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.