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*Gene*GEO Profiles
*HomoloGene*UniGene
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Medline Plus Health Information
*Diabetes Type 1
*Islet Cell Transplantation
The Journal of Immunology, 2003, 171: 3878-3885.
Copyright © 2003 by The American Association of Immunologists

Islet Allograft Rejection in Nonobese Diabetic Mice Involves the Common {gamma}-Chain and CD28/CD154-Dependent and -Independent Mechanisms1

Gulcin Demirci, Terry B. Strom and Xian Chang Li2

Department of Medicine, Harvard Medical School, Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Once nonobese diabetic (NOD) mice become diabetic, they are highly resistant to islet transplantation. The precise mechanism of such resistance remains largely unknown. In the present study we tested the hypothesis that islet allograft survival in the diabetic NOD mouse is determined by the interplay of diverse islet-specific T cell subsets whose activation is regulated by CD28/CD154 costimulatory signals and the common {gamma}-chain ({gamma}c; a shared signaling element by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). We found that common {gamma}c blockade is remarkably effective in blocking the onset and the ongoing autoimmune diabetes, whereas CD28/CD154 blockade has no effect in suppressing the ongoing diabetes. However, CD28/CD154 blockade completely blocks the alloimmune-mediated islet rejection. Also, a subset of memory-like T cells in the NOD mice is resistant to CD28/CD154 blockade, but is sensitive to the common {gamma}c blockade. Nonetheless, neither common {gamma}c blockade nor CD28/CD154 blockade can prevent islet allograft rejection in diabetic NOD mice. Treatment of diabetic NOD recipients with CD28/CD154 blockade plus {gamma}c blockade markedly prolongs islet allograft survival compared with the controls. However, allograft tolerance is not achieved, and all CTLA-4Ig-, anti-CD154-, and anti-{gamma}c-treated diabetic NOD mice eventually rejected the islet allografts. We concluded that the effector mechanisms in diabetic NOD hosts are inherently complex, and rejection in this model involves CD28/CD154/{gamma}c-dependent and -independent mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The nonobese diabetic (NOD)3 mouse spontaneously develops diabetes via a disease process similar to human type I diabetes (1). One of the key features in these mice is the progressive and selective destruction of insulin-producing pancreatic {beta} cells, and by the time hyperglycemia becomes evident, destruction of the {beta} cells is often >90% complete (2). Thus, it has long been envisioned that transplantation of islet allografts into diabetic NOD mice would be a preferred choice of treatment.

However, diabetic NOD mice have proven notoriously resistant to islet transplantation. The islet allografts are often rapidly destroyed by the diabetic hosts, in some cases even before the islet allografts have a chance to resume primary function (3). Moreover, certain tolerizing protocols that consistently induce transplant tolerance in conventional mice often completely fail to prevent islet allograft rejection in autoimmune diabetic NOD hosts (3). The precise nature of such hyper-resistance to islet engraftment in diabetic NOD mice remains largely unknown. It has been proposed that activation of islet-specific autoreactive T cells and the alloantigen-specific alloreactive T cells coexist in diabetic NOD hosts, and the dual action of autoreactive T cells and alloreactive T cells toward islet transplants may form a formidable barrier to islet engraftment. It is also believed that diabetic NOD mice may have developed an unusually large pool of memory effector T cells that can readily attack the islet transplants even before the activation of autoreactive and alloreactive T cells (3, 4). Furthermore, certain evidence suggests that the NOD mice may have an inherent defect in the acquisition of peripheral tolerance (5, 6, 7). Clearly, such complexity is not equally represented in nonautoimmune mice, all of which may potentially contribute to the difficulty of islet transplantation in diabetic NOD hosts.

