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Dendritic cells (DC) play important roles in the initiation of immune responses and maintenance of self-tolerance. We have been studying the role of DC in the ...

Immunologic Research 2006;36/1–3:167–173

Dendritic Cell Immunotherapy for Autoimmune Diabetes

Abstract Dendritic cells (DC) play important roles in the initiation of immune responses and maintenance of self-tolerance. We have been studying the role of DC in the pathogenesis of type 1 diabetes and exploring the ability of specific DC subsets to prevent diabetes in non-obese diabetic (NOD) mice. DC subsets that prevent diabetes in this model have a mature phenotype and induce the production of regulatory Th2 cells. We review here recent advances in this area and highlight the importance of optimizing culture conditions and purification methods in the isolation of therapeutic DC.

Maryam Feili-Hariri1,3 Rafael R. Flores3 A. Cecilia Vasquez3 Penelope A. Morel2,3

Key Words Dendritic cells Type 1 diabetes Autoimmunity Immunotherapy

Introduction Over the last several years the work in our laboratory has focused on the study of dendritic cells in the murine model of type 1 diabetes (T1D), the non-obese diabetic (NOD) mouse. T1D is an autoimmune disease char-

Departments of 1Surgery, 2 Medicine, and 3Immunology, University of Pittsburgh, Pittsburgh, PA

acterized by the destruction of the insulinproducing β cells of the islets of Langerhans (1). Prior to the development of diabetes, the islets become heavily infiltrated with lymphoid cells, including CD4+, CD8+ T cells, dendritic cells, and monocytes (2,3) and by the time diabetes appears over 90% of the

Penelope A. Morel, MD Department of Immunology, University of Pittsburgh, 200 Lothrop Street, BST E1048, Pittsburgh, PA 15261. E-mail: [email protected] and Maryam Feili-Hariri, PhD Departments of Surgery and Immunology, University of Pittsburgh, 3550 Terrace Street, 666 Scaife Hall, Pittsburgh, PA 15261. E-mail: [email protected]

© 2006 Humana Press Inc. 0257–277X/ (Online)1559-0755/06/ 36/1–3:167–173/$30.00

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islets have been destroyed. Therapeutic interventions to prevent diabetes have to be aimed at the period of insulitis during which most of the destruction takes place. The NOD mouse is a good model of human T1D as it shares many of the genetic and immunological features of the human disease (4). NOD mice (females>males) spontaneously develop diabetes between 15 and 20 wk of age (4). Genetic analysis of diabetes susceptibility in the NOD mouse has revealed that a minimum of 15 genes are implicated, and one of these is mapped to the MHC (5). Many of the other genes associated with T1D have important functions in the immune system, and include genes important in T cell differentiation and function (5). It has been known for some time that effector Th1 cells and CTL are responsible for the autoimmunity in NOD mice (6–8). This effect seems to be both at the level of the initiation of the autoimmune response and at the stage of islet cell destruction. The importance of the Th1 pathway has been demonstrated in various knockout systems in which it was shown that IRF1–/– (9) and Stat4–/– NOD mice (10) were protected from the development of diabetes. In contrast Stat 6–/– NOD mice developed diabetes more rapidly than wild-type NOD suggesting that endogenous Th2 cells can protect mice to some degree (10). We have been interested in determining whether specific DC subsets play a role in the development of the type 1 skewed immune response in NOD mice and we have explored the use of specific DC subsets in the prevention of diabetes onset. Do DC Defects Contribute to the Development of Autoimmunity in NOD Mice? In T1D, several groups have reported abnormalities in DC phenotype and function

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in both the mouse and human, which may result in the skewing of the response toward pathogenic Th1 cells (11–15). Studies on bone marrow (BM)–derived DC have been somewhat controversial with some groups reporting increases in IL-12p70 production (11–13,15), a cytokine important for Th1 differentiation, while others including ourselves have reported reduced IL-12p70 production by NOD BMderived DC (16–18). These discrepancies led us to the detailed examination of the phenotype and function of BM DC in NOD mice. There is agreement among all investigators that there is a defect in the number of DC generated from NOD BM (13,14,16,18), which appears to be related to a defect in the responsiveness of NOD BM cells to the cytokine GM-CSF (19). However, there is disagreement as to whether NOD DC are more or less prone to drive Th1 differentiation, or T cell activation. Some of these discrepancies could be due to differences in the culture conditions used in the generation of BM-derived DC, which vary in terms of cytokine concentrations used, length of the culture, and method of purification. We have found that all of these factors can have a profound influence on DC phenotype and function. We have conducted experiments in which we have tested different culture systems and maturation conditions. In these experiments we compared DC from NOD and BALB/c mice generated either in the presence of GM-CSF (GM DC) alone or GMCSF + IL-4 (GM4 DC). The GM DC were induced to mature with either CpG or TNF-α + PGE2. Following maturation the DC were stimulated with J-558 cells expressing CD40L in the presence of IL-4 and the resulting supernatants were analyzed for the presence of IL12p70 (Fig. 1). It was apparent that some conditions, such as CpG, led to increased IL12p70 production by NOD DC, whereas other conditions, such as GM4 DC, resulted in increased IL-12p70 production by BALB/c

