Improving Long‐Term Renal Allograft Survival via a Road Less Traveled By

It has become cliché to state that improvements in early renal allograft survival over the past two decades have not led to increased long‐term renal allograft survival. However, it is not clear how long‐term graft survival can be improved. Here, we present the viewpoint that the road forward does not involve searching for new and more ideal immunosuppressive regimens, but rather detailed patient follow‐up to identify specific causes of late renal allograft loss and the development of new therapy designed to address these problems before allograft damage becomes irreversible.     

Extracellular Hmgb1 Functions as an Innate Immune-Mediator Implicated in Murine Cardiac Allograft Acute Rejection

Hmgb1, an evolutionarily conserved chromosomal protein, was recently re-discovered to be an innate immune-mediator contributing to both innate and adaptive immune responses. Here, we show a pivotal role for Hmgb1 in acute allograft rejection in a murine cardiac transplantation model. Extracellular Hmgb1 was found to be a potent stimulator for adaptive immune responses. Hmgb1 can be either passively released from damaged cells after organ harvest and ischemia/reperfusion insults, or actively secreted by allograft infiltrated immune cells. After transplantation, allografts show a significant temporal up-regulation of Hmgb1 expression accompanied by inflammatory infiltration, a consequence of graft destruction. These data suggest the involvement of Hmgb1 in acute allograft rejection. In line with these observations, treatment of recipients with rA-box, a specific blockade for endogenous Hmgb1, significantly prolonged cardiac allograft survival as compared to those recipients treated with either rGST or control vehicle. The enhanced graft survival is associated with reduced allograft expression of TNFalpha, IFNgamma and Hmgb1 and impaired Th1 immune response.     

CD154 Deficiency Uncouples Allograft CD8+ T‐Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long‐term success of lung transplantation, while T‐cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8⁺ T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8⁺ T cells, as airway rejection has been reported to be CD154‐dependent. Airway allografts from CD154^−/− recipients had significantly lower day 28 OAD scores compared to wild‐type (WT) recipients, and adoptive transfer of CD8⁺ T cells from WT recipients, but not CD154^−/− recipients, were capable of airway rejection in fresh CD154^−/− allograft recipients. Intragraft CD8⁺ T cells from CD154^−/− mice showed similar expression of the surface markers CD69, CD62L^low CD44^high and PD‐1, but markedly impaired IFN‐γ and TNF‐α secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8⁺ T cells from CD154^−/− recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8⁺ T cells required for murine OAD.     

Role of Lentivirus‐Mediated Overexpression of Programmed Death‐Ligand 1 on Corneal Allograft Survival

To investigate the role of lentivirus‐mediated overexpression of programmed death‐ligand 1 (PD‐L1) on rat corneal allograft survival. A fully allogeneic rat cornea transplant model was used for in vivo studies. Lentiviral (LV) vectors are efficient tools for ex vivo genetic modification of cultured corneas. LV vector encoding for PD‐L1 (LV.PD‐L1) and LV vector encoding for eGFP (LV.eGFP, as control) were constructed and tested. PD‐L1 or eGFP expression was increased on corneal cells upon LV.PD‐L1 and LV.eGFP transduction, respectively. Both allogeneic controls and allogeneic LV.eGFP transduced corneas were uniformly rejected (MST: 13.8 ± 1.7 days and 12.3 ± 1.9 days, respectively). In contrast, allogeneic LV.PD‐L1 transduced corneas showed a high percentage (83%) of graft survival (MST > 30 days, n = 5, 15 days, n = 1). Graft opacity of PD‐L1 transduced corneas was present but was significantly reduced compared to control or eGFP expressing corneas. Flow cytometric analysis revealed that percentages of CD3⁺CD8⁺CD161⁺ and CD3⁺CD8⁺CD161^– lymphocytes were decreased in animals receiving LV.PD‐L1 transduced corneas compared to animals grafted with LV.eGFP transduced corneas. Moreover, reduced expression of proinflammatory cytokines (IFN‐γ and IL‐6) in PD‐L1 transduced corneas compared to allogeneic controls was also observed. Local PD‐L1 gene transfer in cultured corneas is a promising approach for the prolongation of corneal allograft survival and attenuation of graft rejection.     

Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

T cell receptor transgenic (TCR‐Tg) T cells are often used as tracer populations of antigen‐specific responses to extrapolate findings to endogenous T cells. The extent to which TCR‐Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR‐Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4⁺ or CD8⁺ TCR‐Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4⁺ TCR‐Tg T cells accelerated rejection and, unexpectedly, led to a dose‐dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4⁺ TCR‐Tg T cells exhibited enhanced endogenous donor‐specific CD8⁺ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4⁺ TCR‐Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4⁺ TCR‐Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4⁺ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.     

