Transplantation of solid organs currently relies upon chronic immunosuppressive (IS) agents to prevent graft rejection. These agents have significant cost, side effects, and toxicities, highlighting the need to investigate transformational approaches that promote full IS withdrawal and donor-specific tolerance.1 Interestingly, the liver appears to be the most immunoregulatory organ transplanted. Donor-specific immunoregulatory effects, clonal deletion, alloantibody dilution, circulating and resident regulatory T (Treg) cells, Vδ1 γδ T cells, tolerogenic dendritic cells (DC), and donor-recipient hematopoietic chimerism are components of liver immunoregulation.2-15 This is seen clinically by the lower importance of HLA matching, lower rejection incidence, reduced IS required, and the immunological protection conferred by the liver on other organs. Although the percentage of liver transplantation (LT) recipients able to undergo simple IS withdrawal is the highest of all organ recipients, the rates are still clinically unacceptable and low when attempted early after LT, emphasizing the need for more optimal strategies.16-19
Hematopoietic stem cell (HSC) infusion leading to chimerism is the only tolerance-inducing approach that has been successfully achieved in all species. However, the widespread clinical application of HSC in organ transplantation has been constrained by graft-versus-host disease (GVHD), the need for HLA matching, and the toxicity of recipient conditioning.20-22 Our group recently developed a protocol to induce tolerance and durable chimerism without GVHD using a donor facilitating cell product at the time of mismatched living donor kidney transplantation.23 However, the clinical application of these approaches are not as practical or safe in the LT setting, given the toxicities of conditioning sick liver failure patients at the time of LT and that most LT are performed with deceased donors. Thus, delaying induction of tolerance after recipients have recovered postoperatively, in a period of clinical and immunological quiescence, would be more desirable and could be extended to both living and deceased donor recipients.
Since first reported in the early 1970s, rat LT models have been widely used to investigate mechanisms of liver rejection and tolerance induction.24-26 Liver allografts from PVG (RT1c) or Lewis (RT1l) are spontaneously accepted when transplanted into Dark Agouti (DA, RT1a) and are referred to as low-immune responder combinations.27,28 On the contrary, high-immune responder combinations such as ACI (RT1av) or DA to Lewis transplants develop robust acute rejection,29,30 making them more relevant models for investigations of therapeutic interventions. Several strategies have tested donor-derived cell infusions before or at LT in these preclinical models, such as bone marrow (BM),31,32 mesenchymal stem cells,33,34 blood transfusion,35 Treg cells,36 and immature DC.37,38 While these strategies prolonged graft survival (mostly only up to 100 days post-LT), their effects on longer-term graft protection and the phenotypic immune regulation have not been thoroughly examined. Furthermore, it has not been investigated whether tolerance induction could be achieved in delayed fashion.
Our goal was therefore to adapt our successful kidney transplant protocol to the LT setting in a novel, delayed approach, first in a preclinical high-immune responder animal model (this work) to eventually translate to human early phase clinical trials.
MATERIALS AND METHODS
Ten- to 14-week-old (280-350 g) male Lewis (RT1l), ACI (RT1av1) and BN (RT1n) rats were used for this study. The breeder rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and were bred and maintained in the specific-pathogen-free facility at Center for Comparative Medicine of Northwestern University. All the surgical procedures were approved by the institutional animal care and use committee of Northwestern University.
Orthotopic LT and Postoperative Monitoring
Liver grafts from ACI rats were transplanted orthotopically into allogeneic rats using a modified 2-cuff technique similar to previously described.29,39 Briefly, liver grafts were harvested and perfused thoroughly via the portal vein with cold heparinized (50 U/mL) lactated Ringer solution. The recipient's native liver is removed and followed by the suprahepatic vena cava anastomosis with a 10-0 running suture. The cuff technique was used for anastomosis of both the intrahepatic vena cava and the portal vein. The graft arterial supply was not reconstructed. The common bile duct reconstruction was achieved by using a polyethylene stent. The recipients were monitored daily for clinical signs and symptoms of allograft rejection, which include a gradual weight loss, hunched posture, ruffled fur, reduced mobility, and jaundiced skin. Blood samples were collected periodically for multi-lineage phenotyping, and liver function analysis (by Charles River Laboratories, Wilmington, MA). The day of transplantation was referred to as day 0, where the endpoint of study was defined as greater than 180 postoperative days (POD) of survival, recipient death or when recipients developed clinical signs of liver failure from rejection. Tissues samples were collected at the end of the study for histological examination. Additional transplant recipients were euthanized at preselected earlier time points for sequential functional, immunological, and histological analysis.
