Pancreatic islet transplantation (Tx) has considerable potential as a clinical therapeutic option to replace β cell function in patients with type 1 diabetes (T1D).1-4 Practically all clinical islet Tx have been performed by infusion into the liver, even though it is not an optimal microenvironment because of immunologic,5,6 anatomic7-9 and physiologic factors leading to a significant graft loss within the first hours and days after the infusion.10-12 This is mainly due to the induction of an instant blood-mediated inflammatory reaction associated with intraportal islet infusion that triggers the activation of the coagulation cascade, platelets adhesion, leukocytes recruitment, and the formation of ischemic regions.13 Altogether, this nonspecific inflammation, mediated predominantly by site-dependent innate inflammatory events at the time of graft infusion,14 may heighten the intensity of the subsequent adaptive immune response.15 Other disadvantages of the hepatic site include both the relatively hyperglycemic microenvironment and the elevated concentration of immunosuppressive drugs that are well known to be toxic for the islets.16-18
The recognition of these problems has renewed the interest in the search of alternative sites for islet Tx to avoid liver-specific hurdles.19 We have recently shown that the bone marrow (BM) may represent a valid alternative site for islet infusion.20 Using a syngeneic marginal mass model of islet Tx, we demonstrated that islet infusion in the BM is safe and more efficient than the liver in restoring normoglycemia.20 Besides, the pilot clinical study performed supported the clinical feasibility and safety of intra-BM islet Tx demonstrating that autologous islets successfully survive and engraft in BM.21 Because β-cell replacement recipients are usually patients with T1D, it is mandatory to understand the influence of the transplant site on the development of de novo alloimmune response and its effect on the rechallenge of antigen-specific memory immune response.22,23
The aim of this study was to characterize the immune response against intra-BM or intraliver transplanted islets by using preclinical mouse models. The hypothesis is that the transplant site could potentially influence the adaptive immune response toward the graft both qualitatively and quantitatively. Our results show that in the absence of an immunosuppression therapy or in the presence of a maintenance immunosuppressive therapy as MMF/FK-506 combination, the transplant site does not impact the timing nor the kinetics of the alloimmune and the antigen-specific memory T cell responses. However, the transplant site influences the immune response against allogeneic islets in the presence of a T cell–depleting agent as anti-CD3 treatment, since islets infused in the BM appears less protected from the adaptive immune response.
MATERIALS AND METHODS
Less (C57BL/6 in Balb/c) and highly (Balb/c in C57BL/6) stringent major histocompatibility complex (MHC) fully mismatched mouse models were used to evaluate the role of the transplant site in the stimulation of an alloimmune response: C57BL/6 or Balb/c mice (8 weeks old; Charles River Laboratories, Calco, Italy) were made diabetic with intravenous alloxan injection (70-75 mg/kg; Sigma-Aldrich, Italy) 1 week before islet Tx. Single antigen-mismatched mouse model of islet Tx was used to evaluate the role of the transplant site in the rechallenge of an antigen-specific immune response: (1) primary response (C57BL/6 rat insulin promoter [RIP]-GP in C57BL/6): recipient mice, made diabetic with alloxan injection, were infected with 103 pfu of lymphocytic choriomeningitis virus (LCMV) Arm 8 weeks after islet Tx and (2) memory response (C57BL/6 RIP-GP in LCMV-infected C57BL/6 RIP-GP): C57BL/6 RIP-GP recipient mice (8 weeks old, inhouse breeding) were rendered diabetic with LCMV infection24 1 month before islet Tx. For all recipient mice, diabetes was defined as blood glucose levels greater than 450 mg/dL.25 Blood glucose measurements were performed using a Glucometer Elite (Bayer, Milan, Italy). All mice were maintained under specific pathogen-free conditions at San Raffaele Scientific Institute and handled in accordance with the Institutional Animal Care and Use Committee approved protocol (IACUC number: 558).
