Recent studies have demonstrated that intrathymic

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1 Intrathymic Islet Cell Transplantation Reduces p-cell Autoimmunity and Prevents Diabetes in NOD/Lt Mice IVAN C. GERLING, DAVID V. SERREZE, SHERRI W. CHRISTIANSON, AND EDWARD H. LEITER Intrathymic transplantation of syngeneic islets into adolescent NOD/Lt mice was performed to establish whether the thymus would serve as an immunoprivileged site for p-cell engraftment, and whether this treatment would prevent the development of diabetes by eliciting tolerance to islet antigens. Intrathymic injection of cells from 200 NOD islets into 4-wk-old female NOD/Lt mice produced a significant reduction in the severity of insulitis at 24 wk of age. Furthermore, diabetes development was strongly suppressed (11% incidence) compared with controls (100% incidence). Both thymus histology and thymic insulin content revealed a rapid loss of the implanted p-cells with <1% remaining 1 wk posttransplantation. Despite the rapid loss of thymus-implanted islet cells, evidence for tolerance induction to islet cell antigens was obtained by adoptive transfer of splenic leukocytes from these mice into NOD-scidlscid recipients. After adoptive transfer of splenic leukocytes from 24-wk-old untreated prediabetic donors, 4 of 5 MOD-scidlscid recipients developed diabetes within 4 wk, and none of the recipients became diabetic after transfer of splenocytes from intrathymic islet-implanted donors. Intrathymic islet transplantation did not lead to reduction of sialitis in females with reduced severity of insulitis, indicating that the protective effect was tissue specific. This also was reflected in adoptive transfer experiments, because equal severity of sialitis was observed in NOD-sc/d/sc/d recipients of spleen cells from either islet transplanted or control NOD/Lt mice. In conclusion, the data suggest that intrathymic injection of islet cells prevents diabetes by stimulating immunological tolerance to p-cells. Diabetes 41: ,1992 From The Jackson Laboratory, Bar Harbor, Maine. Address correspondence and reprint requests to Edward H. Leiter, The Jackson Laboratory, Bar Harbor, ME Received for publication 1 September 1992 and accepted 17 September STZ, streptozocin; APC, antigen-presenting cell; PBS, phosphate-buffered saline; RIA, radioimmunoassay. Recent studies have demonstrated that intrathymic transplantation of allogeneic islets can reduce hyperglycemia in STZ-induced diabetic rats (1). Moreover, intrathymic engraftment of syngeneic islets into prediabetic, diabetes-prone BB rats protects against diabetes development (2,3). T-cells maturing within the thymus normally are rendered tolerant to self-antigens through interactions with both hematopoietically derived APCs and thymic epithelial cells (4). The generation of p-cell autoreactive effectors in BB rats results in part from defects expressed at the level of intrathymic APCs (5). Hence, intrathymic transplantation of prediabetic BB rats with syngeneic islets may override this defect by rendering T-cells that are maturing in the thymus tolerant to p-cell autoantigens, thus blocking the development of diabetes (3). Indeed, intrathymic transplantation of allogeneic islets allows overtly diabetic BB rats to retain donor-matched islets that are subsequently engrafted intrahepatically (1). However, these studies in BB rats did not conclusively demonstrate that the mechanism of protection was by specific induction of T-cell tolerance to pancreatic p-cells. Indeed, the finding that 8 of 23 diabetes-prone BB rats still became diabetic, despite the retention of syngeneic islets engrafted into the thymus (2), suggested the possibility that the thymus simply may be an immunoprivileged site in BB rats, from which normoglycemia is maintained by insulin secreted from the intrathymic islet grafts. Although the BB rat is severely T-lymphopenic, whereas the NOD mouse has an abundance of peripheral T-cells, APCs from NOD mice also fail to present p-cell autoantigens in a completely tolerogenic fashion (6,7). Hence, APC dysfunctions likely underlie the generation of p-cell autoreactive effectors in both models. Therefore, this study was conducted to evaluate if the thymus also is an immunoprivileged site for engraftment of syngeneic islets in NOD/Lt mice, and if intrathymic 1672 DIABETES, VOL. 41, DECEMBER-1992