BM Cell Isolation and Infusion
Hind leg bones were harvested from anesthetized and euthanized ACI rats and BMCs were collected based on standard procedures.40 The BMCs were manipulated by T cell depletion by using pan T cell MicroBeads according the manufacturer's instruction (Miltenyi Biotec, San Diego, CA). For BM infusion (BMI), recipients received an intravenous infusion of BMCs (100 × 106) at POD24 through penile vein injection under anesthesia with a 25-gauge needle.
Immunosuppression Regimens and Experimental Design
Immunosuppression (IS) regimens included (1) Tacrolimus (TAC) (Astellas Pharma US, Inc.), 1 mg/kg, daily subcutaneously from day 0 to POD21, then followed by once every other day, (2) T cell depletion with anti-rat T cell receptor (TCRαβ) monoclonal antibody (TCR mAb, Clone: R73, BD Pharmingen, San Diego, CA),41-43 500 μg/each, intravenously at POD22 and (3) total body irradiation (TBI) in which rats were exposed to a 60Co[γ]-ray source at a single dose of 300 cGy at POD23.
Recipients were randomly selected into the groups as listed in Table 1. The overall treatment protocol and follow-up is shown in Figure 1A. All rats, except syngeneic transplanted (group 1) and nonimmunosuppressed rejection (group 2), received TAC. At 3 weeks post-LT, 3 groups underwent: (group 3) TAC withdrawal alone at 4 weeks, once every other day until the endpoint; (group 4) nonmyeloablative conditioning (mAb + TBI) followed by TAC withdrawal; (group 5) nonmyeloablative conditioning + donor BMI (100 × 106 T cell–depleted BM cells) followed by TAC withdrawal.
In long term surviving recipients, skin grafts from either liver donors (ACI) or third-party Brown Norway strains were performed at 20 weeks post-LT to confirm donor-specific unresponsiveness. For skin transplantation, 2 square graft beds (approximately 1 cm2) were prepared by removing shaved rat skin. Full thickness skin grafts of similar size were placed onto the rat graft beds. The animals were monitored for skin survival for an additional 6 weeks after skin transplantation. The endpoint of skin graft rejection was defined as three fourths of the skin graft exhibited dark discoloration, scabbing, necrotic degeneration, or detachment.
Donor-Specific Antibody Analysis
Donor specific antibodies (DSAs) were measured as described previously.44 Splenic lymphocytes from naïve ACI donors were isolated and the cells (0.5 × 106/tube) were first blocked with 0.5 μg Fc blocker (BD Biosciences, San Jose, CA) followed by incubation with plasma samples from recipients (1:10 dilution) for 1 hour at 4°C. Subsequently, cells were washed and stained with PE-conjugated mouse antirat CD45R, FITC-conjugated goat antirat IgG (BioLegend, San Diego, CA), and FITC-conjugated mouse antirat IgM, IgG1, IgG2a, IgG2b (BD Biosciences) Abs for 35 minutes in dark at 4 °C, respectively, and then analyzed by FACS. Levels of DSAs were represented by mean fluorescence intensity (MFI). Negative controls were provided by incubation with naïve rat sera.