Mouse Islet Isolation and Culture
Pancreatic islets were isolated from C57BL/6 or Balb/c (8 weeks-old; Charles River Laboratories) or C57BL/6 RIP-GP (8 weeks old; inhouse breeding) mice by a collagenase digestion method as previously described.26,27 The islets (250 islets/ml) were cultured freely floating (37°C, 5% CO2) overnight in RPMI 1640 medium supplemented with 1% L-glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum (Euroclone, Milan, Italy) before Tx. Islet purity was more than 90%, and islet mass was expressed as islet equivalent (IEQ).28
Islets were transplanted in the liver (liver-Tx)26 or in the BM (BM-Tx)20 as previously described. We performed a preliminary study in syngeneic model (C57BL/6 in C57BL/6; Fig. S1, SDC,http://links.lww.com/TP/B340) to evaluate the number of islets needed in both sites to have the maximum number of recipients achieving normoglycemia within the adequate time (5 days). Seventy three alloxan-induced severely diabetic C57BL/6 mice (not-fasting glycemia 562 ± 48 mg/dL) were transplanted with marginal islet mass (median, 150 IEQ; ranging from 100 to 237 IEQ) or full islet mass (median, 378 IEQ, ranging from 370 to 380 IEQ) in the liver (liver-Tx: marginal n = 20; full: n = 13) or in the BM (BM-Tx: marginal n = 27; full: n = 13). As previously reported,20 marginal islet mass infusion in the BM is more efficient than the liver in restoring normoglycemia, but in both sites only few mice gain normoglycemia within 5 days (<30%; Fig. 1SA, SDC,http://links.lww.com/TP/B340). On the other hand, full islet mass infusion showed a time to engraft compatible with the possibility to monitor graft rejection in allogeneic model (Figure 1SB, SDC,http://links.lww.com/TP/B340). Consequently, even if with full islet mass infusion, the probability to reach the normoglycemia was lower in BM-Tx than that in liver-Tx (likely be due to the limited space of the femur cavity) 450 IEQ were transplanted in MHC fully mismatched models to evaluate the time to graft rejection.
Evaluation of Graft Function
Blood glucose levels were measured daily for the first week and then every second day after islet Tx. Surgical death was defined as death within the first 7 days after Tx. Islet engraftment (ie, normoglycemia achievement) was defined as not-fasting glycemia less than 250 mg/dL within 5 days after islet Tx. Graft rejection was evaluated only in mice that have previously achieved normoglycemia and defined as 2 consecutive measurements greater than 350 mg/dL.
To evaluate whether in the presence of immunosuppression, the time to graft rejection is site-dependent, C57BL/6 or Balb/c recipient mice were treated daily starting from day −1 to +14 or from day −1 to +30 after allogeneic islet Tx. Tacrolimus (FK-506; Astellas Pharma S.p.a., Milan, Italy) was diluted in saline solution and administered by intraperitoneum at 0.1 mg/kg per day. Mycophenolate mofetil (MMF; Roche, Nutley, NJ) was diluted in saline solution and administered orally at 60 mg/kg per day.18 InVivoMAb anti-mCD3e (50 μg/injection per day, clone 145-2C11 f(ab′)2 fragments; BioXCell, West Lebanon, USA) was administered i.v. from day −1 to +3 for 5 consecutive days peritransplant.29
Evaluation of Intra-BM Infiltrating Leukocytes and Graft Morphology After Islet Tx
Alloxan-induced severely diabetic C57BL/6 mice transplanted with 450 Balb/c IEQ in the BM were sacrificed at days 0, 1, 3, 5, 7, 10, 14 after Tx (1) for the characterization of Intra-BM infiltrating leukocytes (IBL) by flow cytometry analysis and (2) for the evaluation of graft morphology by histology.
