Injection Leads to Islet Regeneration in Autoimmune Diabetes
                    Study points to unexpected treatment for type 1 diabetes

A possible cure for insulin-dependent diabetes is in sight following a major medical breakthrough. Scientists in the United States have not only halted the disease in mice mimicking human type 1 diabetes, but reversed it. Plans are now under way to conduct patient trials which, could lead to the first ever curative treatment for the disorder. Massachusetts General Hospital researchers have harnessed newly discovered cells from an unexpected source, the spleen, to cure juvenile diabetes in mice, a surprising breakthrough that could soon be tested in local patients and open a new chapter in diabetes research.  The current study builds on the team's earlier work in which they found that beta cell-directed autoimmunity in NOD mice could be reversed and pancreatic islet cell function could be restored by injection of donor splenocytes and complete Freund's adjuvant, which induces expression of tumor necrosis factor (TNF)-alpha. The MGH scientists injected diabetic mice with the spleen cells. The cells migrated to their pancreases, prompting the damaged organs to regenerate into healthy, insulin-making organs, ending their diabetes. This is among the few documented cases of a major organ regenerating itself in an adult mammal. The research also finds a potential use for the spleen, long considered an organ with no apparent purpose. "This shows there might be a whole new type of therapy that we haven't tapped into," said Dr. Denise Faustman, MGH immunology lab director and lead author of the new study, which appears today in the journal Science. "We've figured out how to regrow an adult organ."  Dr. George King, Joslin Diabetes Center research director, who was not involved in the research, said: "That you could just take spleen cells, infuse them, and somehow the pancreas is regenerated, that's exciting . . . The next step is to see if it can be done in humans." Mass. General's Diabetes Center has received approval from the US Food and Drug Administration to try the techniques pioneered by Faustman in humans. The center's director, Dr. David M. Nathan, stressed it remains uncertain whether they will work in humans. The hospital's team has not yet raised enough money to proceed with a 40-person clinical trial, which Nathan estimates would cost about $10 million. The research was funded by the Boston-based Iacocca Foundation, a diabetes charity begun by then-Chrysler executive Lee Iacocca two decades ago after his wife succumbed to the disease. The foundation's resources are not nearly enough to bankroll the proposed clinical trial. In juvenile, or Type 1, diabetes, victims' immune systems attack the insulin-making cells in the pancreas early in life. Insulin moves sugar, a crucial energy source, from blood into cells. The new MGH findings build on research first reported two years ago, when Faustman's team found it could retrain the immune system of diabetic mice not to attack the pancreas by injecting the mice with spleen cells, along with a protein that tames the immune system, from healthy mice. Faustman's team expected to have to transplant new insulin-making cells into the pancreas to give the mice the lifetime ability to produce insulin. In humans, such transplants are risky and debilitating surgeries.  Instead, it turned out that a select subpopulation of the spleen cells grew and nurtured the damaged pancreases into health. The pattern repeated in 11 mice.  "We've found that [pancreas] regeneration was occurring and that cells were growing from both the recipient's own cells and from the donor cells," Faustman said.  The results, published in the journal Science, confirmed that the pancreas was being reconstructed in the sick mice. That finding means for the first time there is a prospect of curing patients with type 1 diabetes

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Islet Regeneration During the Reversal of Autoimmune Diabetes in NOD Mice

Shohta Kodama, Willem Kühtreiber, Satoshi Fujimura, Elizabeth A. Dale, Denise L. Faustman^*

Nonobese diabetic (NOD) mice are a model for type 1 diabetes in humans. Treatment of NOD mice with end-stage disease by injection of donor splenocytes and complete Freund's adjuvant eliminates autoimmunity and permanently restores normoglycemia. The return of endogenous insulin secretion is accompanied by the reappearance of pancreatic ß cells. We now show that live donor male or labeled splenocytes administered to diabetic NOD females contain cells that rapidly differentiate into islet and ductal epithelial cells within the pancreas. Treatment with irradiated splenocytes is also followed by islet regeneration, but at a slower rate. The islets generated in both instances are persistent, functional, and apparent in all NOD hosts with permanent disease reversal.

Immunobiology Laboratory, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Room 3602, Charlestown, MA 02129, USA.

^* To whom correspondence should be addressed. E-mail: faustman@helix.mgh.harvard.edu

The NOD mouse exhibits spontaneous autoimmunity that causes diabetes through destruction of insulin-secreting pancreatic islets. A lymphoid cell–specific proteasome defect in these mice interrupts the presentation of self antigens by major histocompatibility complex (MHC) class I molecules that is required for negative selection of autoreactive naïve T cells (1, 2). The proteasome defect also impairs activation of the transcription factor nuclear factor–B in pathogenic memory T cells, increasing their susceptibility to apoptosis induced by tumor necrosis factor– (TNF-) (3–5). Reselection of peripheral autoimmune naïve T cells is possible by the introduction of matched MHC class I–self peptide complexes, whereas self-directed autoimmune memory T cells can be reselected by treatment with TNF- or by the induction of the endogenous TNF- with complete Freund's adjuvant (CFA) (5, 6). Simultaneous treatment of severely diabetic NOD mice with both TNF- and normal splenocytes partially or fully matched for MHC class I antigens thus restores self-tolerance and eliminates T cells directed against islets, resulting in permanent reversal of established diabetes (7). This "cure" is accompanied by the reappearance of insulin-secreting islets in the pancreas which can control blood glucose concentration in an apparently normal manner.

The new pancreatic islets in such treated NOD mice might arise from several sources, either endogenous or donor-derived sources. Donor nonlymphoid cells administered to mice or humans can undergo rare transdifferentiation events (8–25), although these findings remain controversial (26, 27). Alternatively, the regenerated islet cells in NOD mice might be the products of fusion between donor and host cells, in a mouse model of liver damage (28, 29). Such fusion events generate cells with marked chromosomal abnormalities (30, 31).

To investigate the origin of the new pancreatic islet cells in NOD mice, we examined the relative abilities of live versus irradiated donor splenocytes to restore normoglycemia (32). We injected CFA and either live or irradiated male CByB6F₁ mouse splenocytes into severely diabetic NOD females, which were used to ensure the absence of visible islets and insulitis that could obscure dead or dying islets (table S1). We controlled blood glucose concentration with a temporary (40-day) implant of syngeneic islets under the capsule of one kidney, which improved treatment efficacy. Similar to our previous data (7), six (67%) of the nine NOD mice that received live splenocytes remained normoglycemic after removal of the islet implant (Fig. 1A). In contrast, none of the eight animals that received irradiated splenocytes remained normoglycemic; they all rapidly developed severe hyperglycemia. (See supporting online text and fig. S1.) In another experiment, the islet transplant was maintained for 120 days before graft removal, to allow a longer period for islet regeneration. Of the 12 NOD mice that received live splenocytes, 11 (92%) remained normoglycemic for >26 weeks after disease onset or beyond 52 weeks of age. Moreover, 11 (85%) of the 13 animals that received irradiated splenocytes also remained normoglycemic for >27 weeks after disease onset or beyond 48 weeks of age (Fig. 1A, table S2). Both live and irradiated splenocytes could thus effect permanent disease elimination, and with a longer period of imposed normoglycemia greatly increasing the frequency of functional islet recovery in both groups.

Fig. 1. Effects of treatment with live or irradiated splenocytes on the restoration of normoglycemia and pancreatic histology in diabetic NOD mice. (A) Kaplan-Meier plot for normoglycemia. Diabetic NOD females were treated with a single injection of CFA and biweekly injections for 40 days of either live (circles) or irradiated (squares) splenocytes from CByB6F1 males. Syngeneic female islets transplanted subrenally at the onset of treatment were removed after either 40 days (left panel) or 120 days (right panel). Blood glucose concentration was monitored at the indicated times after islet graft removal, and the percentage of animals that remained normoglycemic was plotted. Data are from 9 and 8 (left panel) or from 12 and 13 (right panel) animals that received live or irradiated splenocytes, respectively; P = 0.0002 (left panel), P = 0.68 (right panel) for comparison between the two treatment groups. (B) Pancreatic histology. Three NOD mice successfully treated with either irradiated (top panels) or live (bottom panels) splenocytes were killed 9 weeks after removal of the 120-day islet graft. Sections of each pancreas were stained with hematoxylin and eosin. Pronounced peri-insulitis was apparent only in the NOD mice treated with irradiated cells. [View Larger Version of this Image (37K GIF file)]

Mice treated with irradiated splenocytes that exhibited persistent normoglycemia for 9 weeks after nephrectomy (table S2) exhibited the reappearance of pancreatic islets without invasive insulitis (autoreactive cells within the islets) but with pronounced peri-insulitis (circumferential lymphoid cells that do not progress to invasion) (Fig. 1B, table S3). In contrast, the pancreas of NOD mice that received live splenocytes exhibited the reappearance of pancreatic islets without invasive insulitis and with minimal or no peri-insulitis. The live splenocytes were thus necessary for reduction of peri-insulitis but not for the growth of new islets. Functionally, the restoration of long-term normoglycemia was indistinguishable between animals with disease reversal due to live or irradiated splenocytes.

We next tested mice that had been treated with live or irradiated splenocytes for the presence of live donor cells in blood, pancreas, and other tissues. Peripheral blood lymphocytes (PBLs) from NOD mice treated with irradiated CByB6F₁ splenocytes showed only background staining for H-2K^b (an indicator of live donor cells), indicating that no donor hematopoietic cells remained (Table 1; table S2 and fig. S2). In contrast, 4.4 to 12.6% of PBLs from NOD mice treated with live CByB6F₁ splenocytes were of donor origin. PBLs from an untreated NOD mouse contained only cells expressing H-2K^d, and those from a CByB6F₁ mouse contained exclusively cells coexpressing H-2K^b and H-2K^d. NOD mice treated with live splenocytes thus exhibited a persistent low level of blood chimerism with semiallogeneic cells that was achieved without continuous immunosuppression or lethal preconditioning.

Flow cytometry also revealed between 3.5 and 4.7% of cells positive for both H-2K^d and H-2K^b among splenocytes from five NOD mice successfully treated with live splenocytes; this confirmed the persistence of donor CByB6F₁ cells in all recipients (Table 1). Splenocytes from an untreated control NOD mouse showed a background level of 0.3% double-positive staining for both markers. CByB6F₁ donor splenocytes also contributed to T cells (CD3⁺), monocytes (CD11b), and B cells (CD45R⁺) (data not shown).

We then examined parenchymal tissues for chimerism by fluorescence in situ hybridization (FISH) analysis for detection of the Y chromosome of the male donor cells in two long-term normoglycemic NOD mice (Fig. 2A). Staining of serial pancreatic sections with antibodies to insulin revealed a homogeneous insulin content in the large islets (Fig. 2B; Table 1), consistent with the restored normoglycemia. Single-color FISH analysis revealed abundant nuclei positive for the Y chromosome within the islets (Fig. 2B; Table 1). In contrast, the exocrine portions of the pancreas were largely devoid of male cells. In these five animals, 29 to 79% of islet cells were of donor origin. No islets solely of host origin were detected.

