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The Journal of Immunology, 2004, 173: 4331-4341.
Copyright © 2004 by The American Association of Immunologists

Antisense Oligonucleotides Down-Regulating Costimulation Confer Diabetes-Preventive Properties to Nonobese Diabetic Mouse Dendritic Cells1

Jennifer Machen*, Jo Harnaha*, Robert Lakomy*, Alexis Styche*, Massimo Trucco*,{dagger} and Nick Giannoukakis2,*,{ddagger}

* Diabetes Institute, {dagger} Division of Immunogenetics, Department of Pediatrics, and {ddagger} Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phenotypically "immature" dendritic cells (DCs), defined by low cell surface CD40, CD80, and CD86 can elicit host immune suppression in allotransplantation and autoimmunity. Herein, we report the most direct means of achieving phenotypic immaturity in NOD bone marrow-derived DCs aiming at preventing diabetes in syngeneic recipients. CD40, CD80, and CD86 cell surface molecules were specifically down-regulated by treating NOD DCs ex vivo with a mixture of antisense oligonucleotides targeting the CD40, CD80, and CD86 primary transcripts. The incidence of diabetes was significantly delayed by a single injection of the engineered NOD DCs into syngeneic recipients. Insulitis was absent in diabetes-free recipients and their splenic T cells proliferated in response to alloantigen. Engineered DC promoted an increased prevalence of CD4+CD25+ T cells in NOD recipients at all ages examined and diabetes-free recipients exhibited significantly greater numbers of CD4+CD25+ T cells compared with untreated NOD mice. In NOD-scid recipients, antisense-treated NOD DC promoted an increased prevalence of these putative regulatory T cells. Collectively, these data demonstrate that direct interference of cell surface expression of the major costimulatory molecules at the transcriptional level confers diabetes protection by promoting, in part, the proliferation and/or survival of regulatory T cells. This approach is a useful tool by which DC-mediated activation of regulatory T cells can be studied as well as a potential therapeutic option for type 1 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Type 1 diabetes mellitus (T1DM), 3 a disorder of glucose homeostasis, is the consequence of the autoimmune targeting of pancreatic insulin-producing {beta} cells. At the cellular level, the {beta} cells are subjected to cytokine-induced impairment by the actions of infiltrating macrophages and T cells with subsequent T cell-mediated destruction (1, 2). There is no question that defects in T cell selection at the central level in the thymus and impaired peripheral regulation of {beta} cell Ag-specific T cells underlie the etiopathogenesis of T1DM (1, 2). Dendritic cells (DC) have proven to be integral participants in the initiation and propagation of T1DM at multiple levels including the regulation of diabetes onset (3, 4, 5, 6, 7, 8). DC are the primary APCs of the immune system and as such, they control the activation of naive T cells (4, 9, 10, 11). For full activation of naive CD4+ T lymphocytes to occur, two signals are required. The first is the presentation of the Ag to the TCR in the context of class II MHC on DC. This will cause the responding T cell to up-regulate the CD154 molecule (CD40 ligand) to its cell surface, thereby activating the initiation of the second signal. In this process of coactivation, CD154 will interact with the CD40 molecule at the surface of the APC resulting in the up-regulation of CD80 and CD86 at the cell surface of the APC. Immediately thereafter, CD80 and CD86, acting as the second signal, in the process of costimulation, will engage the CD28 molecule on the T cell resulting in its full activation. In the absence of the interactions between CD80, CD86, and CD28, the T cell will either enter a state of functional silence, termed anergy, or will be primed for apoptosis, perhaps in a CD95-CD95L (Fas-FasL)-dependent manner (12, 13, 14). Converging lines of evidence indicate that the phenotype of the DC cell surface can play an important role in tolerance to self-Ags and can be manipulated to promote allogeneic as well as autoimmune hyporesponsiveness (11, 15).

The first use of DC to prevent T1DM in NOD mice was documented by Clare-Salzler et al. (8) who demonstrated that transfer of pancreatic lymph node DC derived from 8- to 20 wk-old NOD mice into prediabetic NOD mice conferred significant protection from T1DM, insulitis, and adoptive transfer of T1DM. The authors suggested that acquisition of islet Ags by DC during insulitis may have resulted in them acquiring a phenotype, once in the pancreatic lymph nodes, that was able to result in the stimulation of regulatory immune cells which attenuated the insulitic process. Interestingly, while transfer of DC isolated from nonpancreatic lymph nodes to NOD mice was unable to affect T1DM incidence, transfer of these DC pulsed with sonicated islets did confer protection (8). More recently, Morel and colleagues (16, 17) have shown prolongation of a diabetes-free state in NOD recipients of bone marrow-derived syngeneic DC. Other methods of generating diabetes-suppressive DC include vitamin D receptor ligands, Ag pulsing, and IFN-{gamma} treatment (18, 19, 20).

