Type 1 diabetes (T1D) occurs when the body’s T cells attack and destroy insulin-producing β cells in the pancreatic islets, which leads to insulin deficiency and subsequent hyperglycemia. Insulin therapy is burdensome for those with brittle T1D causing frequent hypoglycemic episodes and hypoglycemia unawareness, which is associated with poor quality of life, premature death, and considerable health care costs (1). To this end, transplantation of allogeneic pancreatic islets provides an option to restore physiologic insulin secretion and euglycemia in patients with brittle T1D (1–3).
One major challenge for successful islet transplantation is the T-cell-mediated islet allograft rejection. For instance, in a trial of the Edmonton protocol with 36 T1D patients who were treated with corticosteroid-free immunosuppression, only five patients achieved insulin independence at 2 years after islet transplantation (4). Bellin et al. have recently proposed that potent induction immunotherapy will facilitate the long-term protection of transplanted islet allografts in T1D patients (5). Indeed, 5-year insulin independence has been achieved in about 50% patients by transiently using potent induction immunotherapy in addition to maintenance immunosuppression (5). These findings highlight the significance of selecting an optimal induction immunotherapy for islet transplantation.
The T-cell receptor (TCR) binds to specific peptide and MHC expressed on antigen-presenting cells and other cells, and in turn delivers the essential signal via CD3 molecules to control T-cell activation and function (6). CD3 was a frequently used target of induction immunotherapy for transplantation (7) and is currently being investigated with renewed interest (5, 8). In contrast, less attention has been paid to the therapeutic potential of targeting the TCR. We have recently shown that transient anti-TCRβ mAb (H57-597) treatment was more potent than an anti-CD3 mAb (145-2C11) in inducing long-term cardiac allograft survival in full MHC-mismatched recipient mice (9). We propose that anti-TCRβ mAb can also inhibit islet allograft rejection and represents an important option for induction immunotherapy in islet transplantation.
Transient anti-TCR mAb Treatment Induces Long-Term Islet Allograft Survival in STZ-Induced Diabetic Mice
We investigated the efficacy of an anti-TCRβ mAb (H57-597) in preventing islet allograft rejection. STZ-induced diabetic B6 mice were transplanted with 300 BALB/c islets and then left untreated or treated with a brief course of anti-TCR. All diabetic B6 mice transplanted with BALB/c islets achieved normoglycemia within 1 week after transplantation. However, recipients in the untreated group showed recurrence of diabetes within 20 days post-grafting (Fig. 1A, left panel). In contrast, all anti-TCR mAb-treated B6 recipients maintained normoglycemia for more than 64 days, and two of seven recipients did not become diabetic again (Fig. 1A, right panel). Therefore, transient anti-TCR treatment significantly prolonged islet allograft survival (mean survival time [MST]: 93.1±30.0 days) compared to the untreated group (MST: 15.6±1.7 days, P<0.001) (Fig. 1B).
Data from IPGTT indicated a robust improvement in glucose tolerance in anti–TCR-treated recipients (days 19 and 64 post-grafting) compared to the untreated group (Fig. 1C). Moreover, histology showed that insulin-producing cells were barely detectable under the kidney capsule of the untreated group by day 19 whereas all the islet allografts from the anti–TCR-treated group at 19 and 64 days after transplantation remained intact and expressed insulin (Fig. 1D, top three panels). Representative histology of a normoglycemic recipient at 152 days after transplantation as well as a rejected islet allograft (day 81) in the anti–TCR-treated group is also shown (Fig. 1D, fourth and fifth panels).
In a mixed lymphocyte reaction assay, splenocytes from anti–TCR-treated, graft-accepting recipients (19 and 64 days after islet transplant) exhibited reduced cell proliferation when compared to the splenocytes from untreated recipients with rejected grafts (Fig. 2A). Furthermore, the level of serum anti-donor IgG was significantly lower in the anti–TCR-treated recipients at day 19 and day 64 after transplant compared to graft-rejected mice (untreated group; day 19 after transplantation) (Fig. 2B). Taken together, these results demonstrate that transient anti-TCR treatment reduced anti-donor immune responses resulting in long-term protection of islet allografts.