Fig. 2. Long-term restoration of normoglycemia and the direct contribution of live donor splenocytes to islet regeneration in successfully treated NOD female mice. (A) Blood glucose concentrations during the lifetime of two NOD females (top and bottom, nos. 789 and 790 in Table 1, respectively) successfully treated with CFA and CByB6F1 male splenocytes, as well as with a temporary subrenal transplant of syngeneic islets. (B) Immunofluorescence and FISH analyses of serial pancreatic sections from the successfully treated NOD females 789 (left) and 790 (right). The two top panels show immunofluorescence staining of islets with antibodies to insulin (red); the three pairs of images below show FISH signals obtained with a Y chromosome–specific probe (pink dots) and nuclear staining with DAPI (blue) in sections containing islets (arrows), pancreatic ducts (arrowheads), and exocrine pancreas, respectively. [View Larger Version of this Image (61K GIF file)]

Male donor cells also contributed to the epithelium of NOD female pancreatic ducts, although the distribution of male cells in this tissue was more heterogeneous than was that in islets (Fig. 2B, Table 1). Among the five treated NOD females studied in detail, 33 to 75% of the ducts contained genetic material of male origin. Ducts purely of host origin were never associated with an adjacent islet containing male cells. The proportion of male cells in the pancreatic ducts of the five NOD mice ranged from 9 to 41%. Single-color FISH analysis revealed abundant nuclei positive for the Y chromosome within both the exocrine and endocrine portions of the pancreas of control C57BL/6 male mice, whereas the pancreas of control C57BL/6 females was devoid of the Y chromosome (fig. S3A). We detected no evidence of engraftment, transdifferentiation, or fusion of male splenocytes in organs including the brain, liver, and kidney of treated NOD females (fig. S4B), which suggests that, in addition to the low level of hematopoietic chimerism observed, the marked incorporation of donor cells was selective for the diseased pancreas.

To examine whether the new islet cells arose by fusion of donor cells with endogenous islet cells, we evaluated >800 nuclei in ß cells as well as >800 nuclei in adjacent exocrine tissue of the five treated NOD females studied in detail (fig. S3B and Table 2). At three scanning focal lengths, none of the regenerated cells within the islets was enlarged compared with the adjacent native exocrine cells. The ß-cell nuclei were of normal size and did not contain multiple nucleoli. These observations suggest that the regenerated islet cells were not the products of fusion between donor splenocytes and endogenous dying or injured ß cells, since hybrid cells contain markedly enlarged nuclei and multiple nucleoli and are tetraploid (30, 31). We cannot, however, exclude the possible occurrence of fusion followed by rapid and complete expulsion of host chromosomes.

We further examined the ploidy of the sex chromosomes of cells in the regenerated islets of successfully treated NOD mice by two-color FISH analysis with a Y chromosome–specific probe linked to fluorescein isothiocyanate (FITC) (green) and an X chromosome–specific probe conjugated with cyanine 3 (Cy3) (red). A NOD female treated with live male splenocytes exhibited only rare if any islet cells with an apparent XXY or XXXY genotype (Fig. 3). A normal complement of sex chromosomes was also observed in pancreatic duct epithelial cells. These results also indicate that the regenerated islet cells were not likely to be the result of fusion between donor male cells and host female cells. None of the islet cell nuclei examined in a NOD female treated with irradiated male splenocytes contained a detectable Y chromosome; rather, each nucleus yielded two red signals, corresponding to a genotype of XX (Fig. 3). Two-color FISH analysis of the pancreas of untreated female and male NOD mice revealed that, although this methodology can yield false-negative data (female nuclei with no red signal or only one red signal), it almost never yielded false-positive data (a green signal in the nucleus of a female cell or two green signals within an individual male nucleus) (fig. S4A).

Fig. 3. Two-color FISH analysis of sex chromosomes in the pancreas of NOD female mice successfully treated with either live or irradiated male splenocytes. Pancreatic sections from NOD females treated with live (A) or irradiated (B) CByB6F1 male splenocytes were subjected to nuclear staining with DAPI (blue) and to FISH analysis with a Cy3-conjugated X chromosome–specific probe (red dots) and an FITC-conjugated Y chromosome–specific probe (green dots). Purple represents overlap of Cy3 and DAPI signals. Arrows indicate outline of islets. [View Larger Version of this Image (34K GIF file)]

Embryonic mesenchymal cells are able to differentiate into endothelial and endoderm cells, and they lack surface expression of CD45 (33–37). To examine whether nonlymphoid stem cells contribute to the regeneration of pancreatic islets in NOD mice, we injected 12-week-old NOD females with live CD45⁺, CD45^–, or unfractionated CByB6F₁ splenocytes expressing enhanced green fluorescent protein (GFP). These experiments differed from our previous experiments: (i) The NOD females were prediabetic (with residual islet function but with active autoimmunity) at the start of treatment; (ii) they did not receive an islet graft; (iii) the number of cells injected was reduced to 4 x 10⁵ to 5 x 10⁵ administered four times over 2 weeks; and (iv) the regrowth of islets was monitored by detection of GFP immunofluorescence. All of the NOD females that received CD45⁺, CD45^–, or unfractionated splenocytes remained normoglycemic during the monitoring period, whereas all untreated NOD littermates (n = 10) became diabetic.

Immunoblot analysis of cytoplasmic extracts prepared from the pancreas of NOD mice revealed more GFP for those treated >120 days earlier with CD45^– splenocytes than for those treated with CD45⁺ splenocytes (Fig. 4A). In addition, the pancreas of the prediabetic NOD females treated with either CD45^– or unfractionated splenocytes contained islets positive for the GFP marker (Fig. 4B). Furthermore, the newly generated islets lacked invasive lymphocytes and were associated with minimal or no periinsulitis, as revealed by costaining for insulin and CD45 (Fig. 4C). The proportion of islets containing cells of donor origin was markedly smaller for prediabetic NOD hosts treated with CD45^– or unfractionated splenocytes than for severely diabetic NOD females treated with unfractionated splenocytes. This was as expected because the pancreas of the prediabetic mice still contained endogenous islets affected by peri-insulitis. Treatment of prediabetic animals with precursor cells thus rescues damaged islets and also promotes de novo islet regeneration. The islets of prediabetic NOD females treated with CD45⁺ splenocytes were negative for the expression of GFP (Fig. 4B). Moreover, similar to the islet regeneration observed in severely diabetic NOD mice treated with irradiated splenocytes, the newly appearing islets in prediabetic NOD females treated with CD45⁺ splenocytes were devoid of invasive insulitis but exhibited pronounced periinsulitis (Fig. 4C). The donor CD45⁺ splenocytes, although essential for disease reversal, do not include cells able to participate directly in islet generation.

Fig. 4. Effect of treatment of prediabetic NOD mice with CD45– or CD45+ CByB6F1 splenocytes expressing GFP on pancreatic histology. (A) Immunoblot analysis with antibodies to GFP of pancreatic extracts (2 or 5 µg of protein) prepared from the indicated control mice or treated NOD mice. (B) Prediabetic NOD females (12 weeks old) were treated with CFA and either CD45– (lower right), CD45+ (lower left), or unfractionated (upper right) CByB6F1 splenocytes expressing GFP and were monitored for >120 days. Serial pancreatic sections containing islets identified by costaining for insulin and CD45 were then subjected to immunofluorescence analysis with antibodies to GFP (green). Sections from the pancreas of an untreated C57BL/6 mouse are shown as a control (upper left). Arrowheads indicate peri-insulitis. (C) Serial pancreatic sections from a diabetic NOD female, a prediabetic NOD female (12 weeks old), and a C57BL/6 control, as well as from prediabetic NOD females treated with CFA and either unfractionated, CD45+, or CD45– CByB6F1 splenocytes were subjected to immunofluorescence analysis with antibodies to insulin (red) or to CD45 (green), as indicated; merged images are shown in the bottom row. [View Larger Version of this Image (33K GIF file)]

Overall, our data indicate that live male splenocytes injected into female diabetic NOD mice can provide cells (CD45^– mesenchymal precursor cells) for the reconstitution of functional islets. The donor splenocytes also contribute to reversal of autoimmunity, possibly by reeducating naïve T cells through presentation of matched MHC class I molecules and self antigens, yielding islets almost free of any signs of autoimmunity. In contrast, diabetic NOD mice treated with irradiated splenocytes exhibit long-term restoration of normoglycemia as a result of islet regeneration but with markedly slower kinetics than those apparent in NOD animals treated with live splenocytes. Thus, adult diabetic NOD mice contain endogenous precursor cells capable of giving rise to new islet structures after the underlying autoimmune disease is eliminated. These syngeneic islets appear to function normally but succumb to stable, nonprogressive peri-insulitis.

Our findings with the NOD mouse may have implications for treatment of diabetes or other autoimmune diseases in humans. Both the ability of an exogenous population of adult spleen cells to correct established diabetes permanently and the presence of an endogenous population of NOD mouse stem cells able to give rise to new islets suggest that therapies to reverse autoimmune diabetes need not incorporate transplantation of exogenous adult islets. The use of fresh splenocytes eliminates the need for cell culture manipulations that transform stem cells of fetal or adult origin into malignant precursors or fusion hybrids with an abnormal DNA content. Because the cell donors and hosts are adults this system would preclude ethical issues associated with the use of embryonic stem cells, as well as concerns that the transdifferentiation of embryonic stem cells may be incomplete.

References and Notes

Supporting Online Material

www.sciencemag.org/cgi/content/full/302/5648/1223/DC1

Materials and Methods

SOM Text

Figs. S1 to S4

Tables S1 to S3

References

8 July 2003; accepted 10 October 2003
10.1126/science.1088949
Include this information when citing this paper.

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Volume 302, Number 5648, Issue of 14 Nov 2003, pp. 1223-1227.

BOSTON — June 27, 2001 — Researchers at Massachusetts General Hospital (MGH) have shown that an unexpectedly simple treatment cures type 1 diabetes in mice. Published in the July 1 Journal of Clinical Investigation, the findings are distinct from the current theories on how to treat the disease. "With only a brief treatment, we have reversed an established autoimmune disease in a respected animal model," says Denise Faustman, MD, PhD, of the Immunobiology Laboratory at MGH and principal investigator of the study. "Although the results are preliminary, this is an exciting finding for diabetes." Faustman’s treatment involves re-training the immune system in order to halt the disease that causes the destruction of islet cells, the insulin-secreting cells of the pancreas. The apparent permanent reversal of established disease allows the host to remake insulin from the pancreas by regrowth of islet cells that are no longer under attack. Normally, the body’s immune cells attack toxins and foreign invaders, such as bacteria and viruses. In type 1 diabetes, though, the immune cells somehow receive a faulty signal, and their destructive efforts are instead directed toward the insulin-secreting islet cells. This results in a decrease of insulin – a molecule necessary for the conversion of glucose into fuel – and a subsequent increase in glucose. As glucose accumulates in the blood, it can damage organs, leading to conditions such as heart disease, kidney failure and blindness. For the past five years, Faustman and her team have been closely examining the immune cells of diabetic mice and humans. They came across two key findings: the immune cells died when they were exposed to the naturally occurring drug, TNF-alpha, and many of the immune cells were unable to present self-peptides – a process crucial for preventing the development of autoimmune reactions. Because of these two findings, Faustman and her team designed a two-pronged treatment strategy. First, they triggered the expression of TNF-alpha in the mice in an attempt to destroy the immune cells that had gone awry. This approach had never been used for the treatment of type 1 diabetes. On the contrary, TNF-alpha antagonists have been prescribed, an intervention that never permanently reverses or cures the disease. Antagonists of TNF-alpha are often used in the clinic as a general treatment for suppressing inflammation. "According to our findings, future efforts for diabetes treatment should look at using TNF agonists instead of antagonists," says Faustman. In the next step of the two-pronged approach, the researchers addressed the lack of self-peptide presentation. The diabetic mice were injected with donor cells that expressed the self-peptides. This treatment re-educated the newly emerging immune cells of the mouse, ensuring that they do not attack the islet cells.Up to 75% of the treated mice had normal blood glucose levels that persisted beyond 100 days after the treatment was discontinued. Faustman and her group found no need to administer islet cell transplants or to intervene in a pre-diabetic state, the two major treatment approaches used for type 1 diabetic patients today. "With islet cell transplantation you need a tissue source, you need long term immunosuppression and the disease recurs," says Faustman. She also says that trying to attack the disease before it actually develops is expensive, especially when it comes to pinpointing "potential" diabetics by looking for a genetic link. The only other current option for diabetic patients is to treat the complications, says Faustman. In essence, individuals monitor their glucose levels and inject themselves with insulin. Faustman is working with David Nathan, MD, of the Diabetes Unit at MGH, to bring her new treatment approach to the clinic. Funding for the current study was provided by The Iacocca Foundation.The Massachusetts General Hospital, established in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $300 million and major research centers in AIDS, the neurosciences, cardiovascular research, cancer, cutaneous biology, transplantation biology and photo-medicine. In 1994, the MGH joined with Brigham and Women’s Hospital to form Partners HealthCare System, an integrated health care delivery system comprised of the two academic medical centers, specialty and community hospitals, a network of physician groups and nonacute and home health services.

