in a 72-h proliferation assay (left panel); circles denote mcDCs and squares CD11b+ DCs; filled symbols denote exposure to apoptic islets overnight prior to use in assay. Both subsets were equally effective at presenting peptide Ags to BDC2.5 T cells (right panel). Representative of three independent experiments. ‡p , 0.001.
2002
peptide Ag (Fig. 2F). These data suggest that mcDCs were more effective at presenting self-Ag from irradiated cells. In addition, the mcDCs’ enhanced priming capacity may be due to their ability to sustain Ag presentation, as we have recently found that B6 mcDCs stored merocytosed material in nonacidic vesicles that functioned as a long-term depot for Ags, which allowed prolonged Ag presentation by mcDCs (E.M. Janssen, manuscript in preparation). mcDCs break CD8+ T cell tolerance by cross-presentation of islet Ags in vivo
To assess the ability of mcDCs to break CD8+ T cell tolerance to islet Ags, we used the well-established OT-I model (14, 26). In this model, naive CD8+ OT-I T cells (OVA-specific), but not control CD8+ T cells, undergo tolerance by deletion when transferred into mice expressing membrane-bound OVA on the pancreatic b cells (RIP-mOVA). When OT-I T cells encountered their Ag in these mice, they first showed signs of proliferation (CFSE decay) followed by deletion [total cell loss (27)]. This is likewise true when freshly purified mcDCs were transferred 1 d after the OT-I T cells (Fig. 3A, top panels). However, when the transferred mcDCs were first exposed to irradiated cells from Kb2/2 mice expressing mOVA (mOVA/ Kb2/2), the OT-I T cell did not undergo deletion in the RIP-mOVA host, but rather showed significant proliferation in both the spleen and PLNs, resulting in OT-I T cell accumulation and persistence as demonstrated by the increase in the ratio of OT-I to control CD90.1 T cells (Fig. 3A, bottom panels). Moreover, the activated OT-I T cells induced substantial insulitis by day 12 posttransfer (Fig. 3A). Importantly, CD8a+ DCs failed to rescue OT-I T cells from deletion, even when the CD8a+ DCs were preincubated with irradiated mOVA/Kb2/2 cells (Fig. 3A, middle panels). The rescue of OT-I T cells by mcDCs was the result of cross-priming as the Ag-donating mOVA cells came from mice lacking MHC class I (Kb2/2) and thus could not supply preloaded Kb complexes for representation. Thus, these results, with those from Fig. 2F, suggest that mcDCs can present Ags from apoptotic cells to both naive CD4+ and CD8+ T cells and can drive immunopathology.
CUTTING EDGE: mcDCs IN T1D IN THE NOD MOUSE Transfer of mcDCs induces diabetes in NOD mice
To assess whether mcDCs were able to break tolerance and induce diabetes in NOD mice, we performed transfer studies in which DCs from NOD mice were incubated with irradiated islet cells overnight, then sort-purified and transferred into 28-d-old NOD recipients. Eight to nine days posttransfer, all NOD mice that received Ag-loaded mcDCs were diabetic (Fig. 3B). In contrast, the pooled Ag-exposed CD11b+ DCs and CD8a+ DCs (other cDC in Fig. 3B) were significantly poorer in transferring disease, exhibiting a statistically slower time to disease with fewer diabetic mice. The overall severity of insulitis was, likewise, markedly enhanced in mice that received Ag-loaded mcDCs (Fig. 3B). Until now, the mcDCs from FLT3L-treated NOD were exposed to irradiated islet cells ex vivo. Therefore, it became critical to determine if mcDCs could naturally acquire Ag in vivo and likewise transfer diabetes to young NOD mice. To this end, cDC subsets, either mcDCs or a pool of CD8a+ DCs and CD11b+ DCs, were sort-purified from PLNs of recent-onset diabetic WT NOD females ($18 wk) and immediately transferred without additional manipulation into 28-d-old female NOD recipients. Importantly, the recipients had normal levels of mcDCs at time of transfer. All NOD mice that received mcDC from the PLNs of diabetic mice developed rapid-onset diabetes (Fig. 3C). The tempo and incidence of disease were similar to those seen when in vitroloaded mcDCs were used (Fig. 3B). None of the NOD mice that received pooled (other cDC) or were left untreated developed diabetes within the assay period (Fig. 3C). Importantly, these data suggested that the mcDC subset: 1) acquires islet Ags naturally in vivo; 2) was a competent APC when transferred to young disease-free recipients; 3) was capable of breaking of self-tolerance to islet cell Ags; and 4) was capable of hastening diabetes development in vivo. Moreover, mcDCs were not only present but also elevated in NOD mice compared with other strains and accumulated in NOD mice with time. Together, these results lead us to conclude that mcDCs are the likely CD11c+ DC subset responsible for the priming of both diabetogenic CD4+ and CD8+ T cells and the functional break in immune tolerance to islet Ags observed in NOD mice.
FIGURE 3. mcDCs break T cell tolerance, induce insulitis, and transfer diabetes in vivo. A, RIP-mOVA (CD45.2) mice received 106 CFSE-labeled CD8+ OT-I T cells (CD45.1) together with CD8+ irrelevant control T cells (CD90.1) on day 0 and then either mcDCs or CD8a+ DCs exposed to irradiated cells from mOVA/ Kb-deficient mice on day 1. On day 3, proliferation and accumulation of OT-I and control T cells was determined. Only mcDCs exposed to mOVA broke T cell tolerance by cross-priming OT-I and induced insulitis (day 12) (H&E, original magnification 340). B, A pool of CD11b+ DCs, CD8a+ DCs, and mcDCs from NOD mice were exposed to irradiated islet cells in vitro, purified, and transferred (5 3 105) to 4-wk-old NOD recipients. Recipients that received mcDCs but not the other cDCs rapidly developed T1D. Mice receiving mcDCs had more advanced insulitis; six to nine mice per group. Peri-insulitis, white bars; moderate insulitis, gray bars; severe insulitis, black bars (H&E, original magnification 340). C, Rapid onset of T1D and insulitis in NOD mice given sort-purified mcDCs isolated from the PLNs of overtly diabetic female NOD mice. The mixed CD8a+/CD11b+ cDC subset did not induce T1D; three to five mice per group.
The Journal of Immunology
Disclosures The authors have no financial conflicts of interest.
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