NOD DC exhibit strong immunostimulatory capacity, underlied by hyperactivation of NF-{kappa}B (21, 22, 23). Therefore, we proposed and very recently showed that inhibition of NF-{kappa}B using short, double-stranded transcriptional decoys could render NOD DC less immunostimulatory and that administration of these engineered DC into NOD prediabetic mice could prevent the development of diabetes (24). Attractive as this approach is, we nevertheless do have concerns that are under investigation that NF-{kappa}B blockade may interfere with functions crucial to DC survival in vivo that may impact on the persistence of the immunosuppressive effect of these DC in NOD mice. Hence, as a second, and complementary approach, we decided to engineer DC in a way where the expression of only the costimulatory molecules CD40, CD80, and CD86 would be suppressed at the cell surface. We based our rationale on the many studies demonstrating the effectiveness of CD80/CD86-CD28 blockade in generating immune hyporesponsiveness to alloantigens and in preventing autoimmunity (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52). Unlike the use of anti-CD40L Abs and CTLA4-Ig, our approach limits the cell population that is targeted, because the treatment is performed ex vivo and does not involve systemic dissemination of a protein which, in the instance of CTLA4-Ig and anti-CD40L have exhibited nonspecific and toxic effects (53, 54). Herein we report that ex vivo treatment of bone marrow-derived NOD DCs with a mixture of antisense oligonucleotides (AS-ODN) targeting the CD40, CD80, and CD86 transcripts confers specific suppression of the respective cell surface proteins. We further demonstrate that a single injection of these engineered DC into syngeneic prediabetic female NOD mice significantly delays the incidence of T1DM without affecting the response of T cells from diabetes-free DC recipients to alloantigen. Furthermore, there was no evidence of insulitis in the diabetes-free recipients. In NOD-scid recipients, we show that ODN-treated NOD DC administration in cotransfer with T cells promotes an increased prevalence of CD4+CD25+CD62L+ T cells. The use of AS technology specifically targeting the transcripts of key DC cell surface proteins involved in T cell activation and regulation could be a useful technique to study DC:T cell interactions promoting immunoregulatory cell networks and as a potential means of T1DM cell therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female C57BL/6, NOD/LtJ (H2g7), NOD-scid, and C3H/HeJ (H2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under pathogen-free conditions. The {alpha}-actin-GFP transgenic mouse was bred on the C57BL/6 background and was propagated in our mouse colony. All animal experimentation was conducted in compliance with the Animal Research Care Committee of the Children’s Hospital of Pittsburgh.

Reagents

Abs to immune cells were purchased from BD Biosciences (San Diego, CA) and were used as the direct FITC, PE, CyChrome, or allophycocyanin fluorochrome conjugates. The clones used were as follows: CD40 (clone 3/23), CD11c (clone HL3), CD86 (clone GL1), CD80 (clone 16-10A1), CD4 (clone RM4-5), CD25 (clone 7D4), and CD62L (MEL-14). Anti-insulin and anti-glucagon Abs were purchased from DakoCytomation (Carpinteria, CA). Isotype- and species-matched irrelevant monoclonal or polyclonal Abs (where appropriate) were used as controls. The NIT-1 cell line was obtained from American Type Culture Collection (CRL-2055; Manassas, VA) and propagated as described by the repository. Phosphorothioate-modified ODNs were synthesized by the University of Pittsburgh DNA Synthesis Facility and HPLC purified. Cell culture reagents (serum and media) were purchased from Invitrogen Life Technologies (Gaithersburg, MD). Beadlyte multiplex fluorescence cytokine detection kits were purchased from Upstate Biotechnology (Lake Placid, NY) and fluorescence-based proliferation as well as phagocytosis probes from Molecular Probes (Eugene, OR). Recombinant murine cytokines and immune cell enrichment columns were purchased from R&D Systems (Indianapolis, IN). All other biochemicals were purchased from Sigma-Aldrich (St. Louis, MO).

DC propagation and treatment with AS-ODN

DC were propagated from bone marrow progenitors of 5- to 8-wk-old female C57BL/6 or NOD mice in GM-CSF and IL-4 as outlined by Ma et al. (24) and originally described in Fu et al. (55, 56). Briefly, bone marrow was obtained from the femurs and tibiae of female NOD mice. The RBC were lysed using a commercially available reagent (Red Blood Cell Lysing Buffer; Sigma-Aldrich) and the bone marrow cells were plated in 24-well multiwell plates at 2 x 106 cells/ml in R-10 medium (RPMI 1640/10% heat-inactivated FBS/50 µM 2-ME/1% sodium pyruvate/1% nonessential amino acids/1% penicillin-streptomycin solution (Invitrogen Life Technologies) with the addition of 4 ng/ml GM-CSF and 1000 U/ml IL-4 (R&D Systems). Two days later, the nonadherent cells were removed and a 1:1 volume of conditioned medium, fresh R-10 medium, and cytokines were added to the adherent cells. Three days later, the loosely adherent cells were gently agitated and harvested. By FACS analysis, >85% of these cells are routinely DC with class II MHC, CD11c, CD80, CD40, and CD86 positivity (57). The AS-ODN mixture consisted of phosphorothioate-modified ODNs each targeting the 5' end of the CD40, CD80, and CD86 primary transcripts. The sequences are: CD40 AS-ODN, 5'-CAC AGC CGA GGC AAA GAC ACC ATG CAG GGC A-3'; CD80 AS-ODN, 5'-GGG AAA GCC AGG AAT CTA GAG CCA ATG GA-3'; CD86 AS-ODN, 5'-TGG GTG CTT CCG TAA GTT CTG GAA CAC GTC-3'. The AS-ODN were HPLC purified and resuspended in PBS. DC were treated 18–24 h in 10% heat-inactivated FBS/RPMI 1640 with a mixture of 3.3 µM CD40 AS-ODN, 3.3 µM CD80 AS-ODN, and 3.3 µM CD86 AS-ODN (the mixture is collectively referred to as AS-ODN in this study). The cells were then washed extensively in PBS and subsequently used in culture or in vivo. Ag uptake and processing capacity were assessed using the Vybrant Phagocytosis Assay reagent as described by the manufacturer (Molecular Probes). Uptake of fluorescent bioparticles was measured in a fluorescence microplate reader at 480 nm excitation/520 nm emission (Victor2; PerkinElmer Instruments, Boston, MA).