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J Clin Invest, July 2001, Volume 108, Number 1, 63-72
Copyright ©2001 by the American Society for Clinical Investigation

Reversal of established autoimmune diabetes by restoration of endogenous ß cell function

Shinichiro Ryu¹, Shohta Kodama¹, Kazuko Ryu¹, David A. Schoenfeld² and Denise L. Faustman¹

¹ Immunobiology Laboratory, and
² Department of Biostatistics, Harvard Medical School and Massachusetts General Hospital, Charlestown, Massachusetts, USA

Address correspondence to: Denise L. Faustman, Immunobiology Laboratory, Massachusetts General Hospital–East, Harvard Medical School, Building 149, Room 3602, 13th Street, MailStop M1493601, Charlestown, Massachusetts 02129, USA. Phone: (617) 726-4084; Fax: (617) 726-4095; E-mail: denise.faustman@cbrc2.mgh.harvard.edu.

Received for publication January 24, 2001, and accepted in revised form May 14, 2001.

Abstract

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In NOD (nonobese diabetic) mice, a model of autoimmune diabetes, various immunomodulatory interventions prevent progression to diabetes. However, after hyperglycemia is established, such interventions rarely alter the course of disease or allow sustained engraftment of islet transplants. A proteasome defect in lymphoid cells of NOD mice impairs the presentation of self antigens and increases the susceptibility of these cells to TNF-–induced apoptosis. Here, we examine the hypothesis that induction of TNF- expression combined with reeducation of newly emerging T cells with self antigens can interrupt autoimmunity. Hyperglycemic NOD mice were treated with CFA to induce TNF- expression and were exposed to functional complexes of MHC class I molecules and antigenic peptides either by repeated injection of MHC class I matched splenocytes or by transplantation of islets from nonautoimmune donors. Hyperglycemia was controlled in animals injected with splenocytes by administration of insulin or, more effectively, by implantation of encapsulated islets. These interventions reversed the established ß cell–directed autoimmunity and restored endogenous pancreatic islet function to such an extent that normoglycemia was maintained in up to 75% of animals after discontinuation of treatment and removal of islet transplants. A therapy aimed at the selective elimination of autoreactive cells and the reeducation of T cells, when combined with control of glycemia, is thus able to effect an apparent cure of established type 1 diabetes in the NOD mouse.

Introduction

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Autoimmune destruction of pancreatic ß cells is more than 90% complete by the time hyperglycemia becomes clinically evident in individuals with type 1 (insulin-dependent) diabetes mellitus. Prevention of this disease would therefore optimally require arrest of autoimmunity in the prehyperglycemic phase. After hyperglycemia is established, therapies based on islet cell replacement are necessary to restore physiological control of blood glucose. Although islet transplantation has been successful in mice, rats, and nonhuman primates with chemically induced diabetes, sustained survival of allogeneic islet grafts is infrequently observed in spontaneous diabetic hosts such as the NOD (nonobese diabetic) mouse, BB (BioBreeding) rat, and diabetic humans (1-5). Thus, allogeneic or xenogeneic cellular grafts subjected to transient ablation of donor MHC class I antigen expression (6) — achieved either with the use of "masking" Ab’s to MHC class I molecules or by deletion of the ß₂-microglobulin (ß₂M) gene — are capable of permanent engraftment in nonautoimmune recipients, but are minimally protected from recurrent ß cell autoimmunity in NOD mice (7, 8). The immune mechanisms of islet graft rejection and recurrent autoimmunity appear distinct, and protective interventions targeted at these two pathways of islet destruction are nonoverlapping in effectiveness.

Subsets of antigen-presenting cells and T cells of NOD mice that progress to hyperglycemia exhibit a decrease in the production of LMP2, a catalytic subunit of the proteasome, after about 5–6 weeks of age (9, 10). This defect is accompanied by deficient generation of peptides from endogenous proteins for display on the cell surface by MHC class I molecules, a process that is important for T cell memory and tolerance to self antigens (11, 12) and is impaired in various human and murine autoimmune diseases (11, 13, 14). The proteasome also contributes to the processing and activation of NF-B (15-17), a transcription factor that regulates the expression of genes that contribute to cytokine generation, lymphocyte maturation, protection from TNF-–induced apoptosis, and promotion of the processing of antigens for presentation by MHC class I molecules (18-20). The proteasome defect in NOD mice affects lymphoid maturation as a result, at least in part, of impaired activation of NF-B.

In normal humans and other mammals, the continuous expression of MHC class I molecules by peripheral cells, including islet cells (21), maintains peripheral tolerance in the context of properly selected lymphocytes (12, 22). Interruption of exposure to complexes of self peptides and MHC class I molecules results in the aberrant selection of CD8⁺ T cells that exhibit an increased sensitivity to apoptosis (23). Diabetic humans and NOD mice that progress to diabetes thus manifest a paucity of memory cells and an increased susceptibility of T cells to apoptosis, traits that may be secondary, in part, to improper presentation of self antigens by MHC class I molecules and ineffective T cell selection (10, 11).

On the basis of the view that islet transplantation into hyperglycemic NOD mice will require both the prevention of transplant rejection and the elimination of autoimmune T cells, we sought to combine two strategies to achieve these goals. To bypass graft rejection, we used donor islets from C57BL/6 mice in which the ß₂M gene was deleted. MHC class I proteins are re-expressed on graft cells within 24 to 72 hours after transplantation as a result of reconstitution with host ß₂M present in plasma (6, 24, 25). The re-expression of donor MHC class I antigens is important because it is necessary for the development and maintenance of peripheral tolerance.

With regard to interruption of autoimmunity, we hypothesized that the lineage-specific defects both in peptide presentation by MHC class I molecules and in the processing and activation of NF-B might be important in the pathogenesis of diabetes in NOD mice. The NF-B defect in the affected lineages on NOD mice is accompanied by an increased sensitivity of these cells to TNF-–induced apoptosis in vitro (10). The increased susceptibility to apoptosis of misselected T cells that result from improper education by MHC class I peptide complexes suggested that the production of TNF- in vivo might promote the selective death of such poorly educated lineages (10, 26, 27). Furthermore, treatment of diabetic NOD mice or BB rats with CFA, an inducer of TNF- production, both impairs the transfer of disease by T cells from these animals to naive hosts (28-31) as well as prolongs the survival of syngeneic islet grafts in spontaneously diabetic hosts (2, 32). We therefore hypothesized that CFA treatment might eliminate, at least temporarily, the autoreactive lymphoid cells of NOD mice by promoting their apoptosis, in part through the induction of TNF-.

Thus, we both treated hyperglycemic NOD recipients with CFA, seeking to eliminate autoreactive lymphoid lineages, and transplanted islets from ß₂M-deficient donors under the kidney capsule of these animals, seeking to generate graft-specific tolerance. These interventions resulted in the marked reduction or apparent elimination of ongoing ß cell–directed autoimmunity. Unexpectedly, the long-term reappearance of endogenous ß cell function was also observed in the pancreatic islets of the previously hyperglycemic hosts.

Methods

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Animals. Female NOD mice from Taconic Farms (Germantown, New York, USA) and C57BL/6J (C57) mice from The Jackson Laboratory (Bar Harbor, Maine, USA) were maintained under pathogen-free conditions. NOD mice were screened for the onset of diabetes by monitoring body weight and blood glucose; they were diagnosed as diabetic when two consecutive blood glucose concentrations exceeded 400 mg/dl. Before experimental treatments, diabetic NOD mice were maintained for 7–20 days on daily injections of 1.0–1.5 U of NPH human insulin per 100 g of body weight, thereby preventing immediate death and maintaining blood sugar concentration between 200 and 700 mg/dl. The use of such severely diabetic mice, relatively late after disease onset, ensured that endogenous pancreatic islets were completely obliterated before the initiation of experiments. Splenocyte donors included normal C57 mice, C57 mice (C57 ß₂M^–/–) in which the ß₂M gene was disrupted, C57 mice (C57 ß₂M^–/–, TAP1^–/–) in which both the ß₂M and Tap1 genes were disrupted, and MHC class II^–/– mice (C57 class II^–/–) in which the I-A gene was disrupted and the E locus of MHC class II was not expressed because of an endogenous defect in the C57 strain (Taconic Farms). Splenocytes (9 x 10⁶) were injected into NOD recipients through the tail vein twice a week. CFA (Difco Laboratories, Detroit, Michigan, USA) was freshly mixed with an equal volume of physiological saline and injected (50 µl) into each hind-foot pad at the time of islet transplantation or after the first splenocyte injection.

Islet transplantation. Islets were isolated from donor C57 mice or 6- to 8-week-old prediabetic female NOD mice. Density gradient centrifugation followed by hand picking of islets ensured that both preparations were highly enriched in islets and had an accurate determination of islet number. For transplantation, 500–600 freshly isolated islets were grafted beneath the left renal capsule of each diabetic NOD recipient. For islet encapsulation, 900–1,100 islets were enclosed in alginate spheres, which were then surgically inserted into the peritoneal cavity of diabetic NOD mice. Transplantation was considered successful if the nonfasting blood concentration of glucose returned to normal (<200 mg/dl) within 24 hours after surgery. The glucose concentration of orbital blood was monitored three times a week after transplantation with a Glucometer Elite instrument (Bayer Corp, Pittsburgh, Pennsylvania, USA). Body weight was also monitored three times a week. Islet grafts were considered to have been rejected if the blood glucose concentration increased to more than 250 mg/dl on two occasions. Recipients that rejected islet grafts were killed for histological examination and flow-cytometric studies. To assess the contribution of endogenous pancreatic islets to the control of blood sugar concentration, we removed subrenal islet transplants by nephrectomy. Similarly, islets encapsulated in alginate spheres, which were approximately 0.2–0.5 cm in diameter, were removed from the peritoneal cavity by direct visualization under a dissecting microscope. Histological analysis of the pancreata and islet grafts was performed by staining with hematoxylin and eosin for evaluation of lymphocytic infiltrates and with aldehyde-fuchsin for islet insulin content. The entire pancreas from splenic to duodenal stomach attachments was removed, fixed, embedded longitudinally in a paraffin block, and subjected to serial sectioning (10 µm).