Single-cell suspensions were obtained from both BM with the graft and the contralateral sham-operated and from the liver.30 To characterize IBL recruitment, the cells were stained with FITC-labeled anti-CD19 (clone 1D3), PE-labeled anti-CD4 (clone RM4-5), PE-Cy7-labeled anti-CD3 (clone 145-2C11), PB-labeled anti-CD8 (clone 53-6.7), PerCP-Cy5.5-labeled anti-NK1.1 (clone PK136) or FITC-labeled anti-Ly6C (clone AL-21), PE-labeled anti-CD11c (clone HL3), PE-Cy7-labeled anti-CD11b (clone M1/70), PerCP-Cy5.5-labeled anti-Gr1 (clone RB6-8C5) Abs (BD Biosciences, San Diego, CA) for the detection of Gr1+CD11b+Ly6C− (mostly polymorphonuclear cells), CD3+CD4+ (mostly CD4+ T cells), CD3+CD8+ (mostly CD8+ T cells), NK1.1+CD3– (mostly NK cells), NK1.1+CD3+ (mostly NKT cells), Gr1−CD11b+CD11c- (mostly macrophages), CD11c+CD11b+Gr1− (mostly dendritic cells [DC]) and CD19+ (mostly B cells). To characterize intra-BM and intraliver T cell recruitment, the cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen, Carlsbad, CA) and then surface stained with APC-labeled anti-CD45 (clone 30-F11), PB-labeled anti-CD4 (clone RM4-5), PerCP-Cy5.5-labeled anti-CD8 (clone 53-6.7), fluorochromes conjugated with antibodies (FITC)-labeled anti-CD44 (clone IM7), and PE-labeled anti-CD62L (clone MEL-14). Samples were acquired on a BD FACSCantoII instrument. Rainbow calibration particles (BD Biosciences) were used to calibrate and normalize acquisition settings in each experiment. Analysis of flow cytometry data was performed with FCS Express V4 (DeNovo Software, CA).
Femurs were procured, fixed in 10% buffered formalin and placed in Fix Decal (Pro-EKo, Italy) overnight to decalcify the bones before embedding in paraffin. Histologic sections were stained with hematoxylin and eosin, anti-insulin (polyclonal guinea pig, 1:50; Dako, Carpinteria, CA), anti-CD3 (rat antihuman CD3 IgG1 with mouse cross reactivity, clone CD3-12 1:1000; Serotec, UK), anti-F4/80 (rat antimouse F4/80 IgG2b, clone A3-1, 1:200; Serotec). The peroxidase-antiperoxidase immunohistochemistry method (PAP Kit; DakoCytomation, Milan, Italy) or AffiniPure anti-guinea pig tetramethylrhodamine-5 (and 6)-isothiocyanate secondary antibodies (Jackson Immuno Research Laboratories, PA) were used for detection. All immunoreactive cells were analyzed using a Leica DMIRE2 microscope equipped with a color video camera connected to a computer (Hewlett-Packard).
Data were expressed as mean ± standard error or median and interquartile range, according to their distribution. Variables with a normal distribution were compared with Student t test. Variables with a nonnormal distribution were compared with Mann-Whitney U test. Pearson χ2 test was used to compare proportions. The time to normoglycemia achievement or to graft rejection was evaluated by the Kaplan-Meier analysis, and the significance was estimated by the log-rank test. Data were analyzed using the SPSS statistical software, version 13.0 (SPSS Inc., Chicago, IL).
MHC Fully mismatched Islets Transplanted in the Liver or in the BM Are Equally Rejected in the Absence of Immunosuppression
Twenty-seven alloxan-induced diabetic Balb/c mice were transplanted with 450 C57BL/6 IEQ infused in the liver (n = 7) or in the BM (n = 20). The median time to graft rejection was: 8 ± 3.3 days for liver-Tx and 6 ± 0.5 days for BM-Tx (P = 0.981; Figure 1). Similarly, 96 diabetic C57BL/6 mice were transplanted with 450 Balb/c IEQ alternatively infused in the liver (n = 34) or in the BM (n = 62). The median time to graft rejection was 6 ± 0.4 days for liver-Tx and 5 ± 0.7 days for BM-Tx (P = 0.401; Figure 1). To characterize the BM-Tx morphology and leukocyte infiltration, both the phenotype and the kinetics of the recruited inflammatory cell subsets within 14 days from islet Tx were determined by histological analysis and flow cytometry. Histological analysis at days 1 to 5 postinfusion showed that the islets were close to the site of injection, stained strongly for insulin, and preferentially distributed near to the trabecular bone. Stromal and F4/80+ cells were present at the site of islet injection and fibrotic area surrounded the graft in the first days after Tx. CD3+ T cells surrounded and infiltrated the graft at days 5 to 7 postinfusion. At later time points (days 10-14), the graft was completely destroyed and few insulin+ single cells were still present at transplant site (Figure 2). Flow cytometry analysis confirmed that CD4+ and CD8+ T cells were specifically recruited in the BM of femur transplanted with the islets and not in the contralateral one (Figure 3). These findings are not different from those that we previously described for liver-Tx.26,31