Cell Isolation and Immunophenotyping
Leukocytes were isolated from serially collected peripheral blood samples, spleens, or liver grafts. Spleen cells were also isolated using standard methods. To obtain single cell suspensions from liver allografts, livers were cut into small pieces (2 mm) and digested with collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ). The resulting cell suspension was run through a 70-μm filter and washed with PBS. After centrifugation, the leukocytes were purified using lymphocyte separation medium (Fisher Scientific, Pittsburgh, PA). Phenotypical analysis was performed by using several panels of fluorescein-labeled antirat monoclonal antibodies: panel 1: anti-RT1A[a,b]-FITC, anti-CD4-PE, anti-CD3-antigen-presenting cell (APC) (BD Biosciences); panel 2: anti-RT1A[a,b]-FITC, anti-CD8-PE, anti-CD3-APC (BD Biosciences); panel 3: anti-RT1A[a,b]-FITC, anti-CD45R-PE (BD Biosciences), anti-CD11b/c-APC (Biolegend); panel 4: anti-RT1A[a,b]-FITC, anti-CD161-APC (Biolegend). To evaluate the level of mixed chimerism, the antirat RT1A[a, b] antibody was used to distinguish donor cells (ACI) from recipient cells (Lewis) (Figure S1, SDC,https://links.lww.com/TP/B404). Rat IgG2b, κ was used as an isotype control. Frequencies of Treg cells were determined by using APC conjugated CD4, FITC conjugated-CD25, and PE conjugated-Foxp3 (Biolegend) monoclonal antibodies according to the manufacturer's instructions. Cell staining was performed with the antibodies above (1 μg/106 cells) at 4°C for 30 minutes, then washed and analyzed by FACS (BD Biosciences).
All the recipients were euthanized at the endpoint of liver failure/rejection or POD180 for histopathology. At the endpoint of study, liver samples were preserved in phosphate buffered 10% formalin for 24 hours, then transferred into 70% ethanol for further procession including paraffin embedding and section. Serial sections (4 μm thickness/each section) were stained with hematoxylin-eosin (H&E) at the Northwestern mouse histology core. H&E-stained liver sections were examined under light microscopy by a hepatopathologist (G-Y.Y) who was blinded to all other animal data. Histopathological features of abnormal graft histology and acute rejection were assessed with established criteria.45
Mixed Lymphocyte Reactions
Mixed lymphocyte reactions (MLRs) were set up using 1 × 105 T cells (responders, purity > 85%) enriched from peripheral blood naïve or transplanted Lewis recipients. Pan T cell MicroBeads (Miltenyi Biotec, San Diego, CA) was used for T cell purification. 3 × 105 irradiated ACI (donor) or BN (third party) APCs per well from peripheral blood cells were assed to responder T cells and cocultured for 5 days in complete DMEM medium with 10% heat-inactivated FBS. Proliferation was assessed by 5,6-carboxyfluoresceine diacetate succinimidyl ester (CFSE) (Molecular Probes, Life Sciences) dilution. The proliferating rate of responder T cells were determined by FACSCalibur, and reported as percentage of CD4+ or CD8+ cells with decreased fluorescence intensity.
All data are expressed as the mean ± SD. Comparisons between graft survival times were accomplished by using Kaplan-Meier survival curves with the log-rank test. Statistical significances between 2 groups were determined by Wilcoxon nonparametric tests or by an unpaired Student t test. A P value less than 0.05 was considered statistically significant.
Delayed Donor BMI Facilitates Long-Term Liver Allograft Survival
Recipients were untreated or treated with the IS regimens as indicated in Figure 1A or Table 1. While syngeneic grafts (Lewis to Lewis transplants, n = 10) survived indefinitely with normal liver function and histology, untreated ACI to Lewis allografts (n = 8) succumbed to fulminant liver dysfunction within 12 PODs indicated by significantly elevated levels of alanine transaminase (ALT), aspartate transaminase (AST), bilirubin, and typical features of acute rejection including massive mixed mononuclear cell infiltration, diffuse hepatocyte necrosis and lobular disorganization (Figures 1B-D). As expected, treatment with TAC alone prolonged the allograft survival, but after TAC withdrawal (n = 10), the recipients experienced weight loss, jaundice, significantly elevated ALT, AST, and bilirubin, acute rejection on histology, and 60% graft loss occurred by POD180 (Figures 1B-D). Conditioning with TCR/TBI alone resulted in 78% long-term survival (POD180); however, all recipients (n = 9) displayed impaired liver function and somewhat milder degrees of acute rejection on histology (Figures 1B-D). In contrast, all recipients treated with the conditioning + BMI (n = 11) had long-term TAC-free survival with preserved liver function and graft histology (Figures 1B-D).