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Fig. 1. Maturation cocktail influences ability of DC to produce IL-12p70. BM DCs were generated in the presence of GM-CSF for 4 d. On d 4 GM DCs were matured with TNF-α + PGE2 or CpG 1668 (PS) overnight. Simultaneously, BM DCs were also generated in the presence of GM-CSF and IL-4 for 5 d. On d 5 all DCs were harvested using an anti-CD11c Mini-MACS column. GM DCs were transferred into 48 well plates at a concentration of 1× 106 cells/mL and co-cultured for 24 h with CD40L expression J-558 cells at a concentration of 1× 106 cells/mL plus IL-4 (2 ng/mL). The supernatants were analyzed for the presence of IL-12p70 by ELISA. The data presented are representative of two independent experiments.

DC (Fig. 1). These data suggest that the study of BM-derived DC in NOD mice may not be representative of what occurs in vivo, and furthermore the plasticity of DC progenitors from NOD mice can lead to the generation of DCs with either Th1 or Th2 promoting activity (20). Thus, our laboratory and other investigators have performed studies to examine the phenotype and function of DC subsets in vivo. It has been observed that NOD mice exhibit defects in the number of regulatory T cells, including NKT cells, CD4+ CD25+ T cells, and Th2 cells (21–23). Recent studies have implicated several DC subsets in the maintenance and activation of different Treg populations (24). Several groups including our own have determined that NOD mice have an imbalance in the numbers of specific splenic DC subsets. This is characterized by a relative decrease in the number of CD8α+ DC (14,18) and an increase in the number of myeloid DC. We have also observed that splenic DC from NOD mice produce lower levels of IL-12p40 than similar populations from C57BL/6 mice (18). In a study from O’Keeffe et al., the defect in

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CD8α+ DC numbers was mapped to a nonMHC susceptibility locus on chromosome 4 (25), and these investigators also observed a reduction in the levels of IL-12 produced by NOD DC. Recent studies targeting antigens to CD8α+ DC through the use of the antiDEC205 mAb have resulted in either the deletion of antigen-specific T cells (26) or the induction of specific Tregs (27), demonstrating a role for these DCs in the maintenance of steady-state tolerance. We observed that the administration of recombinant human Flt3 ligand (FL) to NOD mice preferentially increased the number of CD8α+ DC (18). We also observed that both CD8α+ and CD8α– DCs could delay the onset of diabetes and induce some degree of protection from diabetes development (18). O’Keeffe et al. followed this up by demonstrating that FL treatment could prevent diabetes in NOD mice (25). These data collectively suggest that NOD mice have a defect in the generation of specific DC subsets that may play an important role in the development and maintenance of self-tolerance.

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DCs Can Be Used to Correct the Autoimmune Response In parallel we have also examined the ability of BM-derived DC to prevent and/or treat diabetes. When we started these studies several years ago, we anticipated that so-called immature DC (iDC), expressing low levels of costimulatory molecules and MHC class II, would be more effective in diabetes prevention than mature/stimulatory DC (mDC). To our surprise, we observed that mDC were able to prevent disease when given to young prediabetic NOD mice, whereas iDC were completely ineffective (28). The therapeutic DCs expressed high levels of CD80, CD86, and CD40 and, when stimulated using CD40L or LPS, produced low levels of IL-12p70 (16). Of note, the most mature DCs were the best at preventing the disease. In a study examining the role of gene-modified DCs using adenoviral vectors, we observed that a single injection of adenovirally infected DCs given at 8 wk of age could protect all NOD mice from developing diabetes (29). DCs infected with adenoviral vectors expressed a high level of maturation markers. Although a few investigators have reported modest success with iDC (30), most reports of successful DC therapy in NOD mice have involved the use of DCs matured using various culture conditions (31–33). This may be explained by the fact that the development and maintenance of Tregs require high levels of co-stimulation and the presence of IL-2. We analyzed the mechanism by which mDC prevent diabetes in this model and our early observation suggested that the DC treatment induced regulatory Th2 cells (34). Indeed, splenocytes from NOD mice that were given DCs 7 wk prior to in vitro activation produced high levels of the type 2 cytokines IL-4 and IL-5, whereas splenocytes from age-matched untreated NOD mice produced undetectable levels of these cytokines

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Fig. 2. Analysis of CD45RBlo CD25+ CD4+ T cells in the spleen of (A) 12-wk-old mice treated with DCpep (DC, n=10) or PBS (n=5), or (B) 32-wk-old nondiabetic mice treated with DCpep (n=4) and mice treated with DCmed (n=4). Seven-week-old prediabetic (Pre, n=3) and 24–28-wk-old diabetic (Diab, n=5) mice were included as controls. Cells were stained with rat anti-mouse FITC-conjugated CD45RB, PE-conjugated CD25, and Cychrome-conjugated CD4 mAbs or isotype controls for 30 min on ice. Small CD4+ T cells were analyzed for the expression of CD45RBlo/CD25+ cells by flow cytometry.