It is likely that activation of different T cell subsets (e.g., alloreactive T cells, autoreactive T cells, memory effector T cells, etc.) and the complex interplay among such T cell subsets may be critically important in determining islet allograft survival in autoimmune diabetic NOD hosts. It is believed that signals delivered by certain costimulatory molecules are required for the activation of naive T cells (8, 9). The fate of activated T cells to become armed effector T cells, to undergo apoptosis, or to evolve as long-lived memory cells, however, is primarily regulated by T cell growth factors (10), a family of cytokines whose receptors use the same IL-2R common {gamma}-chain ({gamma}c) as a signaling element (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) (11, 12). Nonetheless, the question of how CD28 and CD154, two conventional costimulatory molecules (9, 13), and {gamma}c-dependent cytokines regulate the activation of such diverse cytopathic T cell subsets during islet allograft rejection has not been examined in diabetic NOD mice. In the present study we critically examined the individual and collective role of autoreactive T cells, alloreactive T cells, as well as memory effector T cells in mediating islet allograft destruction and the impact of CD28/CD154 costimulatory signals and common {gamma}c signals on the activation of such diverse T cell subsets. We found that CD28/CD154 signals and common {gamma}c signals have overlapping, but also distinct, roles in affecting the alloreactive T cells and autoreactive T cells involved in islet rejection. Furthermore, islet allograft rejection in diabetic NOD mice involves both CD28/CD154/{gamma}c-dependent and -independent mechanisms.


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

Female NOD mice (H-2g7) and NOD/SCID mice (H-2g7) at 4 wk of age were obtained from The Jackson Laboratory (Bar Harbor, ME). DBA/2 (H-2d) and C57BL/6 mice (H-2b), 8–10 wk old, were obtained from Taconic Farms (Germantown, NY). Animal care and use conformed to the guidelines established by the animal care committee at our institution.

Reagents

The following mAbs used for cell surface staining were purchased from BD PharMingen (San Diego, CA). CyChrome anti-mouse CD4 (GK1.5), CyChrome anti-mouse CD8{alpha} (clone 53-6.7), FITC-anti-mouse CD44 (clone IM7), PE-anti-mouse CD62L (L-selectin, clone MEL-14), PE-anti-mouse CCR5 (clone C34-3448, rat IgG2c), PE-streptavidin, and PE-isotype control Abs. A biotinylated mutant IL-15/Fc (14) was provided by Cardion Pharmaceutical (Boston, MA).

Murine CTLA-4Ig was constructed, expressed, and tested as previously described (15). A hybridoma cell line secreting hamster anti-mouse CD40 ligand mAb (MR1, hamster IgG) was obtained from American Type Culture Collection (Manassas, VA). The hybridoma cells were grown in serum-free UltraCulture medium (BioWhittaker, Walkersville, MD), and the mAb was purified from the culture supernatant using protein G columns. Rat anti-mouse {gamma}c mAbs (4G3/3E12) were produced and used as previously reported (16, 17). Control rat IgG and hamster IgG were purchased from Sigma-Aldrich (St. Louis, MO).

The NOD model

Blood glucose levels in female NOD mice were monitored weekly using the Accu-Chek III glucometer (Roche, Indianapolis, IN) starting at 10 wk of age. Diabetes was defined as a rise of blood sugar levels >300 mg/dl. The cumulative incidence of diabetes was calculated as the percentage of mice with clinical diabetes among the total number of mice included in the study and was plotted against age.

Treatment of NOD mice with anti-{gamma}c mAbs consisted of 0.4 mg i.p. on days 0, 1, 3, 5, 7, and 10 with the first injection on day 0 regardless of age. For disease prevention, anti-{gamma}c mAbs were given to NOD mice at 4 wk of age. Otherwise, anti-{gamma}c treatment was delayed until NOD mice were ~8 wk of age, when insulitis was already developed (1). In some experiments NOD mice were treated with CTLA-4Ig and anti-CD154 starting at ~8 wk of age. The CTLA-4Ig and anti-CD154 protocol consisted of 0.25 mg of each, i.p., on days 0, 1, 3, and 5, with the first injection on day 0.

The NOD/SCID model

NOD/SCID mice do not usually develop diabetes because of the lack of lymphocytes, but they remain susceptible to the induction of diabetes. To study the induction of diabetes in NOD/SCID mice by autoreactive T cells, splenic lymphocytes were prepared from diabetic NOD mice (blood glucose, >300 mg/dl) as previously described (18), and 20 x 106 cells were adoptively transferred into 8-wk-old NOD/SCID mice via the tail vein. Blood glucose levels were then determined for evidence of diabetes. In some experiments the same anti-{gamma}c protocol (0.4 mg i.p. on days 0, 1, 3, 5, 7, and 10) or CTLA-4Ig and anti-CD154 protocol (0.25 mg of each reagent i.p. on days 0, 1, 3, and 5) was given to the NOD/SCID hosts starting at the time of adoptive cell transfer.