Flow cytometry analysis

All the FACS analyses were performed in a FACSVantage SE flow cytometer with FACSDiva version 2.2.1, capable of eight-color, multiparameter discrimination (BD Biosciences). In every FACS analysis, cells were stained with propidium iodide to exclude dead cells. The initial cell populations were selected based on forward and side scatter properties specific for DC or T cells. By forward and side scatter, we excluded debris and clumped cells and by propidium iodide we excluded dead cells from all analyses. The initial gate was set around the remaining cells. In T cell populations, wherever we aimed at discriminating putative T regulatory cells, we gated CD4+ cells and analyzed this population for CD25 and CD62L positivity. Results were recorded as dot plots in QuadStat analyses.

In vitro phenotype of DC treated with AS-ODN and Ag loading

AS-ODN-treated DC were stimulated with 25 µg/ml LPS (Sigma-Aldrich) or 50 ng/ml recombinant murine CD40L (R&D Systems) for 18–24 h. The culture supernatant was collected and assayed for NO production using the Griess assay and profiled for cytokine secretion using the Beadlyte multiplex assay system (Upstate Biotechnology) in a Luminex Fluorescence Analyser (Luminex, Austin, TX). In parallel, the cells were stained with fluorescence-conjugated Abs against CD40, CD80, and CD86. T cell proliferation was measured in cocultures of irradiated splenocytes and spleen-isolated and column-enriched (R&D Systems) T cells from 5- to 8-wk-old, diabetes-free, diabetic female NOD or T cells from the spleen of C3H/HeJ females (5–10 wk of age). Proliferation was measured after 5 days in culture using the CyQuant fluorescence reagent (Molecular Probes). Cytokine production was measured in the culture supernatants of the cocultures at 5 days using the Beadlyte-Luminex assay. All assays were performed in triplicate on at least two different occasions.

Examination of DC phenotype following in vivo transfer of CFSE-labeled or GFP-positive (GFP+) DC into immunocompetent recipients

We first labeled control and AS-ODN-treated NOD DC (from 7-wk-old female donors) with CFSE (Molecular Probes) as directed by the manufacturer. Cells (2 x 106) were injected i.p. into age-matched female recipients. At weekly intervals, for 3 wk, we harvested the spleens from individual recipients and examined the levels of CD80 and CD86 in recovered CFSE+ cells by FACS using Abs for CD80 and CD86 (BD Biosciences). As a complementary approach, we obtained DC from the bone marrow progenitors of GFP transgenic mice. In these mice, the GFP transgene is under the control of the chicken {alpha}-actin promoter. These mice were generated on a C57BL/6 background. They express GFP in almost all tissues including monocytes and DC. The DC were treated ex vivo with AS-ODN or PBS vehicle and 2 x 106 cells were injected i.p. into nontransgenic C57BL/6 sex-matched recipients. At weekly intervals, for 3 wk, the spleens of individual recipients were harvested and single cells were stained with CD80 and CD86 Abs. CD80 and CD86 levels were analyzed by FACS in GFP+-gated populations.

DC administration to NOD mice, diabetes monitoring, and immune profiling of DC recipients

NOD DC (2 x 106–3 x 106) (control, AS-ODN-treated, or {beta} cell Ag/AS-ODN cotreated) in PBS were injected by i.p. route into 5- to 8-wk-old female NOD mice. The {beta} cell Ag was in the form of a lysate obtained from the NOD-derived NIT-1 insulinoma cell line. Given that multiple autoantigens have been identified for diabetes, we chose to coadminister NIT-1 lysate which in principle should contain all known (and unknown) autoantigens. The NIT-1 cell line is derived from NOD transgenic mice where the SV40 large T-Ag is expressed from the rat insulin gene promoter. These transgenic NOD mice display {beta} cell adenoma and these {beta} cells have been immortalized as the NIT-1 line (58). The choice of the NIT-1 cell line over NOD islets as source of {beta} cell Ag was also due to the significant logistics we previously experienced in the isolation of islets (24) that involved hundreds of mice to produce enough lysate to realistically provide Ags at reasonable levels (100 islets usually isolated per mouse; 1000 islet cells per islet on average). The cell line was easily grown to very large numbers in a short time period. Second, the NIT-1 cell line shares almost identical phenotype with normal {beta} cells, expresses class I MHC in response to IFN-{gamma}, but does not express class II MHC at the cell surface (58). More importantly, sera from diabetic NOD mice strongly stained NIT-1 cells, but no staining was observed when sera from prediabetic or diabetes-resistant NOD were used (58). Moreover, sonicated NIT-1 membranes injected i.v. into 5-wk-old NOD mice prevented T1DM (59). Finally, CD8+ T cells from NOD mice were able to recognize and destroy NIT-1 cells in vitro (60).