(34). In addition, we observed an increase in the number of CD4+ CD25+, CD45RBlo T cells in the spleen of DC-treated mice (Fig. 2). These cells were sorted and found to secrete type 2 cytokines following activation, whereas they failed to suppress the proliferation of CD4+ CD25– T cells in a classic suppression assay (34). At the time that we performed these studies, the FoxP3 mAb was not available. It is possible that Tregs were also induced in addition to the Th2 effectors and/or expanded by the DC treatment but we were not able to detect them. We are actively pursuing this possibility at the present time. This speculation is supported by the recent demonstration by Tarbell et al. that sorted CD86hi DC from NOD mice can be used to expand sorted CD25+ Treg in vitro and that these expanded Tregs had suppressor activity in

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Fig. 3. The influence of the method of purification on DC phenotype and function. DCs were generated from BM progenitors in the presence of GM-CSF+ IL-4. The GM+ IL-4 DCs were purified on 14.5% metrizamide gradients (solid lines) or DC were purified using CD11c beads (dashed lines). (A) The Purified DCs were stained with the indicated mAbs and were analyzed by flow cytometry on an open gate. (B) Purified DCs were activated with LPS + IFN-γ for 24 h and culture supernatant were collected and analyzed for IL-12p70 by ELISA.

vitro and prevented diabetes development in prediabetic NOD mice (35). The fact that sorted CD86hi DC could be used to expand preexisting Tregs was interesting and prompted us to further examine the therapeutic DCs in our system. The DCs that we used were isolated following a relatively short (4–5 d) culture of BM progenitors in the presence of GM-CSF and IL-4 (28). At the end of the culture the cells were purified on a 14.5% metrizamide gradient and the cells routinely expressed a uniformly high level of CD86 and other costimulatory molecules (16,28,29). More recently, we have compared the use of CD11c microbeads with the metrizamide gradient, and we have observed a significant difference in the phenotype and function of DCs isolated using these two techniques (Fig. 3). When GM4 DCs were purified using CD11c beads, two populations were clearly distinguishable, a minor population that expressed high levels of CD86 representing mDC and a

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larger population of iDC. The metrizamide purification specifically enriched for the CD86hi DCs (Fig. 3). Interestingly, the metrizamidepurified DC produced lower levels of IL-12p70 compared to the DCs isolated using CD11c microbeads. This difference was not due to the ability of metrizamide to induce DC maturation because in our early studies we used a 16% metrizamide gradient to purify the iDC, and these cells maintained a very immature phenotype following the purification step (28). We believe that the use of metrizamide gradients allowed us to “sort” CD86hi DC from the BM cultures, thus isolating a DC population with an increased ability to stimulate type 2 responses, but also perhaps Tregs. Our more recent studies have focused on the identification of other phenotypic markers or secreted cytokines/chemokines that may be playing a role in the therapeutic effect of mDC in NOD mice. Microarray analysis comparing the gene expression profiles of the

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mDC and iDC used in these studies have revealed several additional molecules that are expressed more highly in the therapeutic DC compared to iDC. These include OX40L, CD200, and various chemokine and secreted proteins thought to be associated with the development of Th2 cells or Tregs. Future Prospects for DC Therapy Several issues need to be addressed before DC therapy can be considered for use in human prediabetics. The DC therapies that we have used are most effective when given early during the prediabetic phase, and clearly strategies should be developed such that this could be given later once symptoms have started. We have shown that DCs transduced to express the cytokine IL-4 can be given much later, once destructive insulitis has begun (29). We are presently exploring using DCs that produce IL-10 or TGF-β in addition to IL-4 with the hopes of inducing additional Treg populations, and thereby allowing us to delay treatment even longer. Another is the issue of antigen specificity. In our system we have found that unpulsed DCs are as effective in preventing disease as those pulsed with islet-derived antigens or peptides (28). We have explained this by demonstrating that DCs migrate to both pancreas and pancreatic lymph

node (28,29) and speculating that the DCs acquire specific antigen at these sites, although this remains to be proven. However, a recent publication has demonstrated that this could be related to the use of fetal calf serum (FCS) in the culture medium (36). These investigators demonstrated that DCs grown in FCS induced a systemic immune deviation, whereas the culture of DC in autologous mouse serum could induce antigen-specific immunomodulation (36). The DCs grown in mouse serum were only modestly effective in preventing disease, and this may be due to the low levels of CD86 expressed by these cells. In our own experience the culture of BM cells in the presence of mouse serum does not give rise to DCs with the same degree of maturity as those grown in FBS. In our view it is important to identify those characteristic features of DC that are important for the therapeutic effect and then focus on developing culture conditions in the human to optimize the generation of DC with the ability to stimulate antigen-specific Tregs of various types. Acknowledgments The authors would like to thank Dewayne Falkner for expert technical assistance. This work was supported by NIH grant CA73743 (PAM) and DOD training grant DAMD17-991-9352 (RRF).

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