Flow cytometry. Spleens were removed and gently minced on a stainless steel sieve. Cell suspensions were rendered free of red blood cells by exposure to a solution containing 0.83% NH4Cl. The splenocytes were stained with mouse mAb’s (PharMingen, San Diego, California, USA) to CD8 (FITC-labeled), to CD62L (phycoerythrin-labeled [PE-labeled]), to CD95 (PE-labeled), or to CD45RB (PE-labeled), and were analyzed (>10,000 cells per sample) the same day with an Epics Elite flow cytometer. Spleen cells were incubated for 24 hours in the absence or presence of TNF- (20 ng/ml), after which apoptotic cells were detected by flow cytometry with FITC-conjugated annexin V. Apoptotic cells were defined as cells positive for both propidium iodide (PI) and annexin V staining; numbers within the upper quadrants represent the corresponding percentages of cells.

Adoptive transfer. Adoptive transfer was performed as described (29). Recipient male NOD mice, 4–8 weeks of age, were irradiated (790 rads) with a ¹³⁷Cs source and injected intravenously within 2 hours of irradiation with donor splenocytes (2 x 10⁷ viable cells) suspended in 0.25 ml of serum-free medium. Diabetic spleen cell donors were female NOD mice that typically had exhibited blood sugar concentrations of greater than 400 mg/dl for at least 3 weeks. Multiple diabetic donor spleens were pooled to produce sufficient cells for all of the hosts in a given experiment.

Statistics. Exact algorithm P values were calculated in some instances with multiple comparisons corrected by the number of tested variables. A P value less than 0.05 was considered statistically significant.

Results

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CFA treatment and islet transplantation in NOD mice. Hosts for the transplantation experiments were severely diabetic female NOD mice, usually more than 20 weeks of age, that had exhibited blood glucose concentrations of greater than 400 mg/dl for at least 7 days and had been treated by daily administration of insulin to prevent death. Islet transplants were placed unilaterally under the kidney capsule to facilitate nonlethal removal and histological examination. Islets from 6- to 8-week-old prediabetic NOD females (recipient group A) or from normal C57 mice (recipient group B) were rapidly rejected by diabetic NOD recipients (Table 1, Figure 1a). Although C57 donor islets with transient ablation of MHC class I expression survive indefinitely in nonautoimmune diabetic hosts (6), the survival time of islets from ß₂M^–/– C57 mice in diabetic NOD females (group C) was only about three times that of normal C57 islets. As expected from previous observations (2, 32), treatment with CFA prolonged the survival of syngeneic islet grafts in diabetic NOD hosts (group D) but had a minimal effect on the survival of C57 islets (group E), which were uniformly rejected by 11 days after transplantation. However, the combination of ß₂M^–/– C57 islet transplants with CFA treatment resulted in sustained (>129 days) normoglycemia in 5 of 14 diabetic NOD hosts (group F). Although the duration of hyperglycemia before initiation of therapy varied between 7 and 20 days, the length of this interval was not significantly related to the duration of sustained normoglycemia after treatment (data not shown). The animals that exhibited sustained normoglycemia also demonstrated progressive weight gain, similar to that apparent in NOD female cohorts who never became diabetic (data not shown). With normalization of blood sugar concentration as a measure of treatment success, the success of ß₂M^–/– C57 islet transplantation together with CFA administration was significantly different from that apparent with the other groups combined but did not differ from that of NOD islet transplantation together with CFA treatment.

After the recurrence of hyperglycemia in the NOD mice that had been treated with CFA and syngeneic (NOD) islet transplants, the kidney containing the islet graft was examined histologically. Marked lymphocytic infiltration was apparent under the kidney capsule at the site of transplantation, a characteristic of recurrent autoimmune disease (Figure 1b, group D); moreover, no intact islets were detected in the pancreas, although islet remnants, largely obscured by dense pockets of infiltrating lymphocytes, were evident. Similar histological characteristics of both the transplant site and pancreas were apparent in diabetic NOD mice that had received CFA and islet grafts from C57 donors (Figure 1b, group E). Unexpectedly, for all five NOD mice with long-term normoglycemia after receiving ß₂M^–/– C57 islets and CFA treatment, no surviving islet grafts were detected under the kidney capsule when the animals were killed at more than 129 days after transplantation (Figure 1b, group F). In contrast, the pancreas of each of these five recipients exhibited well-formed islets that appeared completely granulated when stained by aldehyde-fuchsin. The islets were free of lymphocytes or lymphocytes were present only circumferentially; this latter pattern of lymphocyte accumulation, with lymphocytes surrounding but not invading the islets, has been associated with nonprogressive or interrupted ß cell autoimmunity (33). The return to normoglycemia in the absence of detectable transplanted islet tissue, together with the presence of islets in a pancreas largely devoid of infiltrating lymphocytes, suggested not only that autoimmunity had been interrupted but that the function of endogenous ß cells had been restored.

Restoration of near-normal pancreatic islet histology was observed only in the diabetic NOD mice that received both ß₂M^–/– islet grafts and CFA treatment (Figure 2). Pancreatic islets were thus not detected in any diabetic NOD mice treated with CFA and syngeneic NOD islets; the persistence of normoglycemia in such recipients appeared solely due to the transplanted islets, which always exhibited invasive insulitis (Figure 1b, Figure 2). Thus, treatment with CFA together with syngeneic NOD islets may have slowed disease recurrence, but persistent autoimmunity remained.

The relative contributions of restored endogenous pancreatic islets and transplanted islets to the maintenance of normoglycemia in NOD mice treated with CFA and islet grafts from ß₂M^–/– C57 donors were assessed by removal of the kidney containing the islet transplant after 120 days of normoglycemia in a second group of five animals. All five mice remained normoglycemic after nephrectomy until they were killed 3–60 days later (Figure 3). Histological analysis of the kidneys that received the grafts revealed a complete loss of identifiable islet structures. In contrast, the pancreata of all five recipients contained well-formed islets either without lymphocytic infiltration or with circumferentially distributed lymphocytes only. Normoglycemia after nephrectomy was thus maintained solely by endogenous pancreatic islets. In contrast, nephrectomies performed during the posttransplantation period of normoglycemia (day 62, day 85) in two mice who had received CFA plus syngeneic NOD islets resulted in a rapid return to hyperglycemia (data not shown), demonstrating that the control of blood sugar in this treatment group was mediated solely by the transplanted islet tissue.

We also transplanted diabetic NOD females with islets from C57 mice in which the genes for both ß₂M and TAP1 had been deleted. Together with TAP2, TAP1 mediates the transport of endogenous peptides from the cytosol into the lumen of the endoplasmic reticulum for their assembly with MHC class I molecules (34). Islet cells from these mice are more permanently defective in presentation of self antigens by MHC class I than are those from ß₂M^–/– mice. Transplantation of ß₂M^–/–, TAP1^–/– C57 islets combined with injection of CFA resulted in the return of hyperglycemia within 14 days in five of six animals (group G); histological examination of the pancreata revealed a pattern typical of that for untreated diabetic NOD mice (Table 1, Figure 2). Thus, a transient interruption of peptide presentation by donor MHC class I molecules is essential for the abrogation of autoimmunity, whereas a sustained interruption of this process prevents the reestablishment of tolerance and the restoration of endogenous pancreatic islet integrity.

CFA treatment and splenocyte injection in NOD mice. Given that the restoration of normoglycemia in the diabetic NOD mice treated with CFA and ß₂M^–/– C57 islets did not depend on the continuing secretion of insulin by the islet grafts, we next investigated whether C57 donor cell types other than islets might serve a similar therapeutic role. Nine diabetic NOD mice were treated with a single bilateral injection of CFA followed by a 40-day regimen of biweekly intravenous injections of C57 splenocytes. These lymphoid cells express both MHC class I and class II proteins and survive only transiently in NOD hosts because of graft rejection (data not shown). Repeat injections of splenocytes ensure that the host is continuously exposed to intact antigen presentation complexes on the surface of these cells. The recipients were monitored for hyperglycemia every 3 or 4 days, and insulin was administered daily unless normoglycemia returned. A control group of four diabetic NOD mice received daily insulin injections only. All four control mice died on or before day 25 of the experimental period as a result of poor control of blood glucose and consequent ketosis and cachexia (Figure 4a). In contrast, seven of the nine mice injected with CFA and C57 splenocytes were alive after 40 days, and three of these animals had become normoglycemic and insulin independent (Figure 4b). The pancreata of control (insulin treatment only) mice exhibited pronounced lymphocytic infiltrates that obscured any remaining islet structures (Figure 4c). The pancreata of the four NOD mice treated with CFA and C57 splenocytes that remained alive but hyperglycemic and insulin dependent revealed a marked decrease (relative to control animals) in the number of lymphoid infiltrates, which were located either circumferentially or adjacent to the infrequent islet structures (Figure 4d). On killing of each of the three NOD mice treated with CFA and C57 splenocytes that maintained normoglycemia after discontinuation of insulin injections, the pancreata exhibited abundant islets that were free of invasive lymphocytes or were associated only with circumferential lymphocytes (Figure 4e). Thus, treatment with CFA combined with repeated exposure to C57 lymphocytes resulted in complete reversal of diabetes in approximately 30% of NOD recipients and partially restored ß cell function in an additional approximately 40% of recipients.

Influence of glycemic control on restoration of endogenous islet function. The reversal of diabetes in NOD mice by CFA and repeated exposure to C57 splenocytes indicated that restoration of endogenous islet function is achievable without islet transplantation and despite the poor glycemic control attained by insulin injections. The beneficial influence of glycemic control on the growth, survival, and function of cultured islets, as well as of transplanted islets in nonautoimmune settings, has been demonstrated (35, 36). To determine whether the restoration of endogenous ß cell function could be achieved more consistently with better control of blood glucose, we replaced insulin injections with the intraperitoneal implantation of alginate-encapsulated C57 mouse islets. Alginate encapsulation prevents direct contact between donor endocrine cells and host T cells, and such grafts have been shown to provide near-normal glycemic control for 40 to 50 days in approximately 70–80% of autoimmune NOD recipients (37).

Almost all diabetic NOD mice that received alginate-encapsulated C57 islets exhibited improved glucose regulation or normoglycemia. The alginate spheres were removed 40–50 days after implantation, and blood glucose concentration was monitored (Table 2). The seven mice treated only with alginate-encapsulated islets (group A), the six mice that received a single bilateral injection of CFA (group B), and the three mice treated with biweekly injections of C57 splenocytes (data not shown) all exhibited a rapid return to hyperglycemia and early death after removal of the implants (Table 2, Figure 5). The pancreata of NOD mice that received only alginate-encapsulated islets revealed no sign of intact islets or of lymphoid infiltrates (data not shown). The pancreata of NOD hosts treated with CFA and alginate-encapsulated islets exhibited marked invasive insulitis that obscured islet structures (Figure 6). In contrast, seven of the nine (78%) diabetic NOD mice that received CFA and C57 splenocytes (group C) remained normoglycemic for more than 40 days (until killing) after removal of the alginate-encapsulated islets (Figure 5, Table 2). The pancreata of these animals contained large islets with circumferentially distributed lymphocytes (Figure 6). The islet mass after at least 80 days of disease reversal was estimated at approximately 50% of the original value. The pancreata from control BALB/c mice contained approximately 25–35 islets; the pancreata from successfully treated NOD mice contained approximately 12–20 islets with serial histological sections. Thus, maintenance of normoglycemia during the treatment period increased the percentage of diabetic mice cured of hyperglycemia.

