Cell-intrinsic effects of non-MHC NOD genes on dendritic cell generation in vivo
Simon J. Prasad1,2 and
Christopher C. Goodnow1
1 Medical Genome Centre, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia 2 Present address: Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand
Correspondence to: C. Goodnow; E-mail: Chris.Goodnow{at}anu.edu.au
Transmitting editor: A. Kelso
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Abstract
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Genes outside the MHC create a general susceptibility to autoimmunity in non-obese diabetic (NOD) mice. Here we describe marked differences in dendritic cell generation in vivo, caused by non-MHC NOD genes. Analyses of splenic dendritic cells from the autoimmunity-prone NOD.H-2k mice revealed a relative over-representation of the CD8
subsets, in contrast to the level of these subsets observed in the autoimmunity-resistant B10.H-2k congenic strain or other H-2k strains. The imbalance towards CD8
dendritic cells was selectively manifested by NOD.H-2k-derived cells in radiation chimeras reconstituted with equal mixtures of NOD.H-2k and B10.H-2k bone marrow cells. In addition to the cell-intrinsic imbalance in dendritic cell subsets, the myeloid lineage overall was intrinsically altered by NOD genes, as this lineage was disproportionately derived from the NOD.H-2k donor in mixed chimeras. These results identify a striking effect of non-MHC NOD genes upon the balance of dendritic cell subsets that may contribute to the generalized defects in self-tolerance in the NOD strain.
Keywords: autoimmune diabetes, dendritic cell subsets, H-2 congenic NOD mice, myeloid precursors, non-MHC NOD genes
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Introduction
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The non-obese diabetic (NOD) mouse is a model for human autoimmune insulin-dependent diabetes mellitus (IDDM) or type I diabetes (1). In NOD mice, autoimmune diabetes is a triphasic process characterized by an early appearance of macrophages and dendritic cells in pancreatic islets of Langerhans at 3 weeks of age, followed by lymphocytic infiltration at 810 weeks of age and finally the onset of overt diabetes at 1620 weeks of age. Overt diabetes occurs as a result of T cell-mediated destruction of insulin-producing ß cells in the islets. Genetic susceptibility to autoimmune diabetes is complex, conferred by at least 18 susceptibility loci (Idd118) within and outside the MHC in the NOD mouse (2,3). Analyses of MHC congenic NOD strains indicate that non-MHC diabetogenic loci create an overall susceptibility to autoimmunity in the NOD mouse, while particular MHC alleles determine the target tissues for autoimmune destruction (4). Thus, autoimmune pathology may be directed against the pancreas, thyroid or salivary glands, depending on the genotype of the MHC on the NOD background. Mapping studies and reciprocal experiments placing the NOD MHC on other strain backgrounds also show that the unique NOD MHC haplotype, Idd1, is necessary, but insufficient, to cause diabetes. A key issue for understanding the role of non-MHC NOD diabetogenic loci is identification of the primary cell types and cellular functions dysregulated by these genes.
Part of the genetic predisposition to autoimmune diabetes resides in hematopoietic stem cells and their progeny. In radiation chimeric mice, bone marrow-derived cells from NOD mice can reconstitute diabetes susceptibility in disease-resistant strains that carry Idd1 and a subset of non-MHC NOD genes (5,6). It is not yet known to what extent this susceptibility resides in lymphocytes, antigen-presenting cells (APC) or other bone marrow derivatives. The possibility that NOD genes have a primary effect in the APC compartment is raised by the finding that myeloid-derived dendritic cells and macrophages constitute the initial infiltrate into pancreatic islets of young pre-diabetic NOD mice, prior to infiltration by lymphocytes (7). Furthermore, dendritic cells and macrophages have been demonstrated to be the initial and principal producers of the proinflammatory cytokine, tumor necrosis factor (TNF)-
, in pancreatic islets (8). Dendritic cells form a system of APC with a pivotal role in initiating and modulating T cell-mediated immune responses (9). Dendritic cells have a well-established role in promoting T cell responses in processes like rejection of tissue grafts in vivo (10), generation of Th1 type responses in vitro (11) and inducing T cell-mediated insulitis in a transgenic model (12). Here we describe a unique and striking dysregulation of dendritic cell formation caused by non-MHC genes from NOD mice.