Transient anti-TCR treatment resulted in a significant reduction of CD4+ and CD8+ T-cell numbers in the spleen of recipient mice at 19 and 64 days after transplant (Fig. 2C, left panels). In the same anti–TCR-treated recipients, the percentage of CD4+CD25+Foxp3+ regulatory T (Treg) cells within CD4+ splenocytes was significantly increased (P<0.05), but the number of Treg cells in spleen was still much lower when compared to that in untreated recipients (Fig. 2C, right panels). Importantly, the T-cell numbers recovered to normal levels in the anti–TCR-treated recipients with long-term (>120 days, n=2) accepting islet allografts (Fig. 2C, left panels), suggesting that the T cells were unresponsive to alloantigen upon T-cell recovery.
Natural Treg Cells are not Required for Anti-TCR mAb-Mediated Induction of Peripheral Tolerance to Allografts
Treg cells control peripheral T-cell tolerance to self-antigens (10). Herein, we determined whether Treg cells are required for the induction of peripheral T-cell tolerance to alloantigens by anti-TCR mAb. Rag1−/− mice adoptively transferred with Foxp3/GFP− splenocytes obtained from Foxp3/GFP reporter mice and transplanted with BALB/c skin allografts were treated with a brief course of anti-TCR or left untreated. All anti-TCR mAb-treated recipients accepted the skin allografts for more than 120 days, whereas all untreated recipients quickly rejected skin allografts within 10 days (Fig. 3A). Therefore, anti-TCR prevented peripheral T-cell-mediated skin allograft rejection, and this prevention did not require the presence of natural Treg cells.
At 120 days after BALB/c skin transplantation, peripheral blood mononuclear cells (PBMCs) in anti-TCR mAb-treated Rag1−/− recipients contained an average of 9.9% CD4+ and 5.8% CD8+ T cells, which were still lower than the frequencies of CD4+ (27.4%) or CD8+ (17.4%) T cells in PBMCs of naive Foxp3/GFP mice (Fig. 3B). The frequency of Foxp3/GFP+ (0.43%) cells in PBMCs of anti-TCR mAb-treated recipients was low (Fig. 3C). These few Treg cells might be de novo derived from transferred Foxp3/GFP− splenocytes.
Although the T-cell frequency remained low in anti-TCR-treated Rag1−/− recipients, these T cells caused robust rejection of the third-party C3H skin grafts transplanted at 120 days after BALB/c skin grafting (Fig. 3D and E). Thus, transient anti-TCR treatment during initial exposure to antigens (e.g., BALB/c alloantigens) induced peripheral T-cell tolerance specifically towards those antigens, but not the antigens (e.g., C3H third-party alloantigens) that were exposed later.
Anti-TCR Prevents Adoptive Transfer of Diabetes by Diabetogenic T Cells From Overtly Diabetic NOD Mice
T1D patients contain activated autoreactive T cells that have previously recognized islet antigens and destroyed self-islets (11). To determine whether anti-TCR can abrogate the function of activated diabetogenic T cells, we performed adoptive transfer of cells from new-onset or overtly diabetic NOD mice into NSG mice to transfer diabetes. NSG mice that received cells from overtly diabetic mice were either left untreated or injected with anti-TCR. Although cells obtained from overtly diabetic NOD mice transferred disease more rapidly than those from new-onset diabetic NOD mice (P<0.05), transfer of the disease was completely prevented by transient anti-TCR treatment (Fig. 4A and B). Moreover, anti-TCR did not induce long-term T-cell depletion in this model, as it did not reduce the T-cell frequencies in PBMCs of NSG mice at 70 days after cell transfer (Fig. 4C and D). Therefore, anti-TCR mAb is capable of abrogating the function of diabetogenic T cells from overtly diabetic NOD mice.