Role of TNF- in treatment outcome. We attempted to identify features of the successful treatment regimens that are critical to a positive outcome. We had used CFA to induce the endogenous production of TNF- (31). The importance of TNF- in treatment success was therefore investigated by the intravenous administration of a rat IgG1 mAb to this cytokine (clone MP6-X73; Accurate Chemical & Scientific Corp., Westbury, New York, USA) at a dose of 1.5 mg/day for the first 10 days in diabetic NOD hosts treated with C57 splenocytes, CFA, and alginate-encapsulated islets. All five NOD mice so treated exhibited a rapid return to hyperglycemia on removal of the alginate-encapsulated islets 50–70 days after transplantation (Table 2, group F), consistent with the notion that TNF- plays an obligatory role in the beneficial effect of CFA. This effect of the mAb to TNF- was specific, given that administration of a rat IgG1 mAb to the human T cell receptor V_ß1 chain (clone BL37.2; American Type Culture Collection, Rockville, Maryland, USA) at a dose of 1.5 mg/day for 10 days did not affect the success of treatment with C57 splenocytes and CFA (data not shown). Direct administration of TNF- to diabetic hosts was not feasible because of the prohibitive cost.

We next investigated whether the production of TNF- in diabetic NOD mice treated with CFA results in the selective elimination of autoreactive lymphoid cells first by examining the susceptibility of lymphocytes from successfully treated animals to TNF-–induced cell death in vitro. As shown previously (26, 27), incubation of normal C57 spleen cells with TNF- in vitro had no effect on cell viability; for the animal shown in Figure 7a, the proportion of apoptotic cells was 0.01% for splenocytes incubated in the absence or presence of TNF-. In contrast, exposure of splenocytes from an untreated 20-week-old NOD female to TNF- in vitro increased the proportion of apoptotic cells from 0.03 to 38.3%. Such an increased sensitivity to TNF-–induced apoptosis in vitro was no longer evident with spleen cells derived from NOD mice cured of diabetes; thus, splenocytes from a NOD female successfully treated with both CFA and C57 splenocytes (Table 2, group C) exhibited 23.2 and 23.9% apoptosis in the absence and presence of TNF-, respectively (Figure 7a). Successful therapy generated a subpopulation of nonpathologic but TNF-–resistant T cells that exhibited an increased tendency to undergo cell death in culture (Figure 7a). Disease reversal, even 210 days after cessation of treatment, was thus associated with the persistent elimination of TNF-–sensitive T cells, a population of cells with a demonstrated ability to play a role in disease (29, 30). The permanent elimination of these formerly abundant TNF-–sensitive lymphoid cells, presumably in response to TNF- (and, perhaps, to other CFA-induced cytokines), was observed uniformly in successfully treated diabetic NOD mice. Similar complete and stable elimination of TNF-–sensitive cells at various times after treatment has been observed in more than 20 NOD mice.

View larger version (43K): [in this window] [in a new window]   Figure 7. Roles of TNF- and MHC class I peptide complexes in reversal of diabetes in NOD mice. (a) Effect of TNF- on the survival of spleen cells derived from a control C57 mouse or from untreated or successfully treated NOD female mice. (b) Effect of TNF- treatment of splenocytes from diabetic NOD mice on the adoptive transfer of disease and the inability of splenocytes from successfully treated NOD mice to transfer disease. Young male NOD mice were irradiated and then injected with diabetic NOD female splenocytes either immediately after their isolation (left panel, dashed lines) or after incubation for 24 hours in the absence (left panel, solid lines) or presence (middle panel) of TNF- (20 ng/ml); alternatively, four irradiated hosts each received splenocytes from a different NOD donor with long-term normoglycemia restored by CFA and C57 spleen cell injections (right panel). (c) Flow cytometric analysis of the percentages of CD8+CD45RBhigh, CD8+CD62L+, and CD8+CD95+ cells among splenocytes of mice from various treatment groups. Diabetic NOD females were implanted intraperitoneally with alginate-encapsulated C57 islets. They then received no further treatment (group A), a single bilateral injection of CFA only (group B), or CFA treatment plus biweekly intravenous injections of splenocytes from normal C57 mice (group C), ß2M–/–, TAP1–/– C57 mice (group D), or MHC class II–/– C57 mice (group E). Shaded bars represent C57 control mice (group F) or NOD mice that exhibited normoglycemia and disease reversal after removal of alginate-encapsulated islets (groups C and E); open bars represent untreated NOD mice (group A) or NOD mice subjected to treatments that did not result in disease reversal (groups B and D).

We also examined the effect of TNF- on the pathogenesis of autoimmune diabetes in adoptive transfer experiments. Young recipient NOD males were subjected to irradiation followed by an intravenous injection of donor splenocytes either from newly diabetic NOD females or from NOD mice with long-term normoglycemia due to treatment with CFA and C57 splenocytes. The onset of diabetes was observed in all recipients by day 15 after the transfer of diabetic mouse cells that were injected either immediately after isolation or after control culture for 24 hours (Figure 7b, left panel). In contrast, four of the five recipients of diabetic mouse cells that had been cultured with TNF- for 24 hours before adoptive transfer remained normoglycemic for at least 40 days after cell injection (Figure 7b, middle panel). Furthermore, the four NOD hosts each injected with splenocytes from a different NOD female that had experienced reversal of autoimmunity for more than 120 days failed to develop disease during observation periods of more than 60 days (Figure 7b, right panel). TNF-–resistant NOD splenocytes, enriched either in vitro by direct exposure of cells to TNF- or in vivo by disease reversal, are thus incapable of transferring disease to naive hosts.

Role of MHC class I peptide complexes in T cell selection and treatment outcome. Disease reversal in diabetic NOD mice required treatment with both CFA and cells that express MHC class I peptide complexes. Only two of six (33%) diabetic NOD mice that received CFA and biweekly injections of splenocytes from ß₂M^–/–, TAP1^–/– C57 donors remained normoglycemic after removal of alginate-encapsulated islets (Table 2, group D). The pancreata of the four animals that became hyperglycemic after removal of the alginate spheres contained no granulated islets and only a few visible islet structures, which were invaded and obscured by lymphocytic infiltrates (Figure 6). In contrast, 8 of 11 (73%) diabetic NOD mice treated with CFA and splenocytes from C57 donors lacking MHC class II protein expression remained normoglycemic after removal of the alginate-encapsulated islets (Table 2, group E); the pancreata of these animals contained large islets that exhibited only moderate lymphocytic accumulation at the periphery (Figure 6).

Interruption of antigen presentation by MHC class I skews the T cell repertoire to an overabundance of naive cells, a consistent trait of diabetes-prone NOD mice and humans (38-40). Improper T cell selection secondary to interruption of antigen presentation by MHC class I results in overexpression of CD95 by CD8⁺ T cells as well as an increase in the abundance of cells with naive cell markers such as CD62L⁺ and CD45RB^high (12, 23). To investigate whether therapeutic reversal of NOD mouse diabetes was associated with a change in naive T cell selection, we subjected splenocytes to flow-cytometric analysis. Flow cytometry was performed 5 to 26 days after removal of the alginate-encapsulated islets and termination of therapy.

Untreated NOD mice exhibited the expected increases in the abundance of naive CD8⁺CD45RB^high, CD8⁺CD62L⁺, and CD8⁺CD95⁺ cells compared with C57 animals (Figure 7c). In contrast, in NOD female mice that were successfully treated with alginate-encapsulated islets, CFA, and administration of C57 splenocytes, the percentages of each of these cell populations were reduced to normal or near-normal values. The abnormally high numbers of CD8⁺CD45RB^high, CD8⁺CD62L⁺, and CD8⁺CD95⁺ cells remained increased in diabetic NOD females treated with alginate-encapsulated islets and CFA, either alone or together with administration of ß₂M^–/–, TAP1^–/– C57 splenocytes. Data are means plus or minus SEM of values from at least five mice per group. Exact algorithm P values were calculated for comparisons of each cell population between groups C, E, and F versus groups A, B, and D: P = 0.001 for CD8⁺CD45RB^high cells, P = 0.01 for CD8⁺CD62L⁺ cells, and P = 0.05 for CD8⁺CD95⁺ cells. Despite the fact that many comparisons were performed, the P value remained less than 0.05 even when multiplied by the three comparisons. These data showed that the T cell reselection apparent in successfully treated NOD mice was secondary to reexposure to complexes of MHC class I molecules and self peptides. The normalization of T cell phenotype did not require reexposure to MHC class II–peptide complexes, given that the administration together with CFA and alginate-encapsulated islets of MHC class II^–/– splenocytes was as effective as was that of normal C57 splenocytes.

Discussion

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We have demonstrated the effectiveness of a novel therapy for the correction of established autoimmune diabetes in the NOD mouse. Three aspects of this treatment regimen appear to operate in parallel and in a synergistic manner: (a) Injection of CFA, and the consequent induction of TNF-, results in the elimination of TNF-–sensitive cells, which have been shown previously to transfer existing disease (28-31); (b) the introduction of functional MHC class I peptide complexes expressed on the surface of either normal islet cells or normal lymphocytes results in partial but stable reselection of the T cell population of the NOD host, leading to an increase in the abundance of long-term memory T cells (6, 12, 23); and (c) suppression of hyperglycemia, although not obligatory, promotes the functional restoration of endogenous ß cells or their precursors.

We propose that continuous or repeated exposure to parenchymal or lymphoid cells expressing MHC class I molecules and self peptides initiates the reeducation of host T cells, which was apparent in CFA-treated hosts from the loss of cells with an increased sensitivity to TNF-–induced apoptosis and from the restoration of a cell surface phenotype characteristic of long-term memory cells. This reeducation resulted in the establishment of long-term tolerance, as demonstrated by the elimination of both recurrent hyperglycemia and invasive insulitis. Treatment of NOD mice with severe hyperglycemia and islet destruction resulted in the reappearance of pancreatic insulin-secreting cells and normoglycemia. The rate of pancreatic ß cell proliferation is increased during the active phase of disease in NOD mice, and NOD islet stem cells proliferate in culture (41). The interruption of ß cell autoimmunity may promote both the rescue of surviving ß cells in islets as well as the production of new ß cells that are now able to survive in the altered immunological milieu. The expression of MHC class I molecules and self peptides by NOD pancreatic ß cells (21) may be responsible for maintenance of peripheral tolerance after termination of disease by transient therapy.

The application of this therapy to humans with type 1 diabetes may be feasible. As in NOD mice, lymphocytes from type 1 diabetic humans show an increased sensitivity to TNF-–induced apoptosis (10) as well as age-related defects in MHC class I presentation of self peptides for proper T cell selection (11, 13). Moreover, diabetic humans continue to produce auto-Ab’s to islet targets for several years after the onset of frank hyperglycemia, indicating the persistence of islet cell antigen expression. Thus, a proportion of individuals with type 1 diabetes may possess a ß cell mass or islet regenerative potential similar to that of hyperglycemic NOD mice. Even if the regenerative capacity of ß cells is exhausted, a similar immunomodulation approach may provide a less hostile milieu for islet replacement.