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Methods
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Mice
Inbred B10.BR, NOD.H-2k [gift of Dr Linda Wicker, Merck Research, Rahway, NJ (13)], BALB.H-2k, CBA/H, AKR and (B10.BR x NOD.H-2k)F1 hybrid mice were housed under specific pathogen-free conditions in microisolator cages in the Medical Genome Centre or the Animal Services Unit at the JCSMR. BALB.H-2k mice were purchased from the Animal Resources Centre, Perth, Australia. Mice used in this study were 816 weeks of age, and where necessary matched for age and sex. Animals were cared for and used in accordance with principles outlined by the Australian National University Animal Experimentation Ethics Committee and the current Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Flow cytometry
Flow cytometry was performed using the standard argon laser FACScan or BD LSR six-color cytometer (Becton Dickinson, San Jose, CA). Data acquisition and analyses were conducted using the CellQuest or CellQuest Pro software packages. Data from separate individuals from each type were compared using Students t-test, with ratios first converted to logarithms. Dead cells and cellular debris were eliminated from analysis by propidium iodide exclusion or appropriate forward and side scatter gating. The staining reagents used were: 10-3.6.2 (anti-I-Ak)FITC (gift of Professor H. McDevitt, Stanford University, Stanford, CA), CD4 (GK1.5)biotin (PharMingen, San Diego, CA), CD8
(53-6.7)phycoerythrin (PE) (Caltag, South San Francisco, CA), CD8
PerCP (PharMingen), CD11b (Mac-1
, M1/70)FITC or biotin (Caltag), CD19 biotin (PharMingen), CD45R/B220, RA3-6B2FITC, RA3- 6B2PE, RA3-6B2biotin (Caltag), Gr-1 (RB6-8C5)biotin (PharMingen), Ly5a (CD45.1), AS20FITC or PE or biotin; Ly5b (CD45.2)FITC or biotin (PharMingen), N418 (anti-CD11c)biotin (gift from Dr S. Townsend, University of British Columbia, Vancouver, BC, Canada) and Thy1.2 FITC (Caltag). Biotinylated reagents were detected with Streptavidin (SA)PE or SAallophycocyanin.Cy7 (Caltag) or SATriColor (PharMingen). The Ly5 mAb are of mouse IgG2a isotype and serve as internal isotype controls for each other, and for 10-3.6.2 (anti-I-Ak, also of mouse IgG2a isotype), when these mAb are used in combination.
Staining whole blood
Required staining cocktails were prepared by first preincubating primary mAb with appropriate secondary reagents in FACS buffer at 4°C for time required to bleed mice. Where required, other directly conjugated primary mAb were subsequently added to the cocktail. Whole blood (2 µl) was stained at 4°C for 20 min with appropriate mAb in a total 25 µl stain volume. Each well was then diluted with 175 µl RPMI containing 4 x 103 counting beads/100 µl RPMI. Stained blood samples were then transferred to cluster tubes and taken immediately for acquisition. Samples were acquired at a slow flow rate until 400 beads were acquired.
Enriching for splenic and thymic dendritic cells
The protocol to enrich for splenic and thymic dendritic cells was adapted from Vremec and Shortman (14). Briefly, spleens or thymi were cut into small fragments and digested in collagenase (Boehringer Mannheim, Mannheim, Germany) and treated with EDTA. Cells recovered from the digest were layered onto 14.5% (w/v) metrizamide (Nycomed Pharma, Oslo, Norway) and centrifuged at 800 g for 10 min at room temperature to enrich for low-density cells. The dendritic cell-enriched low-density cells were analyzed by flow cytometry for appropriate cell surface markers.
Bone marrow reconstitution assays
Total bone marrow leukocytes (2 x 106) from female B10.H-2k and NOD.H-2k mice (812 weeks old) were i.v. injected into
-irradiated (2 x 5 Gy) B10.H-2k recipients. Control groups of recipient mice received either B10.H-2k or NOD.H-2k bone marrow cells alone while the test group of mice received an inoculum comprising a 1:1 mixture of bone marrow cells from both strains. Recipient mice were given drinking water containing polymyxin B (8.5 x 105 U/ml) and neomycin (1.1 mg/ml) ad libitum for the duration of reconstitution. Recipient mice were screened by flow cytometry for the level of chimerism in peripheral blood 5 weeks post-reconstitution. The allelic variants of Ly5 were used to distinguish between strain-specific cells. Further analysis of recipient mice was done 1012 weeks post-reconstitution by flow cytometric determination of the origin splenic and thymic dendritic cells, and blood monocytes.