Flow cytometry and cell sorting

Three-color cell staining was performed to phenotype peripheral T cells in NOD mice. Briefly, splenic leukocytes were prepared from diabetic NOD mice as previously described (18). Cells were stained with CyChrome anti-mouse CD4 and CyChrome anti-mouse CD8, followed by staining with FITC-anti-mouse CD44 and PE-anti-mouse CD62L, PE-anti-mouse CCR5. For IL-15R{alpha} staining, a biotinylated recombinant fusion protein consisting of a mutant form of IL-15 and mouse IgG (14) was used as a primary staining reagent, followed by staining with PE-streptavidin. After staining, cells were washed twice in PBS/0.5% BSA washing buffer and analyzed by flow cytometry (BD Biosciences, Mountain View, CA). Cells stained with isotype control mAbs were included as a control.

For cell sorting, splenic leukocytes were labeled with CyChrome anti-CD4 and CyChrome anti-CD8. The CD4 and CD8 T cell subsets were then plotted against the forward scatter (FSC). A subset of CD4+ and CD8+ T cells that displayed a higher FSC was sorted out using high speed cell sorter (FACSVantage; BD Biosciences) and was used for adoptive cell transfer.

Islet isolation

Crude islets from donor DBA/2 mice (H-2d) were isolated as previously described (19). Briefly, donor pancreata were perfused in situ with collagenase (2 mg/ml; Worthington Biochemical, Freehold, NJ), and the pancreatic tissue was harvested after the perfusion and further incubated at 37°C for 40 min. Islets were released from the pancreata by gentle vortex and were further purified using discontinuous Percoll gradients. The isolated islets were washed in Hanks’ solution and counted before transplantation.

Islet transplantation

For transplantation into nonautoimmune C57BL/6 recipients (H-2b), the recipient mice were rendered diabetic by a single i.p. injection of streptozotocin (225 mg/kg; Sigma-Aldrich) 5 days before transplantation. The crude islets (500~600 islets) were then transplanted under the renal capsule into each diabetic C57BL/6 mouse. In some experiments diabetic NOD mice (H-2g7) were used as transplant recipients. Islet transplantation was performed on diabetic NOD mice within 1 wk of being diagnosed as diabetic (blood glucose, >300 mg/dl). Graft function was monitored by serial blood glucose measurements. In all cases primary graft function was defined as a blood glucose level <200 mg/dl on day 3 post-transplantation, and failure to achieve this was defined as no primary function. Rejection was defined as a rise in blood glucose >300 mg/dl following a period of primary graft function.

Treatment of transplant recipients

Treatment of transplant recipients with anti-{gamma}c mAbs consisted of 0.4 mg i.p. on days 0, 1, 3, 5, 7, and 10 starting at islet grafting. CTLA-4Ig and anti-CD154 were given (0.25 mg each, i.p.) on days 0, 1, 3, and 5 after transplantation.

Histopathology

Pancreatic tissue in NOD mice or the left kidney bearing the islet allografts in transplant recipients was removed at specified time points, fixed in 10% formalin, and embedded in paraffin. Serial tissue sections (5 µm) were cut and mounted on SuperFrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), fixed in methanol, and stained in H&E for identification of cellular infiltrates.

Statistics

Islet allograft survival and the incidence of diabetes in NOD mice following various treatments were compared using log-rank test, and p < 0.05 was defined as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-{gamma}c blocks ongoing islet-specific autoimmunity in NOD mice

The {gamma}c is a shared signaling component by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (11, 12), a family of cytokines with considerable importance in regulating the fate of activated T cells (10). To study the role of {gamma}c signals in the pathogenesis of autoimmune diabetes, female NOD mice were treated with anti-{gamma}c mAbs (0.4 mg i.p. on days 0, 1, 3, 5, 7, and 10) starting at 4 wk of age (i.e., before the onset of insulitis) to block {gamma}c signals, and the incidence of diabetes was monitored. As shown in Fig. 1A, control Ab-treated mice developed clinical diabetes at ~14 wk of age, and by the time they were 26 wk old, >70% of them were diabetic, with blood glucose levels exceeding 300 mg/dl (n = 14). Treatment with anti-{gamma}c mAbs markedly inhibited the spontaneous onset of diabetes, only one of 10 anti-{gamma}c-treated NOD mice was diabetic at 26 wk of age (p < 0.05).