Parallel to the histological findings, phenotypical analysis revealed that BMI had significantly reduced numbers of recipient inflammatory cells in the spleen and peripheral blood as well as intragraft infiltrates including T cells (CD3+), myeloid cells (CD11b/c), and NK cells (Figure S2, SDC, https://links.lww.com/TP/B404).
Donor-Specific Tolerance Is Achieved By Delayed Donor BMI
To determine whether the tolerance achieved by delayed BMI was donor specific, we performed 2 sets of experiments. We first performed ex vivo allogenic MLR analyses using APCs isolated from either donor or third-party spleens as stimulators, and T cells enriched from PBMCs of long-term surviving recipients as responders. As shown in Figures 2A and B, CD4+ and CD8+ T cells from BMI-treated recipients exhibited significantly lower levels of proliferation in response to APCs, compared with T cells from TAC withdrawn and TCR/TBI conditioned groups (P < 0.001 for both vs the other groups). However, the response of BMI treated T cells to third-party APCs was preserved. These data confirm that the delayed BMI compromised T cell recall responses only to the donor.
To further examine donor-specificity, we transplanted skin grafts from donor-type (ACI) or third-party (BN) rats onto long-term surviving Lewis recipients (n = 3) at the 20th week posttransplant. Although all third-party skin allografts were rejected by day 21, donor-matched skin allografts remained normal in appearance until the end of point of study (6 weeks post skin transplant) in the BMI-treated recipients, supporting a state of donor-specific tolerance (Figures 2C and D).
Delayed Donor BMI Inhibits De Novo Production of Donor-Specific Antibodies
To test our hypothesis that delayed BMI down-regulates DSA production, we analyzed DSA levels in serial plasma samples collected from the allografts using flow cytometry. Our results revealed that DSAs including IgM, total IgG (Figure 3A) and subtypes IgG1, IgG2a, IgG2b (Figure 3B) were significantly increased as early as POD7, compared with the naïve rats and isografts, suggesting that liver allografts trigger an early, robust DSA response. Treatment with TAC significantly reduced DSA, both IgG and IgM production of the untreated allograft to the levels comparable to the isografts at POD7. However, on POD90 and 150, DSA levels (both IgG and IgM) increased significantly in TAC withdrawn groups. Unexpectedly, TCR/TBI conditioning triggered the most robust production of DSAs as indicated by the highest levels of IgM, IgG, and its subtypes IgG1, IgG2a, IgG2b on POD90. In contrast, delayed BMI profoundly decreased level of DSA at both POD90 and POD150 and significantly attenuated the impact of TCR/TBI induced DSA (Figures 3A and B). Collectively, these data suggest that delayed BMI significantly downregulates B cell and plasma cell responses.
Delayed Donor BMI Increases the Frequency of CD4 + CD25 + FoxP3+ Treg Cells
To determine whether our delayed tolerance induction approach promotes Treg cell generation, we performed a serial analysis of peripheral blood Treg cell frequencies after delayed BMI. As in Figures 4A and B, a significantly higher frequency of peripheral circulating CD4+CD25+FoxP3+ Treg cells was observed in BMI recipients compared to that in TAC withdrawn or TCR + TBI recipients (P < 0.05), starting from POD90; this persisted throughout the study. We then further examined whether delayed BMI influenced the accumulation of Treg cells in spleens and liver grafts on POD180. The results shown in Figure 4C revealed that recipients from BMI group exhibited high levels of CD4+CD25+FoxP3+ populations in liver grafts (P < 0.05 vs. TAC withdrawn and TCR/TBI) as well as spleen tissue (P < 0.01 vs. TAC withdrawn and TCR/TBI). These data suggest that up-regulation of CD4+CD25+FoxP3+ Treg cells may have a role in the tolerance induced by the delayed BMI.