Anti-TCR Prolongs Islet Allograft Survival in Overtly Diabetic NOD Mice
The therapeutic effects of anti-TCR in preventing islet destruction by both alloreactive (Fig. 1) and autoreactive T cells (Fig. 4) led us to determine its efficacy in preventing islet allograft rejection in overtly diabetic NOD mice, which is an extremely stringent model that mounts both allo- and autoimmune responses against the transplanted islets. First, we assessed the in vivo effects of anti-TCR on T cells in NOD mice (without transplantation). Five days after injection of anti-TCR, the number of CD4+, but not CD8+, T cells was significantly decreased in the spleen and inguinal lymph nodes of treated mice. The number of splenic Treg cells were also significantly reduced (Fig. 5). Next, we determined the effects of anti-TCR on preventing islet allograft rejection in overtly diabetic NOD mice. These diabetic NOD mice when left untreated rapidly rejected BALB/c islets within 10 days (MST: 8.7±0.6 days). Transient anti-TCRβ treatment significantly prolonged the survival of transplanted BALB/c islets (MST: 28.0±7.7 days, P<0.05 vs. untreated group) (Fig. 5B and C). However, only two of six grafts survived more than 30 days. Histological examination confirmed the graft rejection (Fig. 5D).
We monitored the frequencies and total numbers of T cells in the PBMCs of transplant recipients weekly after anti-TCR treatment. There were proportional increases in CD8+ T-cell frequencies and proportional decreases in CD4+ T-cell frequencies in PBMCs over the first 3 weeks after transplantation (Fig. 5Ei). Treg cell frequency within the CD4+ cell population remained relatively constant in the weeks after islet transplantation in these recipients (Fig. 5Eii). The total number of CD4+ T cells and Treg cells were still reduced by half in the anti–TCR-treated group, whereas the number of CD8+ T cells was not significantly changed when compared to that in non-diabetic NOD mice or the untreated transplant recipients (Fig. 5F). Taken together, these results demonstrate that anti-TCR treatment reduced the frequency of CD4+ T cells and prolonged islet allograft survival in overtly diabetic NOD mice.
In the current study, we investigated the efficacy and mechanisms of action of an anti-TCRβ mAb in preventing islet allograft rejection. Transient anti-TCR treatment initially reduced the peripheral T-cell numbers, inhibited allogeneic T-cell responses, and abrogated the production of anti-donor IgG. Even when the T-cell numbers recovered in the periphery, some BALB/c islet allografts survived long term (>120 days). Therefore, transient anti-TCR treatment is capable of “resetting” the immune system to accept the transplanted tissues. These results were consistent with our previous findings in a cardiac transplantation model (9).
We speculate that new alloreactive T cells generated from the thymus may contribute to the late-stage allograft rejection in some of our islet allograft recipients. To eliminate the influence of newly derived T cells, Rag1−/− B6 recipients were adoptively transferred with Foxp3− B6 splenocytes and transplanted with BALB/c skin allografts. A brief course of anti-TCR completely prevented the BALB/c skin allograft rejection in this model and exhibited lower T-cell frequencies in the PBMCs (∼30% compared to naive mice) at 120 days after BALB/c skin acceptance. However, these T cells robustly rejected the third-party C3H skin allografts within 10 days. This demonstrated the ability of anti-TCR treatment to induce peripheral T-cell tolerance towards the alloantigen that is exposed during the treatment.
Multiple mechanisms exist to induce tolerance in peripheral mature T cells, including functional anergy (12), clonal exhaustion (13), peripheral deletion by apoptosis (14), and suppression by Treg cells (10, 15, 16). In our adoptive transfer model, Foxp3-expressing natural Treg cells are not required for the induction of peripheral tolerance to alloantigen by anti-TCR. We are thus investigating how anti-TCR directly deletes or disables the antigen-specific effector T cells (e.g., anergy, exhaustion, apoptosis, or unidentified therapeutic mechanisms). However, this did not exclude the possibility that Treg cells maintain long-term allograft survival in wild-type B6 recipients. Foxp3-expressing CD4+ Treg cells act as gatekeepers to suppress the peripheral autoreactive T cells that have escaped thymic deletion (10). At the late stage after transplantation in wild-type recipients, Treg cells may also suppress the new thymic-derived or residual peripheral alloreactive T cells (9).