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Results
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Non-MHC NOD genes create a bias towards the CD8
dendritic cell subsets in vivo
To explore the possibility that non-MHC diabetogenic loci from NOD mice act by altering dendritic cell function, splenic dendritic cells from NOD.H-2k, B10.H-2k and other control H-2k haplotype strains were analyzed by flow cytometry. Splenic dendritic cells were prepared by collagenase digestion and enrichment on metrizamide gradient as previously described (14). Dendritic cell characterization experiments were performed by pair-wise analysis of B10.H-2k and NOD.H-2k mice matched for age and sex. Dendritic cells were identified on the basis of high forward scatter (relative size) and N418 (CD11c) (15) staining, and analyzed for CD8
expression. Resolution of the CD8
-bearing dendritic cell subset revealed a marked paucity of this subset, or the relative excess of the CD8
subsets, in the NOD.H-2k strain (Fig. 1A). The CD8
+ dendritic cell subset constituted 12% of all splenic dendritic cells in the NOD.H-2k strain compared with 44% in the B10.H-2k strain. The bias towards the CD8
dendritic cell subsets in the NOD.H-2k strain is clearly depicted when the ratio of CD8
to CD8
+ dendritic cell subsets is compared with the ratio in the B10.H-2k strain (Fig. 1B). The mean ratio of CD8
to CD8
+ dendritic cell subsets is 1.5 in the B10.H-2k strain, while it is 6.5 in the NOD.H-2k strain. The difference between the ratios from the two strains is statistically significant (P = 0.001, Students t-test).
It was important to establish whether the bias towards the CD8
dendritic cell subsets in the NOD.H-2k strain occurred in absolute or relative terms. Splenic and thymic dendritic cells from B10.H-2k and NOD.H-2k mice were prepared in parallel as described in Fig. 1(a). Inclusion of counting beads in the assay enabled absolute quantitation of tissue-derived dendritic cells. Results from a series of separate experiments are expressed as the relative yield of splenic or thymic dendritic cells from the two strains in order to control for variability in dendritic cell preparation from one experiment to another (Fig. 1C). It is clear from this analysis that the ratio is
1 in both the spleen and the thymus, indicating that similar absolute numbers of splenic or thymic dendritic cells are present in B10.H-2k and NOD.H-2k mice.
To examine whether the imbalance towards the CD8
dendritic cell subsets was uniquely conferred by non-MHC NOD genes, three other H-2k strains, AKR, CBA/H and BALB.H-2k were analyzed for splenic dendritic cell subsets. In AKR mice, the ratio of CD8
to CD8
+ dendritic cells (1.6) was comparable to that seen in B10.H-2k mice. CBA/H and BALB.H-2k have an intermediate ratio (
3.5) relative to that seen in NOD.H-2k mice. Interestingly, the CD8
to CD8
+ ratio was also intermediate in (B10.H-2k x NOD.H-2k)F1 hybrids, compared with the parental strains.
To further investigate potential dendritic cell differences, CD11c+ (N418+) cells from B10.H-2k and NOD.H-2k mice were analyzed for cell surface MHC class II expression. Splenic and thymic dendritic cells from NOD.H-2k mice displayed a consistent difference in the cell surface expression of MHC class II molecules when compared with dendritic cells from the B10.H-2k strain. Most splenic dendritic cells from the B10.H-2k strain expressed high levels of cell surface MHC class II molecules (Fig. 2A, gate R3). By contrast,
50% of dendritic cells from the spleen of NOD.H-2k mice displayed low levels of MHC class II (Fig. 2A, gate R2). Notably, the MHC class IIlow cells in the spleen also express a lower level of CD11c. A similar shift towards MHC class IIlow cells was apparent in thymic dendritic cells from NOD.H-2k mice. The mean ratio of MHC class IIhigh- to MHC class IIlow-expressing dendritic cells in the thymus of B10.H-2k mice is 0.75 (Fig. 2B), compared to a value of 0.25 in NOD.H-2k mice (Students t-test, P = 0.002). The ratio in the B10.H-2k and NOD.H-2k spleen is 2.5 and 1 respectively (P < 0.001).

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Fig. 2. Heterogeneity in cell surface MHC class II expression in CD8 dendritic cell subsets. Dendritic cells from B10.H-2k or NOD.H-2k spleens and thymi from individual mice were prepared and analyzed as described in Fig. 1. (A) Representative FACS plots of spleen or thymic cells. Elliptical or rectangular regions depict subsets of dendritic cells with low versus high cell surface MHC class II expression. The percent of low-density cells within these regions is shown. (B) The ratio of MHC class IIhigh to MHC class IIlow dendritic cells in the spleen and thymus of B10.H-2k and NOD.H-2k mice. Note: MHC class IIlow cells in the spleen also show reduced CD11c expression. Circles represent ratio from individual mice and columns show mean ratios. Data is compiled from three separate experiments involving pair-wise analysis of B10.H-2k and NOD.H-2k mice. The difference in splenic or thymic ratios between strains was tested for statistical significance using the Students t-test. *P = 0.002, #P < 0.001. (C) Splenic dendritic cells (identified on the basis of CD11c and MHC class II expression) from B10.H-2k or NOD.H-2k were co-stained with mAb against CD11b, Gr-1, B220, CD4 or CD19 and further analyzed by four-color flow cytometry. In order to minimize background staining seen in (a), cells were preincubated with anti-CD16/32 (2.4G2) mAb to prevent non-specific binding to Fc receptors and SAallophycocyanin.Cy7 was used as secondary reagent instead of SATriColor. Blood leukocytes (bottom histograms) were analyzed in parallel as positive controls for CD11b, Gr-1 and CD4 staining. Representative FACS plots illustrating gates used during analysis are shown on the left. Histograms illustrate expression of indicated markers by cells in shown gates in dot-plots. All panels are representative of three separate experiments.