Increased Mixed Chimerism Is Associated With Delayed Donor BMI-Induced Tolerance
Donor-derived cells regularly persist in liver-grafted patients for some time and may exert beneficial immunomodulatory properties.46-49 Studies have reported that maintenance of immune tolerance after organ transplantation may be due to mixed chimerism after the infusion of allogeneic BM or HSC.50,51 In our experiments, using an anti-donor MHC class I antibody (RT1A[a,b]), we found that all recipients developed early mixed chimerism (POD14) after LT (Figure 5A), likely related to migration of donor leukocytes out of the liver into the circulation under TAC treatment.52-54 The delayed BMI significantly increased the percentage of this donor-derived population, which peaked on POD45 (3 weeks after BMI) (38.47% ± 2.25%, P < 0.001), compared with recipients of TAC withdrawn (21.08% ± 2.11%) or TCR + TBI conditioning (26.54% ± 0.94%). On POD60, recipients in BMI, TCR + TBI and TACwd groups exhibited mean donor chimerism of 30.67% ± 1.22% (P < 0.001, vs. TACwd; P < 0.01, vs. TCR + TBI), 24.02% ± 1.73% (P < 0.05, vs. TACwd) and 17.54% ± 1.06%, respectively. Although no statistical difference was found in chimerism between BMI group and TCR + TBI group on POD120, POD150 and POD180, recipients with BMI still had significant higher chimerism than TAC withdrawal alone recipients on POD150 (P < 0.05) and POD180 (P < 0.05) (Figure 5A).
Further analysis revealed that donor cell populations primarily consisted of 3 cell subsets: CD11b/c+, CD161low and CD161high (Figure 5B). Recipients with BMI had significant higher percentages of chimeric CD11b/c+ (88.63% ± 1.03%) and CD161low (87.28% ± 1.24%) than the TAC withdrawal (78.70% ± 1.67%, P < 0.001; 79.90% ± 1.92%, P < 0.05) and TCR + TBI (56.58% ± 8.42%, P < 0.01; 56.58% ± 8.42%, P < 0.01) groups. We then calculated absolute cell numbers of chimeric-CD11b/c+ and CD161low and found that recipients with BMI had the highest level in the peripheral blood (5.91 ± 0.22 × 106 and 5.86 ± 0.13 × 106 cells per mL, respectively) (Figure 5C). We further found that CD161lowCD11b/c+ cells represented the major population (>90%) in the context of donor-recipient chimerism.
Immunosuppression weaning could address the long term toxicities and cost of IS therapy, although this is mainly only achievable in older patients further out from LT in which the clinical benefit may be negligible.16,17 Therefore, novel immune manipulation approaches are needed to induce tolerance earlier after LT.55 Such strategies have been attempted previously at the time of kidney transplantation and include conditioning regimens followed by donor hematopoietic stem cell transplantation or BMI.20-23 However, they might have even more success and benefit in LT recipients given the tolerogenic environment, less IS required, and the high degree of IS toxicity in this population. That said, even the lower intensity conditioning regimens may be too toxic at the time of LT in patients with liver failure. With this rationale, we have herein demonstrated the efficacy of a reduced intensity conditioning/BMI approach in inducing delayed donor-specific tolerance after LT. If our delayed, lower conditioning approach could be translated to future clinical trials, it might be safer and more applicable to a larger pool of LT recipients.
We also analyzed for differences in chimerism, cell populations, and donor-specific antibodies that may provide clues as to why tolerance was only achieved after BMI, expanding extensively on previous donor-cell infusion studies.31-38 Consistent with the known phenomenon of donor hematopoietic cells entering the periphery after LT,52-54 all recipients displayed an early mixed chimerism (POD14) after LT, while non–BMI-infused recipients undergoing TAC withdrawal with or without conditioning had significantly lower percentages of chimerism that ultimately was associated with graft rejection. In contrast, after delayed BMI, donor chimerism was augmented and remained higher compared to control groups throughout the follow up course, correlating with the lack of rejection or GVHD with IS withdrawal. This indicates that some degree of mixed chimerism may be important in achieving a balance between rejection and GVH responses with early IS withdrawal in LT, as opposed to other organs in which higher degrees of chimerism are needed.23
We also found that the majority of peripheral circulating chimeric cells were myeloid (CD11c/b+ CD161+) and less CD3+ T cell derived. While the origins, phenotypes and precise roles of these chimeric myeloid cells remains to be further elucidated, we speculate that these chimeric cells were derived from both infused donor BM stem cells and liver resident progenitors. Myeloid-derived suppressor cells have been shown to induce Treg cell expansion and inhibit T cell proliferation by down-regulating the TCRζ chain.56,57 Treg cells inhibit activation and proliferation of alloreactive donor and recipient T cells, thereby reducing GVHD and rejection.58-60 Not surprisingly, we found that CD4+CD25+FoxP3+ Treg cells expanded significantly in the blood, liver graft and spleen in BMI versus controls. Our next step is to specifically test the importance of Tregs and chimeric myeloid cells in delayed BMT tolerance, such as breaking tolerance with cell depletion or testing their suppressive capacity in vitro and in vivo.