Single Antigen-Mismatched Islets Transplanted in the Liver or in the BM Are Equally Rejected
C57BL/6 RIP-GP transgenic mice express the glycoprotein (GP) of LCMV under the control of rat insulin promoter (RIP) specifically in the β cells of the pancreatic islets. In this model, LCMV infection induces the development of diabetes by triggering an antigen (GP)-specific CD8+ T cell response able to recognize β cells.24 First of all, we verified that viral antigen (GP) expression per se in the β cells did not lead to GP-specific T cell response and graft rejection after islet infusion. Seventeen alloxan-induced diabetic C57BL/6 mice were transplanted with 450 GP-expressing C57BL/6 IEQ in the liver (n = 11) or in the BM (n = 6). In both transplant sites, RIP-GP islets displayed functional activity and promoted normoglycemia. Concordantly, no GP-specific T cell responses were detected in the peripheral blood 4 weeks after RIP-GP islet Tx in both sites (data not shown). Then, normoglycemic (not-fasting glycemia 222 ± 50 mg/dL) C57BL/6 recipients previously transplanted with RIP-GP islets were infected with LCMV 8 weeks after Tx. The median time to graft rejection was 10 ± 1.1 days for intraliver RIP-GP engrafted islets (n = 9) and 11 ± 4.9 days for intra-BM RIP-GP engrafted islets (n = 6; P = 0.322; Figure 4A). The time to graft rejection was comparable with the time to hyperglycemia observed in the classical RIP-GP virally induced model of T1D (Figure 4A). Thus, primary GP-specific immune responses elicited by a viral infection cause graft loss with the same kinetics in the liver and the BM. Next, we evaluated how the GP-specific memory immune response affects graft survival in BM versus liver. Ten LCMV-infected severely diabetic male RIP-GP mice (not-fasting glycemia 541 ± 81 mg/dL) were transplanted, 30 days after LCMV infection, with 450 GP-expressing IEQ in the liver (n = 4) or in the BM (n = 6). The median time to rejection was 4 ± 0.4 days for liver-Tx and 4 ± 1 days for BM-Tx (P = 0.893; Figure 4B). All in all, these data demonstrate that intra-BM and intraliver single antigen (GP)-mismatched islets are equally rejected during primary and memory GP-specific T cell response.
MHC Fully Mismatched Islets Transplanted in the BM in the Presence of Anti-CD3 Immunosuppression Are Less Protected From the Adaptive Immune Response Than in the Liver
Less and highly stringent MHC fully mismatched mouse transplant models have been used to evaluate the alloimmune response against islets in the presence of MMF/FK-506 therapy as (1) it represents the combination of immunosuppressive drugs having limited toxicity on islet engraftment and proved efficacy in prolonging graft rejection in an intrahepatic murine model of islet Tx18 and (2) it was mostly and widely used as maintenance immunosuppression regimen in the last years.32 Twenty-nine alloxan-induced diabetic Balb/c mice were transplanted with 450 C57BL/6 IEQ alternatively infused in the liver (n = 11) or in the BM (n = 18). Recipient mice were treated daily starting from day −1 to +14 with MMF and FK-506. The median time to graft rejection was: 12 ± 3 days for liver-Tx and 8 ± 2.2 days for BM-Tx (P = 0.464; Figure 5). Similarly, 36 diabetic C57BL/6 mice were transplanted with 450 Balb/c IEQ alternatively infused in the liver (n = 18) or in the BM (n = 18). The median time to graft rejection was: 8 ± 0.6 days for liver-Tx and 7 ± 1.9 days for BM-Tx (P = 0.168; Figure 5). Because MMF/FK506 treatment marginally prolongs islet survival in MHC fully mismatched islet transplant model as previously described,18 in a second set of experiments, diabetic C57BL/6 mice were transplanted with 450 Balb/c IEQ and treated with anti-mCD3e mAb (anti-CD3) alone (liver-Tx n = 23, BM-Tx n = 30) or in combination with MMF/FK506 (liver-Tx n = 12, BM-Tx n = 15). The median time to graft rejection was: 28 ± 5.2 and 16 ± 2.6 days for liver-Tx and BM-Tx respectively in anti-CD3–treated group (P = 0.14) and 50 ± 12.5 and 10 ± 1.3 days for liver-Tx and BM-Tx, respectively, in anti-CD3 + MMF/FK-506-treated group (P = 0.003; Figure 6).