Acknowledgments

 
This work was supported by The Iacocca Foundation. We thank Biohybrid Technologies (J. Hayes, D. Wolf, and C. McGrath) for assistance with islet preparation and encapsulation; S. Thompson (Bayer Corp.) for providing surplus blood glucose monitoring strips; M. Contant for preparation of specimens for histological analysis; NICHD for funding to study autoimmune PDF patients; and J. Avruch and D. Nathan for critical review of the manuscript.

Footnotes

 
Shinichiro Ryu and Shohta Kodama contributed equally to this work.

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30. Qin, H.Y., Sadelain, M.W., Hitchon, C., Lauzon, J., and Singh, B. 1993. Complete Freund’s adjuvant-induced T cells prevent the development and adoptive transfer of diabetes in nonobese diabetic mice. J. Immunol. 150:2072-2080.[Abstract]

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33. Gazda, L.S., Charlton, B., and Lafferty, K.J. 1997. Diabetes results from a late change in the autoimmune response of NOD mice. J. Autoimmun. 10:261-270.[Medline]

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37. Lanza, R.P. et al.1992. Transplantation of encapsulated canine islets into spontaneously diabetic BB/Wor rats without immunosuppression. Endocrinology. 131:637-642.[Abstract]

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Copyright © 2001 by the American Society for Clinical Investigation.

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J Clin Invest, July 2001, Volume 108, Number 1, 63-72
Copyright ©2001 by the American Society for Clinical Investigation

Reversal of established autoimmune diabetes by restoration of endogenous ß cell function

Shinichiro Ryu¹, Shohta Kodama¹, Kazuko Ryu¹, David A. Schoenfeld² and Denise L. Faustman¹

¹ Immunobiology Laboratory, and
² Department of Biostatistics, Harvard Medical School and Massachusetts General Hospital, Charlestown, Massachusetts, USA

Address correspondence to: Denise L. Faustman, Immunobiology Laboratory, Massachusetts General Hospital–East, Harvard Medical School, Building 149, Room 3602, 13th Street, MailStop M1493601, Charlestown, Massachusetts 02129, USA. Phone: (617) 726-4084; Fax: (617) 726-4095; E-mail: denise.faustman@cbrc2.mgh.harvard.edu.

Received for publication January 24, 2001, and accepted in revised form May 14, 2001.

Abstract

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In NOD (nonobese diabetic) mice, a model of autoimmune diabetes, various immunomodulatory interventions prevent progression to diabetes. However, after hyperglycemia is established, such interventions rarely alter the course of disease or allow sustained engraftment of islet transplants. A proteasome defect in lymphoid cells of NOD mice impairs the presentation of self antigens and increases the susceptibility of these cells to TNF-–induced apoptosis. Here, we examine the hypothesis that induction of TNF- expression combined with reeducation of newly emerging T cells with self antigens can interrupt autoimmunity. Hyperglycemic NOD mice were treated with CFA to induce TNF- expression and were exposed to functional complexes of MHC class I molecules and antigenic peptides either by repeated injection of MHC class I matched splenocytes or by transplantation of islets from nonautoimmune donors. Hyperglycemia was controlled in animals injected with splenocytes by administration of insulin or, more effectively, by implantation of encapsulated islets. These interventions reversed the established ß cell–directed autoimmunity and restored endogenous pancreatic islet function to such an extent that normoglycemia was maintained in up to 75% of animals after discontinuation of treatment and removal of islet transplants. A therapy aimed at the selective elimination of autoreactive cells and the reeducation of T cells, when combined with control of glycemia, is thus able to effect an apparent cure of established type 1 diabetes in the NOD mouse.

Introduction

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Autoimmune destruction of pancreatic ß cells is more than 90% complete by the time hyperglycemia becomes clinically evident in individuals with type 1 (insulin-dependent) diabetes mellitus. Prevention of this disease would therefore optimally require arrest of autoimmunity in the prehyperglycemic phase. After hyperglycemia is established, therapies based on islet cell replacement are necessary to restore physiological control of blood glucose. Although islet transplantation has been successful in mice, rats, and nonhuman primates with chemically induced diabetes, sustained survival of allogeneic islet grafts is infrequently observed in spontaneous diabetic hosts such as the NOD (nonobese diabetic) mouse, BB (BioBreeding) rat, and diabetic humans (1-5). Thus, allogeneic or xenogeneic cellular grafts subjected to transient ablation of donor MHC class I antigen expression (6) — achieved either with the use of "masking" Ab’s to MHC class I molecules or by deletion of the ß₂-microglobulin (ß₂M) gene — are capable of permanent engraftment in nonautoimmune recipients, but are minimally protected from recurrent ß cell autoimmunity in NOD mice (7, 8). The immune mechanisms of islet graft rejection and recurrent autoimmunity appear distinct, and protective interventions targeted at these two pathways of islet destruction are nonoverlapping in effectiveness.

Subsets of antigen-presenting cells and T cells of NOD mice that progress to hyperglycemia exhibit a decrease in the production of LMP2, a catalytic subunit of the proteasome, after about 5–6 weeks of age (9, 10). This defect is accompanied by deficient generation of peptides from endogenous proteins for display on the cell surface by MHC class I molecules, a process that is important for T cell memory and tolerance to self antigens (11, 12) and is impaired in various human and murine autoimmune diseases (11, 13, 14). The proteasome also contributes to the processing and activation of NF-B (15-17), a transcription factor that regulates the expression of genes that contribute to cytokine generation, lymphocyte maturation, protection from TNF-–induced apoptosis, and promotion of the processing of antigens for presentation by MHC class I molecules (18-20). The proteasome defect in NOD mice affects lymphoid maturation as a result, at least in part, of impaired activation of NF-B.

In normal humans and other mammals, the continuous expression of MHC class I molecules by peripheral cells, including islet cells (21), maintains peripheral tolerance in the context of properly selected lymphocytes (12, 22). Interruption of exposure to complexes of self peptides and MHC class I molecules results in the aberrant selection of CD8⁺ T cells that exhibit an increased sensitivity to apoptosis (23). Diabetic humans and NOD mice that progress to diabetes thus manifest a paucity of memory cells and an increased susceptibility of T cells to apoptosis, traits that may be secondary, in part, to improper presentation of self antigens by MHC class I molecules and ineffective T cell selection (10, 11).

On the basis of the view that islet transplantation into hyperglycemic NOD mice will require both the prevention of transplant rejection and the elimination of autoimmune T cells, we sought to combine two strategies to achieve these goals. To bypass graft rejection, we used donor islets from C57BL/6 mice in which the ß₂M gene was deleted. MHC class I proteins are re-expressed on graft cells within 24 to 72 hours after transplantation as a result of reconstitution with host ß₂M present in plasma (6, 24, 25). The re-expression of donor MHC class I antigens is important because it is necessary for the development and maintenance of peripheral tolerance.

With regard to interruption of autoimmunity, we hypothesized that the lineage-specific defects both in peptide presentation by MHC class I molecules and in the processing and activation of NF-B might be important in the pathogenesis of diabetes in NOD mice. The NF-B defect in the affected lineages on NOD mice is accompanied by an increased sensitivity of these cells to TNF-–induced apoptosis in vitro (10). The increased susceptibility to apoptosis of misselected T cells that result from improper education by MHC class I peptide complexes suggested that the production of TNF- in vivo might promote the selective death of such poorly educated lineages (10, 26, 27). Furthermore, treatment of diabetic NOD mice or BB rats with CFA, an inducer of TNF- production, both impairs the transfer of disease by T cells from these animals to naive hosts (28-31) as well as prolongs the survival of syngeneic islet grafts in spontaneously diabetic hosts (2, 32). We therefore hypothesized that CFA treatment might eliminate, at least temporarily, the autoreactive lymphoid cells of NOD mice by promoting their apoptosis, in part through the induction of TNF-.

Thus, we both treated hyperglycemic NOD recipients with CFA, seeking to eliminate autoreactive lymphoid lineages, and transplanted islets from ß₂M-deficient donors under the kidney capsule of these animals, seeking to generate graft-specific tolerance. These interventions resulted in the marked reduction or apparent elimination of ongoing ß cell–directed autoimmunity. Unexpectedly, the long-term reappearance of endogenous ß cell function was also observed in the pancreatic islets of the previously hyperglycemic hosts.

Methods

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Animals. Female NOD mice from Taconic Farms (Germantown, New York, USA) and C57BL/6J (C57) mice from The Jackson Laboratory (Bar Harbor, Maine, USA) were maintained under pathogen-free conditions. NOD mice were screened for the onset of diabetes by monitoring body weight and blood glucose; they were diagnosed as diabetic when two consecutive blood glucose concentrations exceeded 400 mg/dl. Before experimental treatments, diabetic NOD mice were maintained for 7–20 days on daily injections of 1.0–1.5 U of NPH human insulin per 100 g of body weight, thereby preventing immediate death and maintaining blood sugar concentration between 200 and 700 mg/dl. The use of such severely diabetic mice, relatively late after disease onset, ensured that endogenous pancreatic islets were completely obliterated before the initiation of experiments. Splenocyte donors included normal C57 mice, C57 mice (C57 ß₂M^–/–) in which the ß₂M gene was disrupted, C57 mice (C57 ß₂M^–/–, TAP1^–/–) in which both the ß₂M and Tap1 genes were disrupted, and MHC class II^–/– mice (C57 class II^–/–) in which the I-A gene was disrupted and the E locus of MHC class II was not expressed because of an endogenous defect in the C57 strain (Taconic Farms). Splenocytes (9 x 10⁶) were injected into NOD recipients through the tail vein twice a week. CFA (Difco Laboratories, Detroit, Michigan, USA) was freshly mixed with an equal volume of physiological saline and injected (50 µl) into each hind-foot pad at the time of islet transplantation or after the first splenocyte injection.

Islet transplantation. Islets were isolated from donor C57 mice or 6- to 8-week-old prediabetic female NOD mice. Density gradient centrifugation followed by hand picking of islets ensured that both preparations were highly enriched in islets and had an accurate determination of islet number. For transplantation, 500–600 freshly isolated islets were grafted beneath the left renal capsule of each diabetic NOD recipient. For islet encapsulation, 900–1,100 islets were enclosed in alginate spheres, which were then surgically inserted into the peritoneal cavity of diabetic NOD mice. Transplantation was considered successful if the nonfasting blood concentration of glucose returned to normal (<200 mg/dl) within 24 hours after surgery. The glucose concentration of orbital blood was monitored three times a week after transplantation with a Glucometer Elite instrument (Bayer Corp, Pittsburgh, Pennsylvania, USA). Body weight was also monitored three times a week. Islet grafts were considered to have been rejected if the blood glucose concentration increased to more than 250 mg/dl on two occasions. Recipients that rejected islet grafts were killed for histological examination and flow-cytometric studies. To assess the contribution of endogenous pancreatic islets to the control of blood sugar concentration, we removed subrenal islet transplants by nephrectomy. Similarly, islets encapsulated in alginate spheres, which were approximately 0.2–0.5 cm in diameter, were removed from the peritoneal cavity by direct visualization under a dissecting microscope. Histological analysis of the pancreata and islet grafts was performed by staining with hematoxylin and eosin for evaluation of lymphocytic infiltrates and with aldehyde-fuchsin for islet insulin content. The entire pancreas from splenic to duodenal stomach attachments was removed, fixed, embedded longitudinally in a paraffin block, and subjected to serial sectioning (10 µm).