Diabetes incidence was ascertained twice weekly in tail vein blood by electronic sampling (One-Touch; LifeScan Technologies, Milpitas, CA). Confirmation of diabetes was noted upon two consecutive readings of blood glucose >280 mg/dL. At various time points, DC recipients were euthanized, pancreata, lymph nodes, and spleens were isolated. Pancreata were fixed in 4% paraformaldehyde and embedded in paraffin. Multiple sections (4 mm) were subsequently stained with anti-insulin and glucagon Abs (DakoCytomation) followed by secondary probing with biotin-conjugated secondary Abs followed by diaminobenzidine chromogen visualization. Parallel sections were also stained with H&E. T cells from mesenteric and inguinal lymph nodes as well as spleen were isolated using column enrichment (R&D Systems) and used in coculture proliferation/cytokine profiling experiments with NOD (5–8 wk of age) or C3H/HeJ (5–8 wk of age) irradiated bone marrow-derived DC as stimulators. T cells from DC-treated, diabetes-free NOD mice were cultured overnight in the presence of Con A (5 µg/ml) and the supernatants were then probed for cytokine secretion profiles using the Beadlyte assay (Upstate Biotechnology) in the Luminex multiplex fluorescence-based detection system.

NOD DC and T cell cotransfer into NOD-scid recipients

PBS- or AS-ODN-treated NOD DC (1 x 106–2 x 106) (from 5- to 8-wk-old females) were injected i.p. into sex and age-matched NOD-scid recipients. Twenty-four to 48 h later, an equal amount of splenic T cells from 5- to 8-wk-old female NOD mice was injected i.v. Five days later, the mesenteric and popliteal lymph nodes and spleen were harvested. The lymph nodes were pooled and single cells were isolated (from spleen and pooled lymph nodes) over a T cell enrichment column (R&D Systems). The cells were cultured overnight and the supernatant collected for cytokine profiling using the Beadlyte assay system. In parallel, T cell phenotype was analyzed by FACS where CD4+-gated cells were reanalyzed for CD25 and CD62L presence.

Statistics

GraphPad Prism version 4.0 (San Diego, CA) was used to analyze the data where appropriate. Kaplan-Meier log-rank analysis was used for survival data and unpaired ANOVA or Student’s t test (where appropriate) were applied to the data obtained from in vitro studies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AS-ODN treatment confers a CD80lowCD86low phenotype to NOD DC in vitro and in vivo and prevents production of IL-12p70, TNF-{alpha}, and NO

Because NF-{kappa}B is an important transcription factor in many signaling pathways, perhaps crucial for DC function not involving T cell activation, we chose to assess the potential of a less "global" method of maintaining DC in an immature state characterized by a phenotype of low cell surface levels of costimulatory molecules (CD40, CD80, and CD86). We reasoned that, by using specific short AS-ODN targeting the transcripts for the mouse CD40, CD80, and CD86, we would be able to mimic the immature state conferred by the NF-{kappa}B ODN directly by interfering with crucial regulators of T cell activation (CD40, CD80, and CD86). We first tested the ability of a number of ODN targeting different regions of the CD40, CD80, and CD86 to inhibit NOD DC cell surface expression of these proteins in response to LPS stimulation in culture. Of 27 ODNs each targeting different sequences of the primary transcript (5' end, exon-intron, 3' end), we selected the ones yielding the greatest suppressive effect on cell surface expression, as assessed by FACS, for subsequent studies. Of all ODNs, the ones with the greatest effect were those targeting sequences at the 5' end (Fig. 1A). Despite the presence of LPS stimulation, cell surface expression of CD40, CD80, and CD86 were specifically suppressed in DC treated with each of the ODN. Although LPS is a powerful maturation signal in vitro, the most relevant maturation signal in vivo would be ligation of CD40 by CD40L. To examine the effects of CD40L on the phenotype of DC in culture, we added bioactive recombinant trimeric CD40L (50 ng/ml) to NOD DC treated with PBS or with the AS-ODN. After a period of 24–36 h, the supernatants were collected and examined for cytokine profile and NO production, given that NO production is a feature of maturing DC. The cells were analyzed by FACS for CD80 and CD86 cell surface expression. In Fig. 1, B and C, we demonstrate that CD40L was able to up-regulate CD80 and CD86 in control DC but not in AS-ODN-treated DC. Also, we show that NO, TNF-{alpha}, and IL-12p70 production was significantly suppressed in AS-ODN-treated DC exposed to CD40L compared with untreated DC (Fig. 1C). Although there is no evidence that ODN treatment of DC impairs their capacity to uptake Ag, we proceeded to formally examine this possibility using the Vybrant Phagocytosis assay system (Molecular Probes) where cell fluorescence depends on the uptake and processing of exogenously supplied Escherichia coli bioparticles whose fluorescence is quenched outside the cell due to trypan blue inclusion in the assay buffer. Fig. 1E demonstrates that AS-ODN DC fluorescence is identical to that of untreated DC when the cells are pulsed with the Vybrant bioparticles.