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To firmly establish the identity of the MHC class IIlow- and CD11clow-expressing cells, splenic low-density cells from B10.H-2k or NOD.H-2k were analyzed for the expression of markers for macrophages, granulocytes, B cells or other splenic dendritic cell subsets, using four-color flow cytometry (Fig. 2C). Low-density cells were co-stained for CD11b (Mac-1), Gr-1, B220, CD19 or CD4, in addition to staining for CD11c, MHC class II and CD8
. In contrast to blood leukocytes analyzed in parallel, the MHC class IIlow and CD11clow cells (R3 in Fig. 3C) did not appear to express appreciable levels of CD11b or Gr-1. The majority of these cells (
75% in NOD.H-2k), however, are positive for B220, but almost entirely negative for CD19, a pan-B cell marker. Approximately 50% (in both strains) of the MHC class IIlow and CD11clow cells express CD4. Altogether, results from the analysis of cell surface markers exclude the MHC class IIlow- and CD11clow-expressing cells from being macrophages, granulocytes or B cells. These data suggest that the MHC class IIlow and CD11clow cells represent splenic dendritic cell subset(s) relatively over-represented in the NOD.H-2k spleen.
Given the shift towards dendritic cells with low cell surface expression of MHC class II molecules in NOD.H-2k mice, the next issue was to relate this to the altered balance of splenic dendritic cell subsets described earlier. Furthermore, it was important to know whether this shift to MHC class IIlow dendritic cells reflected a unique combination of non-MHC NOD genes in the NOD.H-2k background. To address these issues, splenic dendritic cells from H-2k strains were prepared and analyzed for CD8
and MHC class II expression. In all the H-2k strains tested, heterogeneity in cell surface MHC class II expression was only evident in the CD8
subsets (Fig. 3A, lower left quadrants in all panels). In contrast, the CD8
+ dendritic cell subset uniformly expressed high levels of MHC class II molecules in all strains (upper right quadrants in all panels). The heterogeneity in cell surface MHC class II expression resulted from the presence of CD8
dendritic cells expressing both high and low levels of MHC class II molecules. The largest proportion of dendritic cells expressing low levels of MHC class II molecules was observed in the NOD.H-2k spleen where it constituted 35% of all splenic dendritic cells (Fig. 3B). The proportion of this subpopulation within the CD8
dendritic cell subsets did not exceed 12.4% in all other H-2k strains tested. The mean proportions in other H-2k strains were 12.1% in B10.H-2k, 3.5% in AKR, 9.8% in CBA/H, 4.3% in BALB.H-2k and 8.9% in (B10.H-2k x NOD.H-2k)F1 hybrids. Overall, these results demonstrate a unique imbalance of dendritic cell subsets resulting from non-MHC NOD genes.
NOD.H-2k myeloid precursors possess a cell-intrinsic bias to produce CD8
splenic dendritic cells in vivo
The effect of non-MHC NOD genes on dendritic cell subsets was further defined in hematopoietic chimeras. Bone marrow reconstitution strategy had the advantage of distinguishing between cell-autonomous traits of NOD.H-2k myeloid precursors and secondary effects of alterations in other cell types, and potentially revealing quantitative differences in myelopoiesis that may be masked when all hematopoietic cells are genetically identical. Four groups of bone marrow chimeric mice were generated. The first group comprised B10.H-2k recipients reconstituted with 2 x 106 B10.H-2k bone marrow cells alone (B
B), the second group comprised B10.H-2k recipients reconstituted with 2 x 106 NOD.H-2k bone marrow cells alone (N
B), the third group comprised NOD.H-2k recipients reconstituted with 2 x 106 B10.H-2k bone marrow cells alone (B
N), and the fourth group comprised B10.H-2k recipients reconstituted with a mixed inoculum comprising bone marrow cells from both B10.H-2k and NOD.H-2k mice (total 2 x 106 cells) at a ratio of 1:1 (B/N
B). Recipient mice were analyzed by flow cytometry for B10.H-2k- or NOD.H-2k-derived leukocytes 812 weeks post-reconstitution. The allelic variants of Ly5 were used to distinguish between B10.H-2k (Ly5b)- and NOD.H-2k (Ly5a)-derived cells (16). Co-staining with mAb against dendritic cells (N418), B cells (B220), T cells (Thy1) and myeloid cells (CD11b, Mac-1) was used to monitor the relative reconstitution of different leukocyte lineages in recipient mice.