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  		FIGURE 1. A, Effect of anti-{gamma}c mAbs on spontaneous onset of diabetes in NOD mice. Female NOD mice were given anti-{gamma}c mAbs or control IgG starting at 4 wk of age. Blood glucose levels were followed, and the cumulative incidence of diabetes (percentage) was determined. B, Effect of anti-{gamma}c mAbs on ongoing diabetes in NOD mice. The anti-{gamma}c mAbs were given to a cohort of NOD mice starting at 8 wk of age. The cumulative incidence of diabetes (percentage) was determined and compared with that in control Ab-treated mice. C, Effect of anti-{gamma}c mAbs on adoptively transferred diabetes in NOD/SCID mice. Splenic leukocytes were prepared from diabetic NOD mice. Cells (20 x 106) were injected into NOD/SCID mice and treated with anti-{gamma}c mAbs at the time of cell transfer. The incidence of diabetes (percentage) in the host mice after adoptive cell transfer is shown.

 
To further test the efficacy of anti-{gamma}c in blocking the ongoing disease, anti-{gamma}c treatment was delayed until NOD mice were ~8 wk age, a time point when peri-insulitis is often present (1). The same treatment protocol was given to a cohort of NOD mice. Remarkably, the development of diabetes was completely inhibited by the anti-{gamma}c mAbs, and none of the anti-{gamma}c-treated NOD mice in this group developed diabetes during the 26-wk follow-up period (n = 10), whereas >70% of the control mice were clinically diabetic (Fig. 1B; p < 0.05). Histological analysis revealed that treatment with anti-{gamma}c mAbs markedly reduced, albeit not completely prevented, cellular infiltration in the pancreatic islets. Mononuclear cells infiltrating pancreatic islets in anti-{gamma}c-treated mice were primarily confined to the periphery of the islets, with no obvious invasion and destruction of the islets (data not shown).

The remarkable effect of anti-{gamma}c in blocking ongoing diabetes suggests that {gamma}c blockade may have a profound effect on committed effector T cells. To examine this possibility, splenic leukocytes from clinically diabetic NOD mice were adoptively transferred into immunodeficient NOD/SCID hosts. The effect of blocking {gamma}c on the development of diabetes in NOD/SCID mice was examined. As shown in Fig. 1C, adoptive transfer of splenic leukocytes uniformly induced diabetes in NOD/SCID hosts 5–7 wk after cell transfer (n = 8). Treatment of the host mice with anti-{gamma}c mAbs at the time of cell transfer markedly delayed the onset of diabetes and reduced the incidence of diabetes by ~50% compared with the control Ab-treated mice (p < 0.05). Thus, the {gamma}c-dependent growth factor signals play an important role in activation of cytopathic autoreactive T cells.

Islet specific memory-like T cells are also sensitive to {gamma}c blockade

It has been suggested that diabetic NOD mice may have developed an unusually large pool of memory T cells that can readily attack the islet transplants (3). In an effort to identify such cells, splenic leukocytes from NOD mice (~30 wk old, regardless of clinical diabetes) were extensively phenotyped using a combination of cell surface markers. As shown in Fig. 2A (R1), when CD4+ T cells, as identified by staining with cychrome-anti-CD4, were plotted against FSC, which is general parameter of cell size, a distinct subset of CD4+ T cells (~7% of total spleen cells) was consistently observed in NOD mice. Similar to naive CD4+ T cells (R2), such cells expressed high levels of CD62L and low levels of CCR5. In contrast to the naive CD4+ T cells, however, IL-15R{alpha} was highly expressed on such cells (Fig. 2A). Analysis of CD8+ T cells in NOD mice showed a similar CD8+ subset with identical cell surface staining (Fig. 2B). The bigger cell size, high CD62L, low CCR5, and high IL-15R{alpha} expression suggest that these cells have features of memory-like T cells (20, 21).