Humeral responses involving B cells, plasma cells and DSA have also been associated with graft rejection in organ recipients but only recently implicated in LT graft injury.61,62 One concern in delaying tolerance induction is that donor alloantigens may have already presensitized the recipient.63 However, flow cytometry performed immediately post-LT demonstrated reduced B cell responses and low level of DSA in all recipients on TAC. Thus, standard IS may promote immunological quiescence that sets the stage for delaying tolerance induction. However, later TAC withdrawal led to DSA resurgence and rejection. In addition, TCR/TBI alone increased DSA generation which has been demonstrated in previous studies. Khiong et al64 demonstrated that lymphopenia-induced expansion of CD4+ T cells can induce systemic autoimmunity coinciding with autoantibody production. Iida et al65 found that transient lymphopenia induced by TBI breaks costimulatory blockade-based tolerance and correlates with the accumulation of B cells in lymph nodes and increased DSA titers. Thus, the increased DSA observed in TCR/TBI alone in our study may be due to transient lymphopenia-induced alloreactive T cell activation and B cell expansion. Interestingly, this was not seen in BMI-treated recipients undergoing TAC withdrawal who had low DSA levels and excellent graft function, histology and survival, similar to isografts. This underscores the importance of minimizing DSA in establishing tolerance even in LT, and approaches such as BMI may promote these mechanisms.
Finally, our BMI-treated recipients developed donor-specific hyporesponsiveness, which may be the most reliable marker of long-standing tolerance.66 After co-culture with donor APCs, recipient T cells in BMI-treated recipients had significantly lower proliferation compared to the non-BMI recipients, while all displayed responses to third-party APCs. These results support previous studies showing lymphocytes from long-term acceptors of kidney or vascularized composite allografts were tolerant to donor APCs but responsive to third-party cells.63 We also demonstrated that BMI-treated LT recipients accepted donor skin grafts but immediately rejected third-party skin, compared to the other groups who rejected all skin grafts.
Our approach is not the first to examine the use of hematopoietic cells in inducing LT tolerance in animal and clinical models but has several aspects that are distinct. First, the delayed approach of inducing tolerance after donor-recipient recognition has not been previously demonstrated and BMI may be required to achieve robust tolerance, as simple TAC withdrawal with or without conditioning led to rejection. Other small clinical series have utilized bone marrow or stem cell infusion immediately or several years after LT, rather than an earlier delayed approach.67,68 Second, tolerance with chimerism and absence of GVHD was seen with our T cell–depleted BMI. Similar findings were seen in our center’s living donor kidney transplant facilitating cell tolerance study.23 Whether this will be translatable to human LT is not known, but these preliminary data will hopefully lead to future clinical investigation given the greater applicability of this approach in LT. Finally, we analyzed cell populations, chimerism, antibodies, and donor-specific alloreactivity together at key study time points. That said, we do not know which of these cells or components are most important to the development of delayed tolerance.
Therefore, the next critical step is to focus in on the key mechanisms of tolerance in our model and more specifically determine if Tregs and chimeric myeloid cells are indeed necessary components. We speculate that augmentation of chimerism generates a tolerogenic environment that promotes host hypo-responsiveness via expansion of regulatory cells and reduction of donor-specific antibodies. Future studies need to test the hypothesis that depleting regulatory cells after conditioning + BMI will break tolerance and lead to rejection or GVHD. Clarifying this might have applications to tolerance approaches in clinical LT, given the increasing utilization of immunoregulatory cells for immune monitoring and as cell therapy.
The authors recognize the Northwestern University Microsurgery Core for performing the surgeries and also thank the Northwestern University Mouse Histology and Phenotyping Laboratory for tissue processing and histology.
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