Transplanted allogeneic islets in T1D recipients are also attacked by diabetogenic T cells that recognize islet autoantigens (17). Over the last few decades, many immunotherapies such as CsA (18, 19), sirolimus (20), FcR-nonbinding CD3 mAbs (21), anti-thymocyte globulin (22, 23), or CTLA4-Ig fusion protein (24) have been tested to control diabetogenic T cells with the objective of preserving residual β cells in patients with new-onset T1D. All of these potent immunotherapies delay the loss of β-cell function in T1D patients but do not provide long-lasting protection for the β cells. Because T1D subjects comprise diabetogenic T cells that have already recognized islet autoantigens and have been activated (11), we propose that an optimal immunotherapy for T1D receiving islet transplants should be able to abrogate the function of activated diabetogenic T cells. Herein, we showed that transient anti-TCR treatment even abrogated the function of diabetogenic T cells from overtly diabetic NOD mice and completely prevented T1D transfer in NSG mice. Importantly, the frequencies of the transferred cells in PBMCs of the anti–TCR-treated group were not reduced when compared to those of the untreated group. Therefore, transient anti-TCR treatment should be able to selectively abrogate the function of activated diabetogenic T cells.
Transplantation of islet allografts into diabetic NOD mice is an extremely stringent model that mounts both allogeneic and autoimmune T-cell responses against the transplanted islets (25, 26). A combination of potent immunotherapies, such as CTLA4-Ig plus anti-CD22 or anti-CD154 plus anti-CD45RB, delayed islet allograft rejection in diabetic NOD mice when administered chronically (25, 26). Herein, we showed that transient anti-TCR treatment alone significantly prolonged BALB/c islet allograft survival in overtly diabetic NOD mice. Therefore, we suggest that anti-TCR may be added to the short list of induction immunotherapies for islet transplantation. Indeed, anti-human TCR mAb clones, T10B9 and BMA031, were shown effective for treating renal and heart transplantations in clinical trials (27–29). With the advancement in technology, new anti-human TCR mAb clones with optimal immunotolerogenic effects could also be identified and tested clinically.
MATERIALS AND METHODS
BALB/c, C57BL/6 (B6), C3H, B6.Cg-FoxP3tm2Tch/J (Foxp3/GFP; B6 background), B6.129S7-Rag1tm1Mom/J (Rag1−/−; B6 background), NOD/ShiLtJ (NOD), and NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD.SCID.γc−/−; NSG) mice were purchased from the Jackson Laboratory. Animals were maintained at the University of Toledo specific pathogen-free or sterile (NOD and NSG mice) animal facility, according to the institutional guidelines.
Pancreatic Islet Transplantation and Anti-TCR mAb Treatment
Diabetes was induced in adult male B6 mice by a single i.p. injection of streptozotocin (STZ, 200 mg/kg; Sigma Aldrich). Blood glucose levels were monitored using a ReliOn Ultima glucose meter. One week after the STZ injection, more than 90% of the mice had non-fasting blood glucose levels above 500 mg/dL. Female NOD mice (12–16 weeks old) with a non-fasting blood glucose concentration greater than 250 mg/dL for two consecutive days were considered as mice with new-onset diabetes. Two weeks after the onset of diabetes, more than 80% NOD mice had non-fasting blood glucose levels above 500 mg/dL.