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  		FIGURE 2. A, Phenotypic analysis of distinct CD4+ T cell subsets in NOD mice. Splenic leukocytes were prepared from ~30-wk-old NOD mice and stained for a set of memory markers. CD4 staining was plotted against FSC, and the expression of CD62L, CCR5, and IL-15R{alpha} on two distinct CD4+ T cell subsets (R1 and R2) was analyzed and compared simultaneously. Representative data from five experiments are shown. B, Phenotypic analysis of distinct CD8+ T cell subsets in NOD mice. Splenic leukocytes were prepared from ~30-wk-old NOD mice and stained for a set of memory markers. CD8 staining was plotted against FSC, and the expression of CD62L, CCR5, and IL-15R{alpha} on two distinct CD8+ T cell subsets (R1 and R2) was analyzed and compared simultaneously. Representative data from five experiments are shown. C, Comparison of memory-like T cells in the periphery after anti-{gamma}c treatment. Splenic lymphocytes were prepared from anti-{gamma}c-treated diabetes-free NOD mice and control Ab-treated NOD mice, and distinct T cell subsets in the CD4 compartment were analyzed and compared. Representative data from three experiments are shown.

 
To determine whether such memory-like T cells are truly autoreactive, cells (both CD4+ and CD8+) were purified by cell sorting and injected into NOD/SCID mice, and the incidence of diabetes in the host mice was followed and compared with that in a cohort of NOD/SCID mice injected with unfractionated splenic leukocytes from diabetic NOD mice. As shown in Table I, similar to NOD/SCID mice with unfractionated splenic leukocytes, all NOD/SCID mice injected with memory-like T cells developed diabetes within 5 wk after cell transfer, suggesting that such memory-like T cells are indeed cytopathic autoreactive cells. Interestingly, parallel comparison of diabetic NOD mice and anti-{gamma}c-treated NOD mice that were free of diabetes at 30 wk of age revealed a marked reduction of such memory-like T cell subset (Fig. 2C), suggesting that a subset of autoreactive memory-like T cells is also sensitive to {gamma}c blockade.


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  		Table I. Induction of diabetes by memory-like T cells

 
The {gamma}c blockade fails to block islet allograft rejection in diabetic NOD mice

Given the potency of anti-{gamma}c in blocking ongoing diabetes in the NOD model (Fig. 1) and in preventing islet allograft rejection in certain mouse strain combinations (17), we reasoned that blocking {gamma}c would be effective in blocking islet allograft rejection in diabetic NOD mice. To test this hypothesis, we transplanted DBA/2 islets into diabetic NOD mice. The recipient mice were treated with the anti-{gamma}c mAbs, and graft survival was determined. As shown in Fig. 3, treatment with anti-{gamma}c mAbs completely failed to prevent islet allograft rejection, and all anti-{gamma}c-treated diabetic NOD mice rejected the islet allografts within 15 days (n = 5). Histological analysis showed massive infiltration and extensive destruction of islet transplants (data not shown). Hence, despite the profound effect of {gamma}c blockade in autoimmune diabetes, blocking {gamma}c alone is insufficient to prevent islet allograft rejection in diabetic NOD recipients.



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  		FIGURE 3. Islet allograft survival in diabetic NOD mice. Crude islets were prepared from DBA/2 donors and transplanted under the renal capsule into diabetic NOD recipients. The recipient mice were treated with anti-{gamma}c mAbs or control rat IgG, and islet allograft survival was determined and shown.

 
CD28/CD154 blockade prevents islet allograft rejection in nonautoimmune mice, but not in diabetic NOD mice

Diabetic NOD mice, like conventional nonautoimmune mice, must have an intact repertoire of alloreactive T cells. Costimulatory signals, especially CD28 and CD154 signals, are clearly critical in the activation of naive T cells involved in the allograft response (9). To examine the impact of CD28/CD154 blockade on islet allograft survival, we first transplanted DBA/2 islets into streptozotocin-induced diabetic C57BL/6 mice and treated them with CTLA-4Ig and anti-CD154 (MR1). Consistent with previous reports (22, 23), treatment with CTLA-4Ig and anti-CD154 uniformly induced permanent islet allograft survival (mean survival time (MST), >100 days; n = 5), while the untreated controls rejected DBA/2 islet allografts with a MST of 14 days (n = 6; Fig. 4A). However, the effects of CTLA-4Ig and anti-CD154 treatment on islet allograft survival in diabetic NOD recipients are strikingly different. Treatment of diabetic NOD hosts with the same CTLA-4Ig and anti-CD154 protocol failed to prevent islet allograft rejection, and all CTLA-4Ig- and anti-CD154-treated diabetic NOD mice rejected DBA/2 islets within 10 days (n = 5; Fig. 4B). Clearly, blocking CD28/CD154 costimulatory signals, although effective in preventing islet allograft rejection in conventional mice, is clearly not sufficient to do so in diabetic NOD mice.