Anti-CD3 Treatment Is More Effective in Reducing Graft-Associated T Cell Responses in Liver Compared With the BM
Intra-BM and intraliver infiltrating T cells and their activation status were analyzed by flow cytometry 7 days after Tx of Balb/c islets in C57BL/6 mice treated with anti-CD3 or vehicle. In liver-Tx anti-CD3 treatment induced a significant decrease of total lymphocytes (5.58 × 105 ± 2.22 × 105 vs 7.52 × 105 ± 1.46 × 105), CD4+ (2.2 ± 0.3% vs 8.9 ± 2.5%; P < 0.05) and CD8+ (8.8 ± 2% vs 15.5 ± 4.4%; P < 0.05) T lymphocytes in comparison to vehicle (Figure 7). No significant modifications were evident in BM-Tx (Figure 7). The interaction of anti-CD3 with T cells is not inert, but may deliver at least a partial TCR signal that contributes to its immunosuppressive activity and, as previously described, CD44 is upregulated after exposure to the anti-CD3.33 Beyond infiltrating T cell decrease, we observed a marked increase in CD44+ on both CD4+ and CD8+ T cells recruited to the liver in the presence of anti-CD3. On the contrary, no significant changes in the activation status of T cells recruited to the BM were observed in anti-CD3-treated mice (Figure 7). To evaluate whether anti-CD3 differentially affect the primed T cells and the naive T cells according to the transplant site, splenocytes isolated from C57BL/6 CD45.1+ mice previously sensitized against Balb/c antigens were transferred in diabetic C57BL/6 CD45.2+ mice receiving 450 Balb/c IEQ in the BM or in the liver in the presence of vehicle or anti-CD3 (BM-Tx n = 2, liver-Tx n = 2 for each group; Figure 8). Anti-CD3 treatment significantly reduced total lymphocytes CD45.1+, CD4+ and CD8+ T cells both in the liver and in the BM 7 days after Tx, whereas was less efficient in reducing total lymphocytes CD45.2+, CD4+, and CD8+ T cells in BM as compared to the liver (Figure 8).
The Low Efficacy of Anti-CD3 in Protecting Islets in BM From the Adaptive Alloimmune Response Is Not Due to Reduced Drug Bioavailability
To evaluate whether anti-CD3 differentially reached BM and liver tissues, leukocytes were isolated from the liver and from the BM of C57BL/6 mice sacrificed 1 hour after the intravenous injection of PE-Cy7–labeled anti-CD3 antibody (anti-CD3 Ab). Flow cytometry analysis detected both in liver and in BM PE-Cy7–labeled T cells. The mice pretreatment with anti-CD3 (Anti CD3 Fab) antagonized the PE-Cy7 labeling of T cell, whereas vehicle did not, demonstrating that intravenously injected anti-CD3 Fab equally reached both tissues (Fig. S2, SDC,http://links.lww.com/TP/B340) binding 74% of T cells in the BM and 56% of T cells in the liver.