Pancreatic islets of BALB/c mice were isolated by Histopaque 1077 (Sigma Aldrich) density gradient separation after in situ digestion with Liberase TL enzyme (Roche). About three to four hundred BALB/c islets were then transplanted under the kidney capsule of diabetic B6 mice at 1 week after STZ treatment or diabetic NOD mice at 2 weeks after the onset of diabetes, respectively (30). Recipient mice were injected i.p. with an anti-TCRβ mAb (H57-597; Bio X Cell; 100 μg/dose on days 0 and 1 post-grafting, 60 μg/dose on day 3, 20 μg/dose on days 7 and 11) or left untreated. Rejection was defined as recurrence of diabetic hyperglycemia exceeding 250 mg/dL for two consecutive days and was confirmed by histological examination.
Histology and Immunohistochemistry
Kidneys containing the islet grafts were fixed in formaldehyde, embedded in paraffin, sectioned, and stained with HE for microscopic evaluation. Some sections were also stained with an anti-insulin antibody as described previously (25). A modified Mayer hematoxylin (Poly Scientific Research & Development) was used for staining cellular nuclei. Imaging microscopy was performed using an Axiovert (Zeiss) microscope with Axiovision imaging software.
Intraperitoneal Glucose Tolerance Test (IPGTT)
At various days after islet transplantation, recipient mice were fasted for 5 hr and then injected i.p. with a 50% glucose solution (1.5 mg/g of body weight). Blood glucose levels were measured at 0, 15, 30, 45, 60, 90, and 120 min after glucose administration.
Mixed Lymphocyte Reaction
Splenocytes from islet-grafted B6 recipients as well as naive B6 mice were used as responders, whereas irradiated (15 Gy) splenocytes from either BALB/c or B6 mice were used as stimulators. Responder cells (3×105) were cultured with 3×105 irradiated stimulator cells in a 96-well culture plate for 72 hr. Cell proliferation was measured using 1 μCi/well [3H] thymidine incorporation during the final 18 hr of culture as described previously (9).
Detection of Anti-Donor IgG
The presence of anti-donor (BALB/c) specific antibodies in sera was assessed using a previously described method (9). Briefly, BALB/c splenocytes were incubated with Fc blocking anti-mouse CD16/CD32 antibody (clone 93; eBioscience) for 30 min at 4°C, washed, and further incubated with 1:10 dilutions of harvested serum for 40 min at 4°C. The cells were then washed and stained with FITC-conjugated anti-mouse IgG (eBioscience) for 25 min in the dark at 4°C, followed by flow cytometric analysis for IgG expression levels (FACSCalibur; BD Biosciences).
Adoptive Cell Transfer and Skin Transplantation
Rag1−/− mice were intravenously injected with 1.5×107 Foxp3/GFP− splenocytes that were purified from Foxp3/GFP mice using a BD FACSAria cell sorter (BD Biosciences). Dorsal ear skins from the BALB/c mice were then grafted onto the flank of Rag1−/− mice as previously described (9). Rag1−/− recipients received the same doses of anti-TCR treatment as for islet transplantation or were left untreated. At 120 days after BALB/c skin grafting, anti-TCR mAb-treated recipients were transplanted with a second skin graft from C3H mice. Grafts were monitored daily for rejection, defined as approximately 80% necrosis of the graft tissue.
Adoptive Transfer of Diabetes
NSG mice were injected i.v. with 2×107 leukocytes from new-onset or overtly diabetic NOD mice (blood glucose >500 mg/dL; 2 weeks after new-onset), respectively. NSG mice that received cells from overtly diabetic mice were left untreated or were injected i.p. with 20 μg/dose of anti-TCR on days 0, 1, 3, 7, and 11 after cell transfers. These NSG mice were monitored for the development of diabetes.
Flow Cytometric Analysis
Single-cell suspensions were obtained from peripheral blood, lymph nodes, and spleen of mice. Cells were stained with fluorescence-conjugated mAbs for CD4, CD8, and CD25 (eBioscience). Foxp3 staining was assessed using an intracellular staining kit from eBioscience. Stained cells were analyzed using a FACSCalibur flow cytometer.
The results of the graft survival data were analyzed by the Mann-Whitney U test. All other statistics were evaluated using the unpaired two-tailed Student t test to document statistical significance. A P value of less than 0.05 was considered statistically significant.
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