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  		FIGURE 1. A, FACS analysis of AS-ODN-treated NOD DC. Bone marrow-derived DC from 8-wk-old female NOD mice were cultured overnight in the presence of 10 µM ODN (mixture of CD40, CD80, and CD86 ODN, each at 3.3 µM final concentration in culture). Untreated DC stimulated with 25 µg/ml LPS overnight exhibited robust up-regulation of CD40, CD80, and CD86 ({square}). AS-ODN pretreatment prevented LPS-triggered up-regulation of costimulatory molecules at the DC cell surface ({blacksquare}). These data are representative of four independently performed experiments. AS refers to AS treatment (shown at the top of each panel). The y-axis indicates mean fluorescence intensity of cells gated on forward and side scatter properties of DC and negative for propidium iodide staining. B, FACS analysis of CD80 and CD86 levels on the cell surface of AS-ODN NOD DC treated with rCD40L. NOD DC obtained from bone marrow progenitors of 5- to 8-wk-old NOD mice were treated with 10 µM ODN (AS-ODN) or PBS vehicle as control. rCD40L was added at a final concentration of 50 ng/ml for a period between 18 and 24 h. CD40L addition was unable to stimulate the cell surface expression of CD80 or CD86 to levels higher than control. The data are representative of three independently performed experiments. C, Graph representation of the data in B. D, Cytokine production by NOD DC treated with CD40L. Bone marrow-derived NOD DC were treated with control PBS vehicle or AS-ODN DC. rCD40L was added subsequently at a final concentration of 50 ng/ml for a period of 18–24 h. Multiplex-based cytokine profiling of the culture supernatants following the CD40L addition demonstrated that AS-ODN-treated DC were refractive to CD40L-stimulated NO production and TNF-{alpha} and exhibited suppressed capacity to produce IL-12p70. These data are representative of three independent experiments. E, Ag uptake and processing capacity of AS-ODN DC. Bone marrow-derived NOD DC (1 x 105) were treated with control PBS vehicle or AS-ODN DC and then pulsed with the Vybrant Phagocytosis reagent. Twenty-four hours later, cell fluorescence was quantitated microfluorometrically. The graph bars denote fluorescence intensity (in arbitrary units) and the error bars denote the SEM of triplicate determinations.

 
A single injection of AS-ODN-treated NOD DC into prediabetic NOD mice prolonged the time to diabetes onset

Our previous studies indicated that ODN-engineered DC were capable of prolonging allograft survival and time to diabetes onset (24, 61). To extend those studies we wanted to determine whether AS-ODN DC could prolong the time to diabetes onset. Indeed, significant prolongation of diabetes onset time was observed in female NOD mice given a single injection of 2 x 106 AS-ODN-treated DC (injection at 5–8 wk of age) but not in untreated mice, untreated-DC recipients, NIT-1 lysate-treated DC, or those administered DC cotreated with AS-ODN and NIT-1 lysate (Fig. 2A). Up to 45 wk following the injection, 4 of the original 20 NOD recipients given a single i.p. injection of AS-ODN-treated DC remained diabetes-free (blood glucose <200 mg/dL).



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  		FIGURE 2. A, Diabetes incidence in NOD recipients of DC. DC from bone marrow progenitors of 6- to 8-wk-old female NOD mice were propagated in GM-CSF/IL-4 and further treated with AS-ODN with or without NIT-1 lysate as described. PBS-resuspended cells (in 200 µl; 2 x 106) were injected i.p. into syngeneic age- and sex-matched NOD recipients. Blood glucose was measured by electronic sampling of tail vein blood beginning at 15 wk of age. AS-ODN DC = DC treated with a mixture of CD40, CD80, CD86 AS-ODN, each oligo at 3.3 µM; AS-ODN + NIT1 DC = DC cotreated with the AS-ODN mixture and NIT-1 lysate. p = 0.012, AS-ODN DC recipients vs AS-ODN + NIT-1 DC recipients, Kaplan-Meier log rank test. B, Immunohistochemistry of pancreata from diabetes-free recipients of AS-ODN DC. For immunohistochemistry, the sections were fixed in paraffin, treated to block peroxidase, and incubated with nonfat dried milk. The slides were then incubated with anti-insulin or anti-glucagon Ab followed by an isotype-reactive biotinylated secondary Ab. Avidin-HRP was then added followed by diaminobenzidine after which a brown color could be observed. No evidence of insulitis was observed with insulin and glucagon readily detectable.

 
Though diabetes, as measured by glucose levels, was considerably delayed in AS-ODN DC-recipient NOD mice, we wished to determine whether this was paralleled by prevention or reduced grade of insulitis—the histological readout of diabetes prevention. We examined the histology of pancreata of NOD recipients of AS-ODN-treated DC who had no signs of diabetes at 35 wk of age. We could find no sign of insulitis in H&E sections (Fig. 2B, left panel) and we were able to visualize normal insulin and glucagon content as well (Fig. 2B, middle and right panels). Twenty-two individual islets were visualized from each one of 47 pancreatic sections of two diabetes-free mice with identical results (no insulitis). These data indicate that injection of the AS-ODN NOD DC was able to prevent the infiltration of immune cells that otherwise impair {beta} cell function and that promote {beta} cell destruction, at least in these mice.

T cells from diabetes-free NOD recipients of AS-ODN DC responded to alloantigens in culture

Many immunoregulatory protocols induce systemic immunosuppression. To determine whether our approach acted at a systemic level, we asked whether T cells from "protected" NOD mice would respond to allogeneic stimulation. In Fig. 3A, we demonstrate the results of T cell proliferation showing that DC from bone marrow progenitors of allogeneic C3H/HeJ mice were able to stimulate the proliferation of T cells from the spleen of "protected" NOD mice to levels identical with proliferation of NOD DC-stimulated C3H/HeJ spleen-derived T cells. These data indicate that alloreactivity was maintained in NOD recipients of AS-ODN DC-treated, "protected" NOD mice and that the diabetes suppression was due to a more precise, yet-to-be fully understood mechanism.