Flow cytometry. Spleens were removed and gently minced on a stainless steel sieve. Cell suspensions were rendered free of red blood cells by exposure to a solution containing 0.83% NH4Cl. The splenocytes were stained with mouse mAb’s (PharMingen, San Diego, California, USA) to CD8 (FITC-labeled), to CD62L (phycoerythrin-labeled [PE-labeled]), to CD95 (PE-labeled), or to CD45RB (PE-labeled), and were analyzed (>10,000 cells per sample) the same day with an Epics Elite flow cytometer. Spleen cells were incubated for 24 hours in the absence or presence of TNF- (20 ng/ml), after which apoptotic cells were detected by flow cytometry with FITC-conjugated annexin V. Apoptotic cells were defined as cells positive for both propidium iodide (PI) and annexin V staining; numbers within the upper quadrants represent the corresponding percentages of cells.

Adoptive transfer. Adoptive transfer was performed as described (29). Recipient male NOD mice, 4–8 weeks of age, were irradiated (790 rads) with a ¹³⁷Cs source and injected intravenously within 2 hours of irradiation with donor splenocytes (2 x 10⁷ viable cells) suspended in 0.25 ml of serum-free medium. Diabetic spleen cell donors were female NOD mice that typically had exhibited blood sugar concentrations of greater than 400 mg/dl for at least 3 weeks. Multiple diabetic donor spleens were pooled to produce sufficient cells for all of the hosts in a given experiment.

Statistics. Exact algorithm P values were calculated in some instances with multiple comparisons corrected by the number of tested variables. A P value less than 0.05 was considered statistically significant.

Results

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CFA treatment and islet transplantation in NOD mice. Hosts for the transplantation experiments were severely diabetic female NOD mice, usually more than 20 weeks of age, that had exhibited blood glucose concentrations of greater than 400 mg/dl for at least 7 days and had been treated by daily administration of insulin to prevent death. Islet transplants were placed unilaterally under the kidney capsule to facilitate nonlethal removal and histological examination. Islets from 6- to 8-week-old prediabetic NOD females (recipient group A) or from normal C57 mice (recipient group B) were rapidly rejected by diabetic NOD recipients (Table 1, Figure 1a). Although C57 donor islets with transient ablation of MHC class I expression survive indefinitely in nonautoimmune diabetic hosts (6), the survival time of islets from ß₂M^–/– C57 mice in diabetic NOD females (group C) was only about three times that of normal C57 islets. As expected from previous observations (2, 32), treatment with CFA prolonged the survival of syngeneic islet grafts in diabetic NOD hosts (group D) but had a minimal effect on the survival of C57 islets (group E), which were uniformly rejected by 11 days after transplantation. However, the combination of ß₂M^–/– C57 islet transplants with CFA treatment resulted in sustained (>129 days) normoglycemia in 5 of 14 diabetic NOD hosts (group F). Although the duration of hyperglycemia before initiation of therapy varied between 7 and 20 days, the length of this interval was not significantly related to the duration of sustained normoglycemia after treatment (data not shown). The animals that exhibited sustained normoglycemia also demonstrated progressive weight gain, similar to that apparent in NOD female cohorts who never became diabetic (data not shown). With normalization of blood sugar concentration as a measure of treatment success, the success of ß₂M^–/– C57 islet transplantation together with CFA administration was significantly different from that apparent with the other groups combined but did not differ from that of NOD islet transplantation together with CFA treatment.

After the recurrence of hyperglycemia in the NOD mice that had been treated with CFA and syngeneic (NOD) islet transplants, the kidney containing the islet graft was examined histologically. Marked lymphocytic infiltration was apparent under the kidney capsule at the site of transplantation, a characteristic of recurrent autoimmune disease (Figure 1b, group D); moreover, no intact islets were detected in the pancreas, although islet remnants, largely obscured by dense pockets of infiltrating lymphocytes, were evident. Similar histological characteristics of both the transplant site and pancreas were apparent in diabetic NOD mice that had received CFA and islet grafts from C57 donors (Figure 1b, group E). Unexpectedly, for all five NOD mice with long-term normoglycemia after receiving ß₂M^–/– C57 islets and CFA treatment, no surviving islet grafts were detected under the kidney capsule when the animals were killed at more than 129 days after transplantation (Figure 1b, group F). In contrast, the pancreas of each of these five recipients exhibited well-formed islets that appeared completely granulated when stained by aldehyde-fuchsin. The islets were free of lymphocytes or lymphocytes were present only circumferentially; this latter pattern of lymphocyte accumulation, with lymphocytes surrounding but not invading the islets, has been associated with nonprogressive or interrupted ß cell autoimmunity (33). The return to normoglycemia in the absence of detectable transplanted islet tissue, together with the presence of islets in a pancreas largely devoid of infiltrating lymphocytes, suggested not only that autoimmunity had been interrupted but that the function of endogenous ß cells had been restored.

Restoration of near-normal pancreatic islet histology was observed only in the diabetic NOD mice that received both ß₂M^–/– islet grafts and CFA treatment (Figure 2). Pancreatic islets were thus not detected in any diabetic NOD mice treated with CFA and syngeneic NOD islets; the persistence of normoglycemia in such recipients appeared solely due to the transplanted islets, which always exhibited invasive insulitis (Figure 1b, Figure 2). Thus, treatment with CFA together with syngeneic NOD islets may have slowed disease recurrence, but persistent autoimmunity remained.

The relative contributions of restored endogenous pancreatic islets and transplanted islets to the maintenance of normoglycemia in NOD mice treated with CFA and islet grafts from ß₂M^–/– C57 donors were assessed by removal of the kidney containing the islet transplant after 120 days of normoglycemia in a second group of five animals. All five mice remained normoglycemic after nephrectomy until they were killed 3–60 days later (Figure 3). Histological analysis of the kidneys that received the grafts revealed a complete loss of identifiable islet structures. In contrast, the pancreata of all five recipients contained well-formed islets either without lymphocytic infiltration or with circumferentially distributed lymphocytes only. Normoglycemia after nephrectomy was thus maintained solely by endogenous pancreatic islets. In contrast, nephrectomies performed during the posttransplantation period of normoglycemia (day 62, day 85) in two mice who had received CFA plus syngeneic NOD islets resulted in a rapid return to hyperglycemia (data not shown), demonstrating that the control of blood sugar in this treatment group was mediated solely by the transplanted islet tissue.

We also transplanted diabetic NOD females with islets from C57 mice in which the genes for both ß₂M and TAP1 had been deleted. Together with TAP2, TAP1 mediates the transport of endogenous peptides from the cytosol into the lumen of the endoplasmic reticulum for their assembly with MHC class I molecules (34). Islet cells from these mice are more permanently defective in presentation of self antigens by MHC class I than are those from ß₂M^–/– mice. Transplantation of ß₂M^–/–, TAP1^–/– C57 islets combined with injection of CFA resulted in the return of hyperglycemia within 14 days in five of six animals (group G); histological examination of the pancreata revealed a pattern typical of that for untreated diabetic NOD mice (Table 1, Figure 2). Thus, a transient interruption of peptide presentation by donor MHC class I molecules is essential for the abrogation of autoimmunity, whereas a sustained interruption of this process prevents the reestablishment of tolerance and the restoration of endogenous pancreatic islet integrity.

CFA treatment and splenocyte injection in NOD mice. Given that the restoration of normoglycemia in the diabetic NOD mice treated with CFA and ß₂M^–/– C57 islets did not depend on the continuing secretion of insulin by the islet grafts, we next investigated whether C57 donor cell types other than islets might serve a similar therapeutic role. Nine diabetic NOD mice were treated with a single bilateral injection of CFA followed by a 40-day regimen of biweekly intravenous injections of C57 splenocytes. These lymphoid cells express both MHC class I and class II proteins and survive only transiently in NOD hosts because of graft rejection (data not shown). Repeat injections of splenocytes ensure that the host is continuously exposed to intact antigen presentation complexes on the surface of these cells. The recipients were monitored for hyperglycemia every 3 or 4 days, and insulin was administered daily unless normoglycemia returned. A control group of four diabetic NOD mice received daily insulin injections only. All four control mice died on or before day 25 of the experimental period as a result of poor control of blood glucose and consequent ketosis and cachexia (Figure 4a). In contrast, seven of the nine mice injected with CFA and C57 splenocytes were alive after 40 days, and three of these animals had become normoglycemic and insulin independent (Figure 4b). The pancreata of control (insulin treatment only) mice exhibited pronounced lymphocytic infiltrates that obscured any remaining islet structures (Figure 4c). The pancreata of the four NOD mice treated with CFA and C57 splenocytes that remained alive but hyperglycemic and insulin dependent revealed a marked decrease (relative to control animals) in the number of lymphoid infiltrates, which were located either circumferentially or adjacent to the infrequent islet structures (Figure 4d). On killing of each of the three NOD mice treated with CFA and C57 splenocytes that maintained normoglycemia after discontinuation of insulin injections, the pancreata exhibited abundant islets that were free of invasive lymphocytes or were associated only with circumferential lymphocytes (Figure 4e). Thus, treatment with CFA combined with repeated exposure to C57 lymphocytes resulted in complete reversal of diabetes in approximately 30% of NOD recipients and partially restored ß cell function in an additional approximately 40% of recipients.

Influence of glycemic control on restoration of endogenous islet function. The reversal of diabetes in NOD mice by CFA and repeated exposure to C57 splenocytes indicated that restoration of endogenous islet function is achievable without islet transplantation and despite the poor glycemic control attained by insulin injections. The beneficial influence of glycemic control on the growth, survival, and function of cultured islets, as well as of transplanted islets in nonautoimmune settings, has been demonstrated (35, 36). To determine whether the restoration of endogenous ß cell function could be achieved more consistently with better control of blood glucose, we replaced insulin injections with the intraperitoneal implantation of alginate-encapsulated C57 mouse islets. Alginate encapsulation prevents direct contact between donor endocrine cells and host T cells, and such grafts have been shown to provide near-normal glycemic control for 40 to 50 days in approximately 70–80% of autoimmune NOD recipients (37).

Almost all diabetic NOD mice that received alginate-encapsulated C57 islets exhibited improved glucose regulation or normoglycemia. The alginate spheres were removed 40–50 days after implantation, and blood glucose concentration was monitored (Table 2). The seven mice treated only with alginate-encapsulated islets (group A), the six mice that received a single bilateral injection of CFA (group B), and the three mice treated with biweekly injections of C57 splenocytes (data not shown) all exhibited a rapid return to hyperglycemia and early death after removal of the implants (Table 2, Figure 5). The pancreata of NOD mice that received only alginate-encapsulated islets revealed no sign of intact islets or of lymphoid infiltrates (data not shown). The pancreata of NOD hosts treated with CFA and alginate-encapsulated islets exhibited marked invasive insulitis that obscured islet structures (Figure 6). In contrast, seven of the nine (78%) diabetic NOD mice that received CFA and C57 splenocytes (group C) remained normoglycemic for more than 40 days (until killing) after removal of the alginate-encapsulated islets (Figure 5, Table 2). The pancreata of these animals contained large islets with circumferentially distributed lymphocytes (Figure 6). The islet mass after at least 80 days of disease reversal was estimated at approximately 50% of the original value. The pancreata from control BALB/c mice contained approximately 25–35 islets; the pancreata from successfully treated NOD mice contained approximately 12–20 islets with serial histological sections. Thus, maintenance of normoglycemia during the treatment period increased the percentage of diabetic mice cured of hyperglycemia.