The liver is currently used as site of choice for islet infusion, although it is far from being ideal. The recognition of these immunologic and nonimmunologic drawbacks has renewed the interest in the search of alternative sites for islet Tx to avoid liver-specific hurdles.19 In this study, we used MHC fully mismatched and single-antigen mismatched mouse models of pancreatic islet transplantation to investigate whether allogeneic islets can efficiently engraft in BM in the presence of naive or memory immune response. In fact, we recently reported both in mice and in humans that autologous islets can efficiently engraft in BM.20,21 Because islet transplant recipients are usually patients with T1D, it was mandatory to understand the influence of the BM on the development of de novo alloimmune and on the rechallenge of antigen-specific memory immune responses. In this study, we demonstrated that the kinetic of the alloimmune response was not site-dependent in the absence of immunosuppression by using both highly and less stringent MHC fully mismatched models.34 Similar results were obtained also when recipient mice were treated with MMF/FK-506 combination, already used as immunosuppressive maintenance regimen in the clinical practice.32 Previous studies have suggested that the BM could provide an immunoprotected microenvironment allowing the survival of allogeneic pancreatic islets without immunosuppression.35,36 Our study rules out any evidences for the BM per se as an immunoprotected microenvironment. Histology and flow cytometry analysis confirm T cell recruitment to the transplant site concurrently with the loss of graft function. In agreement with our results a recent study demonstrated that also xenogeneic islets infused in the BM activate a CD4+ and CD8+ T cell response that finally leads to graft rejection.37
Because in T1D recipients transplanted β cells could be destroyed also by preexisting (auto)antigen-specific memory immune responses, we used a single antigen-mismatched mouse model of islet Tx to study this aspect of the adaptive immune reaction. We reported that memory antigen (GP)-specific CD8+ T cell response leads to the rejection of GP-expressing islets with similar kinetics in both BM-Tx and liver-Tx. Although this mouse model closely mimics patients with long-lasting T1D receiving islets in the presence of a memory β cell-specific immune response, the aggressiveness of the T cell response triggered by LCMV infection could have masked small differences in immune reactivity between BM and liver. Indeed almost 80% of circulating CD8+ T cells acquire an effector phenotype 8 days after LCMV infection and 7% of them are GP-specific (data not shown), whereas circulating CD8+ T cells specific for β cell antigens were in the range of 0.5% to 1% in nonobese diabetic mice38,39 and even less represented in T1D subjects.40
Despite many of our data support the idea that the immune reactivity is not different between BM and liver, the presence of a T cell depleting treatment used to mimic the immunosuppressive induction regimen29 appeared less efficient in preventing graft rejection in BM. Anti-CD3 equally reaches and binds T cells in both transplant site, but it is less efficient in reducing CD4+ and CD8+ T cells in the BM suggesting the presence of a site-dependent resistance to anti-CD3 induced apoptosis. This could be related to the specificity of the microenvironment as BM is a priming site for antigen-specific T cells41-43 and a homing site for memory T cells44-47 highly enriched in cytokines and chemokines able to prevent T cell apoptosis.48,49 Indeed it is becoming clear both in mice and in humans that CD4+ and CD8+ T cells50,51 as well as DCs52 contained in the BM are functionally distinct from those in other compartments.
At the moment, it is difficult to understand if the data obtained in mice has relevance in humans. The ongoing phase II clinical trial (NCT01722682) in which patients with T1D are receiving allogeneic islets randomly either in the liver or in the BM will allow us to clarify the effectiveness of BM as islet transplantation site. Outside this trial, BM should be considered as a site only in people in whom liver is not appropriate due to pre-existing liver disease. A strategy for the future could be the development of immunosuppressive strategies aimed to specifically target BM T cells to achieve longer graft preservation. BM, thanks to its structure and anatomy, represents an appropriate site for local and selective immunomodulation53 and for the co-transplantation of islets with cells having putative immunomodulatory properties54 such as regulatory T cells55,56 or mesenchymal stem cells,57-59 that could benefit from the close proximity of islet, the target of their tolerogenic function, and from the favorable microenvironment of the BM. Although these strategies seem feasible and promising, a better comprehension of the complex immune mechanisms in BM microenvironment is essential for the identification of specific target for BM T cells.
In conclusion, we showed that the transplant site can influence the immune response against allogeneic islets in the presence of anti-CD3 treatment. Despite many of our data support the idea that the immune reactivity is not different between BM and liver, the immunosuppressive induction regimen appeared less efficient in preventing graft rejection in BM than in liver. This result raises some concerns over the potential of the BM as a site for islet allotransplantation. Further research is warranted to develop immunosuppressive strategies specifically aimed to target BM T cells to prevent allograft rejection.
Elisa Cantarelli conducted this study as partial fulfilment of her Ph.D. in Molecular Medicine, Basic and Applied Immunology Program, San Raffaele University, Milan, Italy.
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