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  		FIGURE 4. A, Effect of CTLA-4Ig and anti-CD154 treatment on islet allograft survival. Crude islets from DBA/2 donors were transplanted into streptozotocin-induced diabetic C57BL/6 mice. Islet allograft survival in CTLA-4Ig- and anti-CD154-treated (MR1) and control Ab-treated recipients are shown. B, Effects of CTLA-4Ig and anti-CD154 on islet allograft survival in diabetic NOD recipients. Crude islets from DBA/2 donors were transplanted into overt diabetic NOD mice and treated with CTLA-4Ig and anti-CD154 (MR1). Islet allograft survival in control Ab-treated and costimulatory blockade-treated recipients is shown.

 
CD28/CD154 blockade fails to inhibit ongoing islet-specific autoimmunity

It has been shown that blocking either the B7/CD28 pathway or the CD40/CD154 pathway before the onset of insulitis (i.e., at 4 wk of age) can prevent the spontaneous onset of diabetes (24, 25). It remains uncertain whether blocking both CD28 and CD154 costimulatory pathways could arrest the ongoing diabetes. To test this possibility, NOD mice were treated at ~8 wk of age with combined CTLA-4Ig and anti-CD154, and the incidence of diabetes was determined. As shown in Fig. 5A, treatment with CTLA-4Ig and anti-CD154 to block both B7/CD28 and CD40/CD154 pathways could not prevent the development of diabetes, and nine of 12 treated mice (75%) were diabetic at 26 wk of age. Hence, blocking CD28/CD154 signals, although effective in preventing spontaneous onset of diabetes in NOD mice, has minimal effect on ongoing diabetes, which is in striking contrast to {gamma}c blockade (Fig. 1B). Similarly, adoptive transfer of splenic leukocytes from diabetic NOD mice into NOD/SCID hosts uniformly induced diabetes in NOD/SCID hosts, and treatment with CTLA-4Ig and anti-CD154 at the time of cell transfer had no effect on the incidence of diabetes; all treated NOD/SCID mice were diabetic within 9 wk after adoptive cell transfer (Fig. 5B). Thus, the cytopathic effect of committed islet-specific autoreactive T cells is independent of both CD28 and CD154 costimulatory molecules.



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  		FIGURE 5. A, Effect of combined CTLA-4Ig and anti-CD154 treatment on diabetes in NOD mice. CTLA-4Ig and anti-CD154 were given to a cohort of NOD mice starting at 8 wk of age. The cumulative incidence of diabetes (percentage) was plotted and compared with that in control Ab-treated mice. B, Effect of combined CTLA-4Ig and anti-CD154 treatment on adoptively transferred diabetes in NOD/SCID mice. Splenic leukocytes were prepared from diabetic NOD mice. Cells (20 x 106) were injected into NOD/SCID mice and treated with CTLA-4Ig and anti-CD154 mAb (MR1) at the time of cell transfer. The incidence of diabetes (percentage) in the host mice after adoptive cell transfer is shown.

 
Blocking {gamma}c and CD28/CD154 signals prolongs islet allograft survival in diabetic NOD mice

The above data suggest that activation of alloreactive T cells, autoreactive T cells, or a subset of memory-like T cells in diabetic NOD hosts can cause rapid islet allograft destruction, and stable allograft survival may require simultaneous blockade of CD28/CD154 and {gamma}c signals. To test this hypothesis, DBA/2 islets were transplanted into diabetic NOD mice and treated with CTLA-4Ig, anti-CD154, and anti-{gamma}c, then graft survival was determined and compared with that in control Ab-treated mice. As shown in Table II, control Ab-treated mice rejected islet allografts with an MST of 4 days (n = 5). In contrast, islet allograft survival was markedly prolonged in mice treated with CD28/CD154/{gamma}c blockade, and the MST of islet allograft was ~46 days (n = 6). Nonetheless, long term allograft survival was not achieved in this model, and all treated diabetic NOD mice eventually rejected the islet allografts, suggesting that other CD28/CD154/{gamma}c-independent mechanisms may exist and support the rejection response.