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  		FIGURE 3. A, Alloantigen reactivity of T cells from diabetes-free NOD recipients of AS-ODN DC. NODpr DC, DC obtained from bone marrow of NOD recipients of AS-ODN-treated syngeneic DC; C3H Tc, T cells isolated from spleen of age-matched C3H-HeJ mice; C3H DC, DC obtained from bone marrow of C3H mice; NODpr Tc, T cells isolated from spleen of NOD recipients of AS-ODN-treated DC. Data are from a representative proliferation assay with triplicate cocultures. No significant differences between cocultures (Student’s two-tailed t test). B, Cytokine production by T cells from a diabetes-free NOD recipient of AS-ODN DC. Splenic T cells from 22-wk-old diabetic mice and a 31-wk-old NOD recipient of AS-ODN-treated DC (diabetes-free) were stimulated with Con A for 18 h. The supernatants were probed using the Multiplex Beadlyte assay system for cytokine profile. Key is at the bottom right of the figure. Cytokine identities are at the top of each bar pair. C, B7 levels on exogenously administered DC in NOD recipients. CFSE-labeled DC derived from prediabetic female NOD bone marrow progenitors were recovered 1 wk following i.p. injection (2 x 106 DC originally administered). CD80/CD86 double-positive cells were ascertained in the CFSE+ cell population by FACS analysis of labeled splenocytes. Data are representative of two different DC administrations. D, B7 levels on exogenously administered GFP+ DC in C57BL/6 recipients. DC propagated from bone marrow of GFP+ transgenic C57BL/6 mice were recovered 1 wk following i.p. injection (2 x 106 DC originally administered) into C57BL/6 recipients. CD80/CD86 double-positive cells were ascertained in the GFP+ cell population by FACS analysis of labeled splenocytes. Data are representative of two different DC administrations. E, Persistence of exogenously administered DC in NOD recipients. CFSE-labeled DC derived from prediabetic female NOD bone marrow progenitors were recovered 1, 2, and 3 wk following i.p. injection of 2 x 106 DC from spleen. CD80- and CD86-positive cells were ascertained in the CFSE+ cell populations by FACS analysis of labeled splenocytes. F, Persistence of exogenously administered GFP+ DC in C57BL/6 recipients. DC propagated from bone marrow of GFP+ transgenic C57BL/6 mice were recovered from spleen 1, 2, and 3 wk following i.p. injection of 2 x 106 DC into C57BL/6 recipients. CD80- and CD86-positive cells were ascertained in the GFP+ cell population by FACS analysis of labeled splenocytes.

 
TH1-type cytokine production was suppressed in splenocytes obtained from diabetes-free recipients of AS-ODN DC compared with splenocytes from diabetic NOD mice.

Having ascertained that systemic immunosuppression was not at the root of our AS-ODN DC effect, we asked whether AS-ODN DC in vivo shifted the balance of T cell immune responses from TH1 to TH2. Many studies demonstrate that type 1 diabetes is characterized by a TH1-type immune response and that a shift to TH2 is often associated with prevention or prolongation of time-to-onset (62, 63, 64). To determine whether the protection conferred by the AS-ODN DC was due to the predominance of a TH2-type immune environment, we examined the cytokine secretion profile of splenocytes obtained from diabetes-free NOD recipients of the AS-ODN DC. In Fig. 3B, we show that at 31 wk of age, compared with a diabetic NOD mouse (22 wk of age), there were lower levels of TNF-{alpha} and IFN-{gamma} in the supernatants of Con A-stimulated T cells obtained from the spleen of a "protected" NOD recipient of AS-ODN DC. There were no significant differences in the levels of all other cytokines when compared with a diabetic NOD mouse at 22 wk of age.

Persistence of low level B7 levels on AS-ODN DC in vivo

Throughout these experiments, it was unclear whether the AS-ODN-treated DC maintained the same cell surface levels of CD80 and CD86 in vivo or whether these levels changed following exogenous administration. Two complementary approaches were used to address this question. In the first, we examined the levels of CD80 and CD86 on spleen cells derived from NOD recipients of CFSE-labeled syngeneic DC. The CFSE+ cells obtained from AS-ODN DC-administered ODN recipients exhibited the same levels of CD80 and CD86 as did the CFSE+ cells from control DC-administered recipients 1 wk following injection (Fig. 3C). As a complementary approach, we transferred GFP-transgenic DC that were treated with PBS or with AS-ODN in culture into syngeneic recipients. One week later, we harvested the spleens of the recipients and examined the levels of CD80 and CD86 in GFP+ populations by FACS. In Fig. 3D, we show that there were no changes in the cell surface levels of CD80 and CD86 on AS-ODN-treated GFP+ DC indicating that these cells maintained the same levels of CD80 and CD86 in vivo, as they did before the injection. These studies were followed up and in Fig. 3, E and F, we demonstrate that AS-ODN-treated DC persist for 3 wk while the number of CFSE+ and GFP+ cells recoverable from control DC-treated mice declined by 3 wk after exhibiting a significant increase in CD86 levels by 2 wk in vivo. This increase of CD86 was observed only in NOD recipients and not in the C57BL/6 syngeneic recipients of GFP+ control DC.

An increased prevalence of CD4+CD25+ cells was observed in the splenocytes of diabetes-free AS-ODN DC NOD recipients

Although preliminary, we proposed that the "protective" nature of the AS-ODN-modified DC involved the generation and/or survival of regulatory cell activity in the splenocyte fraction resulting in the suppression of activity and/or modulation of the viability of the diabetogenic immune cell populations. A number of investigators have identified a population of T cells that possess regulatory activity and can prevent a number of autoimmune disorders (65, 66, 67). The cell surface phenotype that these cells all appear to share is CD4+, CD25+. Although it is not yet clear whether these are the specific cells which confer regulation, this population does have activity (cellular or soluble) which fulfills this function (68, 69, 70, 71, 72, 73). Therefore, we wished to compare the prevalence of this cell subtype in the splenocytes of "protected" NOD mice with that in untreated and control DC-treated NOD mice from time of administration to the time of diabetes onset. By FACS analysis using fluorescently conjugated anti-CD4 and anti-CD25 Abs, we have determined the profile in Table I. It appears that the protective effect of the AS-ODN injection in NOD mice may be partially due to the generation/survival/activation of T cells within the CD4+CD25+ compartment. Interestingly, control DC also confer some degree of increased CD4+CD25+ cell prevalence, although these numbers are far less than those obtained in spleen of AS-ODN DC. Furthermore, in the cohort studied, control DC-treated NOD mice all became diabetic. Although these data support a DC-based mechanism for CD4+CD25+ T cell expansion, we cannot yet exclude the possibility that other cell types may be involved in transducing the effects of the AS-ODN-treated DC.