2 I.C. GERLING AND ASSOCIATES implantation of NOD islets renders the developing T-cell population tolerant to p-cell autoantigens and, therefore, prevents development of insulitis and diabetes. D PBS recipients Intra-thymic islet recipients RESEARCH DESIGN AND METHODS Husbandry conditions for the NOD/Lt colony and the development of the NOD congenic stock homozygous for the severe combined immunodeficiency (scid) mutation, now designated NOD/LtSz-sc/tf, have been described previously (8). NOD-scid/scid mice do not develop functional T- or B-cells, and, therefore, are insulitis- and diabetes-free. NOD-scid/scid females at the 10th backcross generation (following outcross from the C.B-17- scid/scid strain) were used as splenocyte recipients in the adoptive transfer studies described below. Preparation and transplantation of islet cells. Islets were isolated from 6-7-wk-old NOD/Lt females as described previously (9) and treated for 5 min with 0.05% trypsin and 0.5mM EDTA in Hanks' balanced salt solution to yield islet cell suspensions. Trypan blue dye exclusion indicated a viability of -90% in the cell suspensions. The cells were washed and resuspended in 0.1 ml PBS/1000 islets, and 0.01 ml of this suspension was injected into each thymic lobe (total of 200 islets/thymus) of day-old female NOD/Lt mice that were anesthesized with tribromoethanol (250 mg/kg, i.p.). Controls were injected with 0.01 ml PBS in each thymic lobe. Analysis of diabetes development. Female NOD/Lt mice were tested weekly for glycosuria by using Tes- Tape (Lilly, Indianapolis, IN). Mice were classified as diabetic when glycosuria of 3 + was detected. Aglycosuric mice were considered prediabetic if, after an overnight fast, their plasma glucose concentration was >27.8 mm, 60 min after injection of 3 g glucose/kg body weight. Plasma glucose levels were determined with a Glucose Analyzer II (Beckman, Palo Alto, CA) using blood from the retro-orbital sinus. At the indicated times, mice were killed; 50% of each pancreas and each thymic lobe were fixed in Bouin's solution, and granulated p-cells were stained with aldehyde fuchsin. All islets sampled from three nonoverlapping pancreatic levels were assigned an insulitis score as follows: 4, end stage; 3, >25% of the islet infiltrated; 2, <25% of the islet infiltrated; 1, peri-insulitis with no islet penetration; 0, no visible lesions. An insulitis index between 0.00 and 1.00 was calculated for each animal by dividing the mean insulitis score at all levels by 4. Half of the serial sections cut from the thymuses were stained for non-p-islet endocrine cells with a mix of antibodies to glucagon, somatostatin, and pancreatic polypeptide, as described previously (9). Submandibular salivary glands were fixed in Tellesniczkys Fekete fixative and stained with hematoxylin/eosin. Insulin was extracted from 50% of each pancreas and thymic lobe, as described previously (10). Thymic insulin extracts were neutralized with ammonium hydroxide (Sigma, St.Louis, MO), vacuum dried, and dissolved in 0.35 ml assay buffer for RIA. Insulin was measured by a rodent insulin RIA kit (Linco Research, St.Louis, MO), which uses rat insulin as standard Weeks of Age FIG. 1. Intrathymic Islet cell transplantation at 4 wk of age reduced diabetes Incidence In NOD/Lt mice. Results for 5 PBS recipients and 9 Intrathymic Islet recipients. Splenocyte preparation and adoptive transfer. Splenic leukocytes were isolated and pooled from 24-wk-old control NOD females or females that had received intrathymic islet transplants at 4 wk of age. Six-week-old NOD-scid/scid female recipients recieved i.v. injections of 2 x 10 7 splenocytes, then were monitored for development of diabetes within 5 wk posttransfer. Statistics. The effect of intrathymic islet cell injection on diabetes incidence was analyzed by x 2 contingency table analysis. Other data were analyzed by Student's t test with significance level given. RESULTS Intrathymic islet implantation prevents diabetes. As shown in Fig. 1, the incidence of diabetes in recipients of intrathymic islet cell transplants was markedly lower (x 2 = 22.9, P = ) than in the vehicle-injected control group. The vehicle-injected control group of 5 females, although small, showed a 100% diabetes incidence by 24 wk, whereas only 1 of 9 (11%) of the intrathymic islet recipients developed diabetes by 24 wk. However, as shown in Table 1, a wide variation in severity of insulitis and pancreatic insulin content characterized intrathymically transplanted mice. Insulitis indexes of females in this group ranged from a minimum of 0.10 to a maximum of 0.90 (mean, 0.44 ±0.13), with pancreatic insulin contents ranging from 548 to 7140 pm/(i,g protein (mean, 2303 ± 713). Interestingly, glucose tolerance testing of the 2 nondiabetic mice in the transplantrecipient group (mice 5 and 6, Table 1) with severe insulitis did not show impaired glucose clearance, indi- 30 DIABETES, VOL 41, DECEMBER