The imbalance towards the CD8
splenic dendritic cell subsets observed in unmanipulated NOD.H-2k was equally clear in the NOD.H-2k-derived cells in B/N
B mixed bone marrow chimeric mice (Fig. 4A). Splenic dendritic cells from mixed bone marrow chimeric mice were prepared by collagenase digestion and enrichment on metrizamide gradient as before. Enriched splenic low-density cells were stained with mAb against CD11c, Ly5a and CD8
, and analyzed by flow cytometry. Large cells positive for CD11c expression were gated, and analyzed for Ly5a and CD8
expression. NOD.H-2k-derived dendritic cells outnumbered B10.H-2k-derived dendritic cells and this over-representation was primarily contributed by the CD8
subsets (P < 0.005). The mean ratio of Ly5a+ to Ly5a CD8
+ dendritic cells in mixed bone marrow chimeric mice was 2.6, whereas it was 5.3 in the CD8
subsets (Fig. 4B).
Analysis of relative reconstitution of different hematopoietic lineages in multiple mixed bone marrow chimeric mice, from two separate experiments, is depicted in Fig. 4(C). The mean ratio of NOD.H-2k to B10.H-2k cells was
1 for B and T cells in blood and spleen, and for T cells in the thymus. In stark contrast, the ratios for monocytes, thymic dendritic cells and splenic dendritic cells are
4, 3 and 6 respectively. Thus, while lymphocytes in blood, spleen and thymus of mixed bone marrow chimeric mice were evenly derived from both hematopoietic stem cell donors, the myeloid lineage of cells was disproportionately derived from the NOD.H-2k component.
The skewing of the myeloid lineage of cells in mixed bone marrow chimeric mice prompted the question whether this skewing occurred in relative ratios or in absolute numbers in the myeloid lineage. To pursue this question, whole blood and red blood cell-depleted (processed) blood leukocytes from bone marrow chimeric mice were stained for B cells, T cells, monocytes and Ly5a, and analyzed by three-color flow cytometry. Counting beads were added to whole blood samples for precise quantitation of leukocytes. Absolute quantitation of blood monocytes in three chimeric mouse groups showed that the skewing towards NOD.H-2k-derived monocytes in mixed chimeras (Fig. 4D) occurs in relative terms, with all three groups of chimeric mice containing comparable total numbers of monocytes. Thus, in mixed chimera analysis, the preferential generation of NOD.H-2k-derived monocytes was primarily due to a contraction in the size of the B10.H-2k-derived component rather than an expansion of the total monocyte pool. Overall, results from the study on bone marrow chimeric mice suggest that non-MHC NOD genes confer a cell-intrinsic change in NOD.H-2k myeloid precursors, resulting in the preferential generation of the CD8
dendritic cell subsets and the myeloid lineage of cells as a whole.
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Discussion
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The results obtained from this study demonstrate unique abnormalities in the dendritic cell system caused by non-MHC genes from the NOD strain. The abnormalities are: (i) imbalance in splenic dendritic cells favoring the CD8
subsets in vivo over the CD8
+ subset, (ii) decreased fraction of MHC class IIhigh dendritic cells in the CD8
subsets and (iii) exaggerated production of myeloid dendritic cells under competitive reconstitution conditions. These results point to the possibility that intrinsic abnormalities in the dendritic cell system contribute to autoimmune disease susceptibility.