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  		Table I. Prevalence/frequency of CD4+CD25+ T cells in NOD splenocytes by FACS analysisa

 
Adoptive cotransfer of NOD AS-ODN DC and NOD T cells into NOD-scid recipients results in an increased prevalence of CD4+CD25+CD62L+ T cells in spleen

As a first approach to understanding the potential mechanism(s) by which the AS-ODN may be acting to prolong the diabetes-free state and whether the association between AS-ODN DC administration and increased numbers of CD4+CD25+ T cells was causally linked, we examined the prevalence of CD25+CD62L+ cells in CD4+ T cell populations from spleen of NOD-scid recipients of control and AS-ODN NOD DC. We first administered 1 x 106–2 x 106 DC from 5- to 8-wk-old female NOD mice into age- and sex-matched NOD-scid recipients i.p. Three days later, we injected 1 x 107 purified splenic T cells from 5- to 7-wk-old female NOD mice. In Fig. 4, we demonstrate a significant increase in the number of total splenic CD4+CD25+ as well as CD25+CD62L+ cells in the splenic CD4+-enriched cell component of NOD-scid recipients of AS-ODN DC compared with untreated DC 1 wk following the T cell transfer. Culture supernatant from splenic and lymph node T cells obtained from all these NOD-scid recipients did not reveal any detectable levels of IL-4 or IL-10 by Beadlyte cytokine profiling (data not shown).



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  		FIGURE 4. A, CD4+CD25+ cell prevalence in a T cell population obtained from spleen of NOD-scid mice reconstituted with NOD DC and prediabetic NOD spleen T cells. Three days following i.p. PBS injection, control DC or AS-ODN DC injection (2 x 106 cells), 1 x 107 splenic NOD T cells were injected i.v. One week later, the recovered splenic T cells were analyzed by FACS for CD4 and CD25 prevalence. The data are representative of prevalence in three different NOD-scid recipients. The cell population was selected based on forward and side scatter. Also shown are the FACS dot plots of the Ab isotypes and the percentage of gated cells that are double positive. B, CD25+CD62L+ cell prevalence in a CD4+ T cell population obtained from spleen of NOD-scid mice reconstituted with NOD DC and prediabetic NOD spleen T cells. Three days following i.p. PBS injection, control DC or AS-ODN DC injection (2 x 106 cells), 1 x 107 splenic NOD T cells were injected i.v. One week later, the recovered splenic CD4+ T cells (magnetic bead column enriched) were analyzed by FACS for CD25 and CD62L prevalence. The data are representative of prevalence in three different NOD-scid recipients. The cell population was selected based on forward and side scatter. Also shown are the FACS dot-plots of the Ab isotypes and the percentage of gated cells that are double positive.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By suppressing costimulatory molecule expression at the cell surface using AS-ODN targeting the primary transcripts of CD40, CD80, and CD86, we have achieved a specific means of engineering host DCs into potentially "tolerogenic" effectors. Our data are comparable with the outcomes presented in a recently published approach targeting the NF-{kappa}B transcriptional pathway in DC using transcriptional decoy ODNs (24). In contrast to the transcriptional decoy approach, direct targeting of the costimulatory transcripts aimed at specific down-regulation of the costimulatory proteins avoids the potential of interfering with NF-{kappa}B-sensitive pathways in DC that may be relevant for survival and in vivo function/persistence of the exogenously administered DC. Although we have not exhaustively determined the effect of the AS-ODN treatment on the transcription of every single gene in DC, preliminary data do not suggest any particular detrimental effects on survival or promiscuous and nonspecific inhibition of cell function/gene transcription (data not shown). Most impressively, by a single administration of AS-ODN DC, we have conferred diabetes protection to NOD mice, although not all recipients remained diabetes-free indefinitely. Interestingly, diabetes incidence was no different in NOD mice administered DC cotreated with NIT-1 lysate and AS-ODN than untreated recipients or mice that received untreated DC. The latter observation is in contrast to data shown by Feili-Hariri et al. (16, 74) and the reasons for this are currently under investigation.