3 INTRATHYMIC ISLET CELL TRANSPLANTATION TABLE 1 Antidiabetogenic effect of intrathymic islet transplantation in 24-wk-old female NOD/Lt mice ne Pancreatic insulin Treatment Mouse Diabetic Insulitis index (pm/jig protein) Intrathymic islets 0.89 ± ± ± ± 0.04f 0.12 ± Ht dt dt dt dt dfc dt dto.oo 0.44 dfco.13tt ±66t ± 713t* Values are means ± SE. *t determined (insulitis index of chronically diabetic females usually is 1.0). tmean of the means for individual sample determinations. jlntrathymic islet-treatment group means are significantly different from nontreated control group at P < 0.05 by Student's t test. eating the presence of significant residual p-cell mass. Because all vehicle-injected controls depicted in Fig. 1 had developed diabetes and were, therefore, necropsied before 24 wk, aglycosuric age-matched NOD/Lt females with impaired glucose tolerance by the criteria described above were identified in our colony and used as controls. The mean insulitis index for this group of age-matched unmanipulated prediabetic NOD Lt females (mean, 0.94 ± 0.04) was significantly higher than in intrathymically transplanted mice, correlating with significantly lower pancreatic insulin contents (Table 1). Intrathymic injection of trypsin/edta dispersed syngeneic submandibular gland cells into 5 28-day-old NOD/Lt females, unlike dispersed islet cells, failed to ==* 800-, 700- H z z ^ 300- co? 200- O % o" 770± T 6.62± ± DAYS POST ISLET TRANSPLANTATION FIG. 2. Insulin content In thymlc extracts from NOD/Lt recipients of Intrathymic Islet cell transplants. The thymus was removed within 5-10 mln (day 0), or at 2, 7, and 14 days postinjection of islet cells. Means ± SE (for 2-4 mice) are Indicated. prevent diabetes, with 3 of 5 recipients (60%) diabetic by 18 wk. Survival of p-cells in the thymus. Marked declines in thymic insulin content within 48 h postimplantation indicated a rapid loss of the injected islet cells from the thymus (Fig. 2). Within 1 wk posttransplantation, the insulin content of the thymus was <1% of the initial (day 0) content. Histological examination confirmed the rapid elimination of the islet syngrafts. At day 0, most islet cells were found to be dispersed within the thymic subcapsule (Fig. 3/4). By 48 h, the numbers of granulated p-cells remaining in this location were markedly reduced (Fig. 38), such that by day 7, only an occasional granulated p-cell was detected (not shown). Elimination was not limited to p-cells, because the non-p-islet endocrine cells, well represented at day 0, also were lost concomitant with the p-cell population (not shown). At 24 wk (20 wk postimplantation), no surviving islet cells were detectable in thymuses of mice described in Fig. 1 and Table 1. The insulin content in the thymus extracts of most of the islet cell transplant recipients was below the detection limit of nmol/ml. Only 1 mouse had a thymus insulin content >0.030 nmol/ml at 24 wk of age. Adoptive transfer of spleen cells to NOD-scidlscid mice. Four of five NOD-scid/scid recipients of pooled splenocytes from 3 prediabetic age-matched controls (control mice 1-3 from Table 1) developed diabetes within 4 wk posttransfer. The one nondiabetic NODscid/scid recipient of control splenocytes exhibited 1 + glucosuria by Tes Tape at wk 5 and an insulitis index of 0.965, indicating severe islet cell damage and incipient diabetes. In contrast, adoptive transfer of pooled splenocytes from 3 of the islet cell-transplant recipients (islet recipient mice 1-3 in Table 1) failed to transfer diabetes 1674 DIABETES, VOL. 41, DECEMBER 1992

4 I.C. GERLING AND ASSOCIATES sialitis was detected between recipients of spleen cells from islet-transplanted mice versus recipients of spleen cells from non-islet-implanted mice (data not shown). FIG. 3. Insulin staining (dark) cells In thymus of Intrathymic Islet cell transplant recipients. Thymus was removed either (A) 5-10 min after Islet cell Injection (x475) or (0) 48 h after Islet cell Injection (x950). to any of 5 NOD-sc/d/sc/d recipients by 5 wk posttransfer. Reduced numbers of p-cell autoreactive T-cells in the spleens of the islet-transplanted group was confirmed by the reduced insulitis index (0.307 ± 0.017) in NOD-sc/d/sc/d recipients of splenocytes from these mice compared with the group receiving splenocytes from nonislet-implanted mice (insulitis index, ± 0.005), P< Inflammation in submandibular glands. Histological examination of the submandibular salivary glands of NOD/Lt females aged to 24 wk revealed widespread, chronic sialitis in all mice without any noticeable difference between recipients of islet transplant and controls (data not shown). Unmanipulated NOD-sc/d/sc/d mice are free of sialitis. All NOD-sc/d/sc/d recipients of splenocytes also developed focal sialitis within the 5-wk postadoptive transfer. difference in the severity of DISCUSSION As in BB rats (2,3), intrathymic transplantation of syngeneic islet cells protects NOD mice from development of diabetes. However, in contrast to BB rats, we found that syngeneic islet cells were rapidly cleared from the thymus of NOD/Lt mice. This finding clearly eliminates ectopic secretion of insulin as the mechanism behind the