The current study demonstrates unique cell-intrinsic effects of non-MHC NOD genes on dendritic cell generation, in vivo, that are not shared by autoimmune-resistant B10.H-2k, BALB.H-2k, CBA/H and AKR strains. There are a number of reasons to suggest genetic dysregulation of dendritic cell function may contribute to autoimmunity in NOD mice. Firstly, dendritic cells and macrophages have been shown to be the first leukocytes recruited to the islets of young pre-diabetic NOD mice (7). Secondly, these cells are the initial and principal producers of TNF-
in pancreatic islets (8). Thirdly, enhanced antigen-presenting capacity of dendritic cells is implicated in NOD mice expressing TNF-
in islets under the rat insulin promotor from neonatal life (17). Ectopic expression of TNF-
in the islets results in accelerated onset of diabetes in neonatal NOD mice. Finally, repeated injection of dendritic cells presenting a transgenic islet-specific antigen is sufficient to precipitate lymphoid tissue neogenesis in islets and diabetes (12). Importantly, dendritic cell abnormalities analogous to that observed in mouse models of autoimmune diabetes have been detected in individuals with type I diabetes and individuals at risk for type I diabetes (18,19).
In studying the potential role of dendritic cells in tissue autoimmunity, it is important to consider the possibility of distinct roles performed by dendritic cell subsets, given the imbalance in splenic dendritic cell subsets observed in this study. Recent evidence suggests that the CD8
and CD8
+ subsets of dendritic cells follow distinct developmental streams (20), and require different growth and survival stimuli. For example, RelB deficiency causes a selective loss of CD8
dendritic cells (21) and granulocyte macrophage colony stimulating factor (GM-CSF) promotes the expansion of this subset preferentially, whereas Flt3 ligand expands both subsets (22). NOD myeloid progenitors have previously been shown to be deficient in their in vitro responses to a range of myeloid growth factors, including GM-CSF (23). In contrast, this study reveals a relative over-production of NOD.H-2k-derived dendritic cells, predominantly the CD8
subsets, under competitive reconstitution conditions. The rapid turnover rates of myeloid progenitors (24) and dendritic cells (20) make it unlikely that the bias towards NOD.H-2k-derived dendritic cells in mixed bone marrow chimeric mice is due to a relative excess in NOD.H-2k progenitors used to reconstitute recipients. Furthermore, the prevalence of the MHC class IIlow dendritic cells, within the CD8
subsets, appears unique to the NOD.H-2k background. Part of these pool of dendritic cells is comprised of the newly described CD4 subset (25). However, most of the MHC class IIlow dendritic cells are B220+, and possible counterparts of B220+ dendritic cells found in the liver (26) and bone marrow (27). Interestingly, the MHC class IIlow-expressing dendritic cells identified in this study lack cell surface Gr-1 expression and are therefore distinct from the recently reported plasmacytoid dendritic cells found in spleen of mice (28).
The selective expansion of the CD8
dendritic cell subsets, on the NOD.H-2k background, and the tendency for these subsets to have more MHC class IIlow cells in vivo, may therefore indicate enhanced transcriptional activity, by RelB for instance, or heightened or aberrant responses to myeloid dendritic cell growth factors.
Evidence is emerging that antigen presentation by dendritic cells may be a key determinant in tilting the balance between tolerance and immunity (29,30). Diminished dendritic cell production in NOD mice could lead to a relative paucity in dendritic cells responsible for presenting antigens derived from self-tissue. Available evidence, albeit indirect, suggests a role for dendritic cells in maintaining peripheral tolerance. It has been demonstrated that high levels of MHC class II complexed with self-peptides are displayed on dendritic cells in T cell zones in lymph nodes (31). Furthermore, peripheral tolerance to model autoantigens, transgenically expressed in the islets, is mediated by bone marrow-derived APC (32,33), likely the CD8
subset of dendritic cells (34). Recently, more direct evidence implicates dendritic cells in tolerance induction (35). This study shows that targeting antigen to dendritic cells via DEC-205, a molecule predominantly expressed by the CD8
subset, induces peripheral T cell unresponsiveness under steady-state conditions in vivo. Tolerance induction by dendritic cells may be mediated by dendritic cells directly or by other cell types, such as regulatory T cells (36) and NK T cells (37). Thus, the propensity for tissue autoimmunity in NOD mice may be underpinned by a global diminution in tolerogenic antigen presentation in the periphery.
Conversely, susceptibility to tissue autoimmunity in NOD mice may reflect a relative excess of immunogenic antigen presentation by dendritic cells. This could drive the generation and polarization of autoreactive Th cells to the Th1 type. Dendritic cells are an important source of IL-12, a key cytokine inducing the differentiation of Th1-type cells (38). Interestingly, a variant allele of IL-12p40 has recently been associated with susceptibility to type I diabetes in humans (39). Overall, production/function of dendritic cells may be altered in such a way to compromise their tolerogenic function or to enhance their immunogenic function, or both. The net effect would be a breach of peripheral tolerance mechanisms and, hence, increased susceptibility to tissue autoimmunity in NOD mice.