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  		Table II. Islet allograft survival in diabetic NOD mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Clearly, the diabetic NOD is an extremely demanding model for the induction of islet allograft tolerance. Understanding precisely the cellular and molecular mechanisms that render those mice so resistant to islet transplants is critically important in designing effective therapeutic strategies. Our data re-enforce the idea that the effector mechanisms developed in diabetic NOD hosts are inherently complex, and activation of multiple T cell subsets involving distinct molecular pathways in this model is a typical feature of islet allograft rejection. Our study also suggests that the {gamma}c-dependent growth factor signals and the CD28/CD154 costimulatory signals regulate certain overlapping, but also distinct, aspects of T cell activation and islet allograft rejection in diabetic NOD hosts.

A key defect in the pathogenesis of autoimmune diabetes in NOD mice is the failure to establish islet {beta} cell-specific self-tolerance, and such defect results in the activation and expansion of islet-specific autoreactive T cells. Our data along with those of others suggest that the {gamma}c-dependent growth factor signals and the CD28/CD154 costimulatory signals play a key role in the initial establishment of the cytopathic autoreactive T cell pool, as blocking the {gamma}c (Fig. 1A), B7/CD28 (24), or CD40/CD154 (25) pathway can prevent the spontaneous onset of diabetes. However, the cytopathic events regulated by CD28/CD154 costimulatory signals and the {gamma}c-dependent growth factor signals are strikingly different once a committed autoreactive repertoire is well established. It is generally believed that infiltration of pancreatic islets by autoreactive T cells (i.e., insulitis) precedes the onset of clinical diabetes, and the process of insulitis is often well underway at ~8 wk of age in NOD mice (1). Treatment with anti-{gamma}c mAbs at this time point can effectively inhibit the development of diabetes, whereas blocking both B7/CD28 and CD40/CD154 costimulatory pathways had no effect. Thus, activation of the committed effector T cells in islet destruction is independent of both CD28 and CD154 signals, but seems to require {gamma}c signals. This idea is further supported by studies using the adoptive transfer model. Splenic leukocytes from diabetic NOD mice can rapidly induce diabetes in NOD/SCID hosts upon adoptive cell transfer, suggesting the presence of islet-specific effector T cells in the inoculum. Blocking the {gamma}c-dependent growth factor signals, but not the CD28/CD154 costimulatory signals, at the time of cell transfer markedly inhibited the development of diabetes in NOD/SCID hosts. Furthermore, a subset of memory-like T cells that is clearly autoreactive (Table I) is also markedly reduced in anti-{gamma}c-treated and diabetic-free mice (Fig. 2). It is important to emphasize that the anti-{gamma}c-treated mice are not grossly immunodeficient, as they can mount a normal delayed-type hypersensitivity response (data not shown) and can reject the skin allograft (26). Thus, in an immunocompetent host, blocking the {gamma}c selectively affects a subset of activated T cells as opposed to genetic mutations of the {gamma}c (27). These data suggest that once T cells are recruited into the autoreactive pool, activation and the effector function of such T cells are resistant to CD28/CD154 costimulatory blockade, but remain sensitive to the {gamma}c blockade.