We have shown herein that the exogenously administered DC are detectable and viable following i.p. administration for up to 3 wk. Although no changes are evident in the cell surface levels of CD80 and CD86 of AS-ODN-treated DC across 3 wk following administration, significant changes especially of CD86, are observed on control DC following exposure to an in vivo autoimmune environment (the NOD mouse). In these recipients, control DC CD86 was observed to be increased at 2 wk following exogenous administration and was dramatically reduced along with CD80 by 3 wk. Its increased level may underlie a time period in which autoimmune processes could be recapitulated in exogenous DC NOD mouse transfer models. Additionally, it appears that untreated DC become scarce by 3 wk following exogenous administration, suggesting a time frame of exogenously supplied DC survival in vivo. In contrast, AS-ODN DC appear to persist at 3 wk at numbers and with a B7 phenotype similar to that observed at 1 wk following exogenous administration. The maintenance of nearly identical B7 levels on AS-ODN DC at 1 and 3 wk following exogenous transfer could also suggest one possible mechanism of immunoregulation where the persistence of DC with low or absent B7 in an environment poised for autoreactivity can engage counterreceptors present exclusively on regulatory T cells promoting their expansion and/or survival. A number of recent studies support such a potential mechanism (75, 76). Indeed, the ability of AS-ODN DC to suppress diabetes onset may involve a direct effect of the AS-ODN DC on the expansion of CD25+CD62L+ cells from CD4+ precursors, and/or their enhanced survival. The failure to observe increases of these same cells in NOD-scid recipients of control DC as well as the absence of any differences in the prevalence and numbers of single CD4+ or single CD8+ cells between NOD-scid recipients of control and AS-ODN DC argues against homeostatic expansion as the basis for the increased prevalence of the CD4+CD25+CD62L+ cells (data not shown).

Despite the prolonged time to diabetes onset in a significant number of AS-ODN recipients, many NOD mice eventually became diabetic. The most obvious reason for failure of persistence with a single injection of AS-ODN DC is the limited lifespan of exogenously administered DC in vivo, a possibility that is supported by the data presented in Fig. 3, E and F. A number of other studies indicate that exogenously supplied DC have a lifespan between 7 and 14 days (77 , 78). Assuming that the DC effect is directly suppressive (i.e., DC:autoreactive T cell interaction), the exhaustion of the exogenous DC population conferring suppressive activity would explain the lack of persistence. If so, multiple dosings could theoretically prolong the effect or achieve indefinite protection. This would also be valid if regulatory T cell expansion was dependent on DC persistence; recent studies appear to support this possibility. Especially exciting are the data by Steinman and colleagues (75, 76) who have just recently shown that DC can directly induce the expansion of CD4+CD25+ T cells in vivo which possess Ag-specific suppressive capacity. We have data that are similar to those published by Yamazaki et al. (76) demonstrating that AS-ODN-treated DC derived from the DO11.10 TCR transgenic mouse promote suppressed Ag-specific T cell proliferation in vivo and a concomitant increase in the prevalence of CD4+CD25+ T cells in vitro that could underlie the in vivo suppression (our unpublished data). Although these and a number of other similar studies favor the induction of regulatory T cells by DC with immunosuppressive activity, a role for NK-T or other cell populations cannot be ruled out (79, 80). Additionally, if the DC effect is indirect, it would be of interest to determine how immunoregulatory DC induce different regulatory immune cell populations and how they promote the persistence of these secondary cellular networks.

Our studies show that AS-ODN DC-treated, diabetes-free NOD mice exhibited a complete absence of insulitis. However, it is possible that this may not be the case in all diabetes-free recipients of AS-ODN. A study of a significantly larger population of diabetes-free mice administered AS-ODN may reveal varying degrees of insulitis and these infiltrating (or peri-islet) cells will need to be phenotyped if such observations are indeed made. AS-ODN DC administration was associated with an increase in CD4+CD25+ cell numbers in the splenocytes of diabetes-free NOD recipients. Furthermore, AS-ODN DC administration to NOD mice was associated with a progressive increase in the prevalence of CD4+CD25+ T cells with increasing age and was not observed in untreated NOD or control DC-treated NOD. Our data in NOD-scid mouse recipients of AS-ODN DC and T cells support the hypothesis that these DC may directly promote the proliferation/survival/activity of CD4+CD25+ cells with immunoregulatory capacity. We are currently determining whether CD4+CD25+ cells generated in NOD-scid mice by AS-ODN DC can suppress adoptive transfer of diabetes with diabetogenic NOD T cells into secondary NOD-scid recipients. The number of reports directly implicating these cells in preventing diabetes onset by regulating immune cell function compel us to further study these specific cell types and their mechanism of action in well-defined in vivo studies (16, 17, 74, 75, 76, 81, 82, 83, 84).

A respectable body of evidence supports the existence of endogenous immunosuppressive DC in vivo and suggests molecular pathways which can be exogenously manipulated to make their immunosuppressive activity persistent in vivo (85, 86). A number of factors, which at this time remain poorly understood or unexplored, could influence the effectiveness of the DC in maintaining such a regulatory DC network along with a regulatory T cell population: 1) the phenotypic nature of the DC; 2) their maturational status at the time of administration, the route of administration; 3) the anatomical site of action; 4) the precise number of cells administered, and the effects of multiple dosings. Addressing the mechanisms of immunoregulation by DC as well as the precise phenotype of the "active" DC and/or regulatory immune cells in appropriate in vivo models, like the NOD mouse, is important because a DC-based approach can be potentially translated to the clinic, for prophylaxis in high-risk individuals or in newly onset cases of T1DM to save residual {beta} cell mass.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Awards DK61328 and DK60183 (to N.G.). Back

2 Address correspondence and reprint requests to Dr. Nick Giannoukakis, Diabetes Institute, University of Pittsburgh School of Medicine, Rangos Research Center, 3460 Fifth Avenue, Pittsburgh, PA 15213. E-mail address: ngiann1{at}pitt.edu Back

3 Abbreviations used in this paper: T1DM, type 1 diabetes mellitus; DC, dendritic cell; AS-ODN, antisense oligonucleotide. Back

Received for publication October 9, 2003. Accepted for publication July 26, 2004.


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 Results
 Discussion
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