In summary, we define here intrinsic abnormalities in the dendritic cell system that are unique to the autoimmunity-susceptible NOD strain background. Identification of these cellular defects will facilitate future work to define genes and biochemical pathways regulating the dendritic cell system, and their role in autoimmune diseases.
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Acknowledgements
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S. P. thanks Dr A. M. Gautam for initiating and encouraging the study of dendritic cells, and Dr S. E. Townsend for useful discussions.
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Abbreviations
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APCantigen-presenting cell
GM-CSFgranulocyte macrophage colony stimulating factor
IDDMinsulin-dependent diabetes mellitus
NODnon-obese diabetic
PEphycoerythrin
SAstreptavidin
TNFtumor necrosis factor
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References
|
---|
- Kikutani, H. and Makino, S. 1992. The murine autoimmune diabetes model: NOD and related strains. Adv. Immunol. 51:285.[ISI][Medline]
- Todd, J. A., Aitman, T. J., Cornall, R. J., Ghosh, S., Hall, J. R., Hearne, C. M., Knight, A. M., Love, J. M., McAleer, M. A., Prins, J. B., et al. 1991. Genetic analysis of autoimmune type 1 diabetes mellitus in mice. Nature 351:542.[ISI][Medline]
- Wicker, L. S., Todd, J. A. and Peterson, L. B. 1995. Genetic control of autoimmune diabetes in the NOD mouse. Annu. Rev. Immunol. 13:179.[ISI][Medline]
- Wicker, L. S. 1997. Major histocompatibility complex-linked control of autoimmunity. J. Exp. Med. 186:973.[Free Full Text]
- Serreze, D. V., Leiter, E. H., Worthen, S. M. and Shultz, L. D. 1988. NOD marrow stem cells adoptively transfer diabetes to resistant (NOD x NON)F1 mice. Diabetes 37:252.[Abstract]
- Wicker, L. S., Miller, B. J., Chai, A., Terada, M. and Mullen, Y. 1988. Expression of genetically determined diabetes and insulitis in the nonobese diabetic (NOD) mouse at the level of bone marrow-derived cells. Transfer of diabetes and insulitis to nondiabetic (NOD x B10)F1 mice with bone marrow cells from NOD mice. J. Exp. Med. 167:1801.[Abstract]
- Jansen, A., Homo-Delarche, F., Hooijkaas, H., Leenen, P. J., Dardenne, M. and Drexhage, H. A. 1994. Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta-cell destruction in NOD mice. Diabetes 43:667.[Abstract]
- Dahlen, E., Dawe, K., Ohlsson, L. and Hedlund, G. 1998. Dendritic cells and macrophages are the first and major producers of TNF-alpha in pancreatic islets in the nonobese diabetic mouse. J. Immunol. 160:3585.[Abstract/Free Full Text]
- Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[ISI][Medline]
- Lafferty, K. J., Prowse, S. J., Simeonovic, C. J. and Warren, H. S. 1983. Immunobiology of tissue transplantation: a return to the passenger leukocyte concept. Annu. Rev. Immunol. 1:143.[ISI][Medline]
- OGarra, A. and Murphy, K. 1994. Role of cytokines in determining T-lymphocyte function. Curr. Opin. Immunol. 6:458.[ISI][Medline]
- Ludewig, B., Odermatt, B., Landmann, S., Hengartner, H. and Zinkernagel, R. M. 1998. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue. J. Exp. Med. 188:1493.[Abstract/Free Full Text]
- Podolin, P. L., Pressey, A., DeLarato, N. H., Fischer, P. A., Peterson, L. B. and Wicker, L. S. 1993. I-E+ nonobese diabetic mice develop insulitis and diabetes. J. Exp. Med. 178:793.[Abstract]
- Vremec, D. and Shortman, K. 1997. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159:565.[Abstract]
- Metlay, J. P., Witmer-Pack, M. D., Agger, R., Crowley, M. T., Lawless, D. and Steinman, R. M. 1990. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J. Exp. Med. 171:1753.[Abstract]
- McClive, P. J., Baxter, A. G. and Morahan, G. 1994. Genetic polymorphisms of the non-obese diabetic (NOD) mouse. Immunol. Cell Biol. 72:137.[ISI][Medline]
- Green, E. A., Eynon, E. E. and Flavell, R. A. 1998. Local expression of TNFalpha in neonatal NOD mice promotes diabetes by enhancing presentation of islet antigens. Immunity 9:733.[ISI][Medline]
- Jansen, A., van Hagen, M. and Drexhage, H. A. 1995. Defective maturation and function of antigen-presenting cells in type 1 diabetes. Lancet 345:491.[ISI][Medline]