Our data strongly suggest that the effector mechanisms of islet allograft rejection in conventional nonautoimmune mice and in autoimmune diabetic NOD mice are strikingly different. In nonautoimmune C57BL/6 mice rendered diabetic by streptozotocin, blocking both CD28 and CD154 costimulatory signals is sufficient to induce islet allograft tolerance (Fig. 4A). However, blocking CD28/CD154-dependent T cell activation in autoimmune diabetic NOD hosts fails to prevent islet allograft rejection (Fig. 4B). Thus, the effector mechanisms that are independent of CD28/CD154 signals remain capable of mediating the rejection response in the diabetic NOD hosts. There is compelling evidence to suggest that activation of CD8+ T cells and certain committed effector/memory T cells may mediate the escape of CD28/CD154 blockade in transplant rejection (28, 29, 30). Indeed, in the NOD model, treatment with CTLA-4Ig and anti-CD154, although effective in preventing spontaneous onset of diabetes (24, 25), has minimal effect in blocking ongoing diabetes in the NOD model (Fig. 5), further supporting this idea. However, our data suggest that the committed effector T cells and certain memory-like T cells that are islet autoantigen-specific are part, but certainly not the entirety, of CD28/CD154 blockade-resistant rejection in the diabetic NOD hosts. Clearly, blocking the {gamma}c signals is remarkably effective in blocking ongoing islet-specific autoimmunity and a subset of islet-specific memory-like T cells (Figs. 1 and 2). Surprisingly, targeting the {gamma}c in combination with CD28/CD154 blockade delayed, but did not prevent, islet allograft rejection in diabetic NOD mice (Table II). Hence, other effector mechanisms that are insensitive to {gamma}c signals can still mediate CD28/CD154 blockade-resistant rejection in diabetic NOD hosts. Blocking the {gamma}c alone can inhibit T cell activation and induce long term islet allograft survival in conventional mice (17), but such an effect was not observed in the diabetic NOD recipients (Fig. 3), further suggesting that the mechanisms mediating islet allograft rejection in conventional mice are different from those in diabetic NOD mice. Analysis of CFSE-labeled T cells proliferating in vivo in the allogeneic hosts revealed that {gamma}c-dependent growth factor signals differentially affect the in vivo activation of CD4+ and CD8+ T cells (26). While activation of CD8+ T cells is particularly sensitive to {gamma}c signals, activation of CD4+ T cells is resistant to the {gamma}c blockade, but appears to require CD28/CD154 costimulation (26). Thus, blocking both {gamma}c-dependent growth factor signals and CD28/CD154 costimulation may be particularly important in models in which activation of both CD4+ and CD8+ T cells are involved in the rejection response. Nonetheless, not all CD4+ T cells require CD28 and CD154 costimulatory signals for activation in vivo; a small subset of CD4+ T cells can still undergo vigorous proliferation in vivo despite combined CD28 and CD154 blockade (26). Similarly, not all memory T cells are dependent on {gamma}c signals for development and survival (20). A detailed understanding of the identity of such cells and their effects in islet allograft rejection in the NOD model is critically important.

Thus, it is possible that other unconventional T cell subsets supported by novel alternative costimulatory molecules (e.g., ICOS, CD27, CD30, 4-1BB, and OX40) may play an important role in mediating the rejection response (31). It is also possible that a subset of memory T cells that are resistant to {gamma}c blockade may mediate islet allograft rejection in NOD recipients. In the NOD/SCID model, blocking the {gamma}c did not completely abolish the disease process, and certain mice still developed diabetes, albeit with delayed kinetics (Fig. 1C). Alternatively, generation of regulatory T cells that may control the escaping alloreactive and autoreactive T cells may be defective in diabetic NOD mice. It has been proposed that NOD mice exhibit certain defect in CD4+CD25+ T cells and NKT cells, both of which are potent suppressor cells (32, 33); such an inherent defect in regulatory cell function may render tolerance induction in diabetic NOD mice a challenging task. Finally, engagement of costimulatory molecules that can transduce negative signals may be required to completely shut down cytopathic T cells. It has been shown that CTLA-4 and PD-1 signals are critical in down-regulating T cell activation (34, 35), and CTLA-4 signals have been shown to play an important role in regulating the progression of diabetes (36). Clearly, their roles in regulating islet allograft tolerance in diabetic NOD mice warrant further study.

In summary, the effector mechanisms in diabetic NOD mice in rejection of islet allografts are far more complex than initially anticipated. Multiple cell types are probably involved in the destruction of islet allografts, and different T cell subsets appear to have distinct activation requirement. Rejection of islet allografts in diabetic NOD mice involves CD28/CD154/{gamma}c-dependent and -independent mechanisms. Our study also provides a key framework to further unravel additional effector mechanisms in diabetic NOD mice.


   Acknowledgments
 
We thank Dr. Thomas Malek (University of Miami, Miami, FL) for the generous gift of anti-{gamma}c mAbs.


   Footnotes
 
1 This work was supported by grants from the Juvenile Diabetic Foundation International 1-2000-47 and 1-2002-620 (to X.C.L.). G.D. was supported by a grant from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Xian C. Li, Department of Medicine, Harvard Medical School, Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215. xli{at}bidmc.harvard.edu Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; {gamma}c, common {gamma}-chain; FSC, forward scatter; MST, mean survival time. Back

Received for publication April 29, 2003. Accepted for publication July 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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