Role of TNF- in treatment outcome. We attempted to identify features of the successful treatment regimens that are critical to a positive outcome. We had used CFA to induce the endogenous production of TNF- (31). The importance of TNF- in treatment success was therefore investigated by the intravenous administration of a rat IgG1 mAb to this cytokine (clone MP6-X73; Accurate Chemical & Scientific Corp., Westbury, New York, USA) at a dose of 1.5 mg/day for the first 10 days in diabetic NOD hosts treated with C57 splenocytes, CFA, and alginate-encapsulated islets. All five NOD mice so treated exhibited a rapid return to hyperglycemia on removal of the alginate-encapsulated islets 50–70 days after transplantation (Table 2, group F), consistent with the notion that TNF- plays an obligatory role in the beneficial effect of CFA. This effect of the mAb to TNF- was specific, given that administration of a rat IgG1 mAb to the human T cell receptor V_ß1 chain (clone BL37.2; American Type Culture Collection, Rockville, Maryland, USA) at a dose of 1.5 mg/day for 10 days did not affect the success of treatment with C57 splenocytes and CFA (data not shown). Direct administration of TNF- to diabetic hosts was not feasible because of the prohibitive cost.

We next investigated whether the production of TNF- in diabetic NOD mice treated with CFA results in the selective elimination of autoreactive lymphoid cells first by examining the susceptibility of lymphocytes from successfully treated animals to TNF-–induced cell death in vitro. As shown previously (26, 27), incubation of normal C57 spleen cells with TNF- in vitro had no effect on cell viability; for the animal shown in Figure 7a, the proportion of apoptotic cells was 0.01% for splenocytes incubated in the absence or presence of TNF-. In contrast, exposure of splenocytes from an untreated 20-week-old NOD female to TNF- in vitro increased the proportion of apoptotic cells from 0.03 to 38.3%. Such an increased sensitivity to TNF-–induced apoptosis in vitro was no longer evident with spleen cells derived from NOD mice cured of diabetes; thus, splenocytes from a NOD female successfully treated with both CFA and C57 splenocytes (Table 2, group C) exhibited 23.2 and 23.9% apoptosis in the absence and presence of TNF-, respectively (Figure 7a). Successful therapy generated a subpopulation of nonpathologic but TNF-–resistant T cells that exhibited an increased tendency to undergo cell death in culture (Figure 7a). Disease reversal, even 210 days after cessation of treatment, was thus associated with the persistent elimination of TNF-–sensitive T cells, a population of cells with a demonstrated ability to play a role in disease (29, 30). The permanent elimination of these formerly abundant TNF-–sensitive lymphoid cells, presumably in response to TNF- (and, perhaps, to other CFA-induced cytokines), was observed uniformly in successfully treated diabetic NOD mice. Similar complete and stable elimination of TNF-–sensitive cells at various times after treatment has been observed in more than 20 NOD mice.

View larger version (43K): [in this window] [in a new window]   Figure 7. Roles of TNF- and MHC class I peptide complexes in reversal of diabetes in NOD mice. (a) Effect of TNF- on the survival of spleen cells derived from a control C57 mouse or from untreated or successfully treated NOD female mice. (b) Effect of TNF- treatment of splenocytes from diabetic NOD mice on the adoptive transfer of disease and the inability of splenocytes from successfully treated NOD mice to transfer disease. Young male NOD mice were irradiated and then injected with diabetic NOD female splenocytes either immediately after their isolation (left panel, dashed lines) or after incubation for 24 hours in the absence (left panel, solid lines) or presence (middle panel) of TNF- (20 ng/ml); alternatively, four irradiated hosts each received splenocytes from a different NOD donor with long-term normoglycemia restored by CFA and C57 spleen cell injections (right panel). (c) Flow cytometric analysis of the percentages of CD8+CD45RBhigh, CD8+CD62L+, and CD8+CD95+ cells among splenocytes of mice from various treatment groups. Diabetic NOD females were implanted intraperitoneally with alginate-encapsulated C57 islets. They then received no further treatment (group A), a single bilateral injection of CFA only (group B), or CFA treatment plus biweekly intravenous injections of splenocytes from normal C57 mice (group C), ß2M–/–, TAP1–/– C57 mice (group D), or MHC class II–/– C57 mice (group E). Shaded bars represent C57 control mice (group F) or NOD mice that exhibited normoglycemia and disease reversal after removal of alginate-encapsulated islets (groups C and E); open bars represent untreated NOD mice (group A) or NOD mice subjected to treatments that did not result in disease reversal (groups B and D).

We also examined the effect of TNF- on the pathogenesis of autoimmune diabetes in adoptive transfer experiments. Young recipient NOD males were subjected to irradiation followed by an intravenous injection of donor splenocytes either from newly diabetic NOD females or from NOD mice with long-term normoglycemia due to treatment with CFA and C57 splenocytes. The onset of diabetes was observed in all recipients by day 15 after the transfer of diabetic mouse cells that were injected either immediately after isolation or after control culture for 24 hours (Figure 7b, left panel). In contrast, four of the five recipients of diabetic mouse cells that had been cultured with TNF- for 24 hours before adoptive transfer remained normoglycemic for at least 40 days after cell injection (Figure 7b, middle panel). Furthermore, the four NOD hosts each injected with splenocytes from a different NOD female that had experienced reversal of autoimmunity for more than 120 days failed to develop disease during observation periods of more than 60 days (Figure 7b, right panel). TNF-–resistant NOD splenocytes, enriched either in vitro by direct exposure of cells to TNF- or in vivo by disease reversal, are thus incapable of transferring disease to naive hosts.

Role of MHC class I peptide complexes in T cell selection and treatment outcome. Disease reversal in diabetic NOD mice required treatment with both CFA and cells that express MHC class I peptide complexes. Only two of six (33%) diabetic NOD mice that received CFA and biweekly injections of splenocytes from ß₂M^–/–, TAP1^–/– C57 donors remained normoglycemic after removal of alginate-encapsulated islets (Table 2, group D). The pancreata of the four animals that became hyperglycemic after removal of the alginate spheres contained no granulated islets and only a few visible islet structures, which were invaded and obscured by lymphocytic infiltrates (Figure 6). In contrast, 8 of 11 (73%) diabetic NOD mice treated with CFA and splenocytes from C57 donors lacking MHC class II protein expression remained normoglycemic after removal of the alginate-encapsulated islets (Table 2, group E); the pancreata of these animals contained large islets that exhibited only moderate lymphocytic accumulation at the periphery (Figure 6).

Interruption of antigen presentation by MHC class I skews the T cell repertoire to an overabundance of naive cells, a consistent trait of diabetes-prone NOD mice and humans (38-40). Improper T cell selection secondary to interruption of antigen presentation by MHC class I results in overexpression of CD95 by CD8⁺ T cells as well as an increase in the abundance of cells with naive cell markers such as CD62L⁺ and CD45RB^high (12, 23). To investigate whether therapeutic reversal of NOD mouse diabetes was associated with a change in naive T cell selection, we subjected splenocytes to flow-cytometric analysis. Flow cytometry was performed 5 to 26 days after removal of the alginate-encapsulated islets and termination of therapy.

Untreated NOD mice exhibited the expected increases in the abundance of naive CD8⁺CD45RB^high, CD8⁺CD62L⁺, and CD8⁺CD95⁺ cells compared with C57 animals (Figure 7c). In contrast, in NOD female mice that were successfully treated with alginate-encapsulated islets, CFA, and administration of C57 splenocytes, the percentages of each of these cell populations were reduced to normal or near-normal values. The abnormally high numbers of CD8⁺CD45RB^high, CD8⁺CD62L⁺, and CD8⁺CD95⁺ cells remained increased in diabetic NOD females treated with alginate-encapsulated islets and CFA, either alone or together with administration of ß₂M^–/–, TAP1^–/– C57 splenocytes. Data are means plus or minus SEM of values from at least five mice per group. Exact algorithm P values were calculated for comparisons of each cell population between groups C, E, and F versus groups A, B, and D: P = 0.001 for CD8⁺CD45RB^high cells, P = 0.01 for CD8⁺CD62L⁺ cells, and P = 0.05 for CD8⁺CD95⁺ cells. Despite the fact that many comparisons were performed, the P value remained less than 0.05 even when multiplied by the three comparisons. These data showed that the T cell reselection apparent in successfully treated NOD mice was secondary to reexposure to complexes of MHC class I molecules and self peptides. The normalization of T cell phenotype did not require reexposure to MHC class II–peptide complexes, given that the administration together with CFA and alginate-encapsulated islets of MHC class II^–/– splenocytes was as effective as was that of normal C57 splenocytes.

Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have demonstrated the effectiveness of a novel therapy for the correction of established autoimmune diabetes in the NOD mouse. Three aspects of this treatment regimen appear to operate in parallel and in a synergistic manner: (a) Injection of CFA, and the consequent induction of TNF-, results in the elimination of TNF-–sensitive cells, which have been shown previously to transfer existing disease (28-31); (b) the introduction of functional MHC class I peptide complexes expressed on the surface of either normal islet cells or normal lymphocytes results in partial but stable reselection of the T cell population of the NOD host, leading to an increase in the abundance of long-term memory T cells (6, 12, 23); and (c) suppression of hyperglycemia, although not obligatory, promotes the functional restoration of endogenous ß cells or their precursors.

We propose that continuous or repeated exposure to parenchymal or lymphoid cells expressing MHC class I molecules and self peptides initiates the reeducation of host T cells, which was apparent in CFA-treated hosts from the loss of cells with an increased sensitivity to TNF-–induced apoptosis and from the restoration of a cell surface phenotype characteristic of long-term memory cells. This reeducation resulted in the establishment of long-term tolerance, as demonstrated by the elimination of both recurrent hyperglycemia and invasive insulitis. Treatment of NOD mice with severe hyperglycemia and islet destruction resulted in the reappearance of pancreatic insulin-secreting cells and normoglycemia. The rate of pancreatic ß cell proliferation is increased during the active phase of disease in NOD mice, and NOD islet stem cells proliferate in culture (41). The interruption of ß cell autoimmunity may promote both the rescue of surviving ß cells in islets as well as the production of new ß cells that are now able to survive in the altered immunological milieu. The expression of MHC class I molecules and self peptides by NOD pancreatic ß cells (21) may be responsible for maintenance of peripheral tolerance after termination of disease by transient therapy.

The application of this therapy to humans with type 1 diabetes may be feasible. As in NOD mice, lymphocytes from type 1 diabetic humans show an increased sensitivity to TNF-–induced apoptosis (10) as well as age-related defects in MHC class I presentation of self peptides for proper T cell selection (11, 13). Moreover, diabetic humans continue to produce auto-Ab’s to islet targets for several years after the onset of frank hyperglycemia, indicating the persistence of islet cell antigen expression. Thus, a proportion of individuals with type 1 diabetes may possess a ß cell mass or islet regenerative potential similar to that of hyperglycemic NOD mice. Even if the regenerative capacity of ß cells is exhausted, a similar immunomodulation approach may provide a less hostile milieu for islet replacement.

Acknowledgments

 
This work was supported by The Iacocca Foundation. We thank Biohybrid Technologies (J. Hayes, D. Wolf, and C. McGrath) for assistance with islet preparation and encapsulation; S. Thompson (Bayer Corp.) for providing surplus blood glucose monitoring strips; M. Contant for preparation of specimens for histological analysis; NICHD for funding to study autoimmune PDF patients; and J. Avruch and D. Nathan for critical review of the manuscript.

Footnotes

 
Shinichiro Ryu and Shohta Kodama contributed equally to this work.

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