prevention of diabetes in NOD/Lt recipients of intrathymic islet cells. Although p-cell rest by prolonged insulin therapy started at an early age can prevent diabetes in NOD mice (11), the rapid clearance of islet cells from the thymus in our experiments precludes this mechanism. The difference in p-cell survival in BB rats and NOD/Lt mice could be attributable to our use of dispersed islet cells instead of whole islets, or it could reflect differences in the intrathymic environment between NOD mice and BB rats. The rapid loss of dissociated islet cells from the thymus of autoimmune NOD mice is not unique to this strain; syngeneic islet cells are eliminated from the thymus of nonautoimmune CBA/J mice, in a time course similar to that for NOD/Lt (data not shown). Although the incidence of diabetes was markedly reduced in NOD/Lt recipients of intrathymic islet cells compared with vehicle-injected controls at 24 wk of age, insulitis was not completely circumvented. The variable levels of both insulitis and pancreatic insulin content in the islet celltransplant recipients indicate that this protocol inhibits, but does not completely block, the development of p-cell-autoreactive T-cells within the thymus. This conclusion is supported further by the finding that although they retained granulated p-cells and remained diabetesfree, NOD-sc/d/sc/d recipients injected with spleen cells from NOD mice that had received intrathymic islet cell transplants were not totally free of insulitis. NOD-sc/d/ scid mice do not develop diabetes or insulitis spontaneously, but diabetes can be transferred adoptively by i.v. injections of splenic T-cells from NOD/Lt mice (12). The severity of sialitis was not affected in recipients of intrathymic islet cell transplantation, indicating that the effect of the intrathymic islet cell transplants was p-cell specific. Similarly, the severity of sialitis was equivalent in NOD-sc/d/sc/d females injected with splenocytes from both islet cell-transplant recipients and NOD/Lt female controls. Because APCs from NOD mice appear to be defective in tolerogenic presentation of p-cell autoantigens to T-cells (6,7), the processing of islet cell antigens directly in the thymus may compensate for the APC functional defects. However, the results of our adoptive transfer experiments indicate that the single implantation of 200 islets into the thymus of prediabetic NOD mice was insufficient to completely eliminate the development of p-cell autoreactive T-cells. Further studies are needed to identify the optimal conditions required to induce and maintain immunological tolerance to p-cells by intrathymic islet transplantation and to identify the autoantigens involved. DIABETES, VOL. 41, DECEMBER

5 INTRATHYMIC ISLET CELL TRANSPLANTATION ACKNOWLEDGMENTS This study was supported by National Institutes of Health Grants DK and DK The authors are grateful for the excellent technical assistance of Anne Higgins, Bruce Regimbal, and Daniel Krull. We are indebted to Dr. Leonard D Schultz for providing the NOD-scid/scid mice. REFERENCES 1. Posselt AM, Naji A, Roark JH, Markman JF, Barker CF: Intrathymic transplantation in the spontaneously diabetic BB rat. Ann Surg 214:365-71, Koevary SB, Blomberg M: Prevention of diabetes in BB/Wor rats by intrathymic islet injection. J Clin Invest 89:512-16, Posselt AM, Barker CF, Friedman AL, Naji A: Prevention of autoimmune diabetes in the BB rat by intrathymic islet transplantation at birth. Science 256: , Sprent J, Webb SR: Function and specificity of T-cell subsets in the mouse. Adv Immunol 41:39-133, Georgiou HM, Bellgrau D: Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat. J Immunol 142: , Serreze DV, Leiter EH: Development of diabetogenic T-cells from NOD/Lt marrow is blocked when an allo-h-2 haplotype is expressed on cells of hematopoietic origin, but not on thymic epithelium. J Immunol 147: , Leiter EH, Serreze DV: Antigen presenting cells and the immunogenetics of autoimmune diabetes in NOD mice. Regional Immunol. In press 8. Prochazka M, Gaskins HR, Shultz LD, Leiter EH: The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency. Proc NatlAcad Sci USA 89: , Serreze DV, Leiter EH, Worthen SM, Shultz LD: NOD marrow stem cells adoptively transfer diabetes to resistant (NOD x NON) F1 mice. Diabetes 37:252-55, Schwizer RW, Leiter EH, Evans R: Macrophage-mediated cytotoxicity against cultured pancreatic islet cells. Transplantation 37:539-44, Atkinson MA, Maclaren NK, Luchetta R: Insulitis and diabetes in NOD mice reduced by prohylactic insulin therapy. Diabetes 39: , Christianson SW, Shultz LD, Leiter EH: Adoptive transfer of diabetes into immunodeficient NOD scid/scid mice: relative contributions of CD4 + and CD8 + T lymphocytes from diabetic versus prediabetic NOD. NON Thy1 a donors. Diabetes. In press DIABETES, VOL. 41, DECEMBER 1992