- Takahashi, K., Honeyman, M. C. and Harrison, L. C. 1998. Impaired yield, phenotype, and function of monocyte-derived dendritic cells in humans at risk for insulin-dependent diabetes. J. Immunol. 161:2629.[Abstract/Free Full Text]
- Kamath, A. T., Pooley, J., OKeeffe, M. A., Vremec, D., Zhan, Y., Lew, A. M., DAmico, A., Wu, L., Tough, D. F. and Shortman, K. 2000. The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J. Immunol. 165:6762.[Abstract/Free Full Text]
- Wu, L., DAmico, A., Winkel, K. D., Suter, M., Lo, D. and Shortman, K. 1998. RelB is essential for the development of myeloid-related CD8alpha dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells. Immunity 9:839.[ISI][Medline]
- Pulendran, B., Smith, J. L., Caspary, G., Brasel, K., Pettit, D., Maraskovsky, E. and Maliszewski, C. R. 1999. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc. Natl Acad. Sci. USA 96:1036.[Abstract/Free Full Text]
- Langmuir, P. B., Bridgett, M. M., Bothwell, A. L. and Crispe, I. N. 1993. Bone marrow abnormalities in the non-obese diabetic mouse. Int. Immunol. 5:169.[Abstract]
- Akashi, K., Traver, D., Miyamoto, T. and Weissman, I. L. 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404:193.[ISI][Medline]
- Vremec, D., Pooley, J., Hochrein, H., Wu, L. and Shortman, K. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164:2978.[Abstract/Free Full Text]
- Lu, L., Bonham, C. A., Liang, X., Chen, Z., Li, W., Wang, L., Watkins, S. C., Nalesnik, M. A., Schlissel, M. S., Demestris, A. J., Fung, J. J. and Qian, S. 2001. Liver-derived DEC205+B220+CD19 dendritic cells regulate T cell responses. J. Immunol. 166:7042.[Abstract/Free Full Text]
- Gandy, K. L., Domen, J., Aguila, H. and Weissman, I. L. 1999. CD8+TCR+ and CD8+TCR cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers. Immunity 11:579.[ISI][Medline]
- Nakano, H., Yanagita, M. and Gunn, M. D. 2001. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J. Exp. Med. 194:1171.[Abstract/Free Full Text]
- Steinman, R. M., Turley, S., Mellman, I. and Inaba, K. 2000. The induction of tolerance by dendritic cells that have captured apoptotic cells. J. Exp. Med. 191:411.[Free Full Text]
- Lesage, S. and Goodnow, C. C. 2001. Organ-specific autoimmune disease: a deficiency of tolerogenic stimulation. J. Exp. Med. 194:F31.
- Inaba, K., Pack, M., Inaba, M., Sakuta, H., Isdell, F. and Steinman, R. M. 1997. High levels of a major histocompatibility complex IIself peptide complex on dendritic cells from the T cell areas of lymph nodes. J. Exp. Med. 186:665.[Abstract/Free Full Text]
- Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F. and Heath, W. R. 1997. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8+ T cells. J. Exp. Med. 186:239.[Abstract/Free Full Text]
- Adler, A. J., Marsh, D. W., Yochum, G. S., Guzzo, J. L., Nigam, A., Nelson, W. G. and Pardoll, D. M. 1998. CD4+ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen-presenting cells. J. Exp. Med. 187:1555.[Abstract/Free Full Text]
- den Haan, J. M., Lehar, S. M. and Bevan, M. J. 2000. CD8+ but not CD8 dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192:1685.[Abstract/Free Full Text]
- Hawiger, D., Inaba, K., Dorsett, Y., Guo, M., Mahnke, K., Rivera, M., Ravetch, J. V., Steinman, R. M. and Nussenzweig, M. C. 2001. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194:769.[Abstract/Free Full Text]
- Salomon, B., Lenschow, D. J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A. and Bluestone, J. A. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immuno regulatory T cells that control autoimmune diabetes. Immunity 12:431.[ISI][Medline]
- Wang, B., Geng, Y. B. and Wang, C. R. 2001. CD1-restricted NK T cells protect nonobese diabetic mice from developing diabetes. J. Exp. Med. 194:313.[Abstract/Free Full Text]
- Moser, M. and Murphy, K. M. 2000. Dendritic cell regulation of TH1TH2 development. Nat. Immunol. 1:199.[ISI][Medline]
- Morahan, G., Huang, D., Ymer, S. I., Cancilla, M. R., Stephen, K., Dabadghao, P., Werther, G., Tait, B. D., Harrison, L. C. and Colman, P. G. 2001. Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele. Nat. Genet. 27:218.[ISI][Medline]