IL‐17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC‐Mismatched Renal Allograft Transplantation

IL‐17 is a pro‐inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL‐17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL‐17 in a fully MHC‐mismatched, life‐sustaining, murine model of kidney allograft rejection using IL‐17 deficient donors and recipients (IL‐17^?/? allografts). IL‐17^?/? allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN‐γ and histological attenuation of acute and chronic allograft rejection, as compared to wild‐type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL‐17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL‐17 showed a trend towards prolongation of survival only when recipients were IL‐17^?/?. Administration of a depleting anti‐CD25 antibody to IL‐17^?/? recipients abrogated the survival advantage conferred by IL‐17 deficiency, suggesting the involvement of a CD4⁺CD25⁺ T cell regulatory mechanism. Therefore, IL‐17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

     

Prolongation of Cardiac Allograft Survival by a Novel Population of Autologous CD117+ Bone Marrow‐Derived Progenitor Cells

Autologous CD117⁺ progenitor cells (PC) have been successfully utilized in myocardial infarction and ischemic injury, potentially through the replacement/repair of damaged vascular endothelium. To date, such cells have not been used to enhance solid organ transplant outcome. In this study, we determined whether autologous bone marrow‐derived CD117⁺PC could benefit cardiac allograft survival, possibly by replacing donor vascular cells. Autologous, positively selected CD117⁺PC were administered posttransplantation and allografts were assessed for acute rejection. Although significant generation of recipient vascular cell chimerism was not observed, transferred PC disseminated both to the allograft and to peripheral lymphoid tissues and facilitated a significant, dose‐dependent prolongation of allograft survival. While CD117⁺PC dramatically inhibited alloreactive T cell proliferation in vitro, this property did not differ from nonprotective CD117^? bone marrow populations. In vivo, CD117⁺ PC did not significantly inhibit T cell alloreactivity or increase peripheral regulatory T cell numbers. Thus, rather than inhibiting adaptive immunity to the allograft, CD117⁺ PC may play a cytoprotective role in prolonging graft survival. Importantly, autologous CD117⁺PC appear to be distinct from bone marrow‐derived mesenchymal stem cells (MSC) previously used to prolong allograft survival. As such, autologous CD117⁺PC represent a novel cellular therapy for promoting allograft survival.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

The Introduction of Human Heme Oxygenase‐1 and Soluble Tumor Necrosis Factor‐α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase‐1 (HO‐1) or soluble tumor necrosis factor‐α receptor type I with human IgG₁ Fc (sTNF‐αR‐Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO‐1, sTNF‐αR‐Fc, sTNF‐αR‐Fc/HO‐1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood–derived CD34⁺ stem cells into NOD‐scid‐IL‐2Rγ^null mice. Both HO‐1 and sTNF‐αR‐Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig‐to‐humanized mice transplantation. The sTNF‐αR‐Fc/HO‐1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor‐α and IL‐6 in treatment groups; however, frequency of pig‐specific interferon‐γ–producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO‐1 or sTNF‐αR‐Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO‐1 or sTNF‐αR‐Fc transgenic pigs have potential for islet xenotransplantation.     

Galectin‐1 Prolongs Survival of Mouse Liver Allografts From Flt3L‐Pretreated Donors

Liver allografts are spontaneously accepted across MHC barriers in mice. The mechanisms underlying this phenomenon remain poorly understood. Galectin‐1, an endogenous lectin expressed in lymphoid organs, plays a vital role in maintaining central and peripheral tolerance. This study was to investigate the role of galectin‐1 in spontaneous tolerance of liver allografts in mice, and to evaluate the therapeutic effects of galectin‐1 on liver allograft rejection induced by donor Flt3L pretreatment. Blockade of the galectin‐1 pathway via neutralizing antigalectin‐1 mAb did not affect survival of the liver allografts from B6 donors into C3H recipients. Administration of rGal‐1 significantly prolonged survival of liver allografts from Flt3L‐pretreated donors and ameliorated Flt3L‐triggered liver allograft rejection. This effect was associated with increased apoptosis of T cells in both allografts and spleens, decreased frequencies of Th1 and Th17 cells, decreased expression of Th1‐associated cytokines (IL‐12, IL‐2 and IFN‐γ), Th17‐associated cytokines (IL‐23 and IL‐17) and granzyme B, in parallel with selectively increased IL‐10 expression in liver allografts. In vitro, galectin‐1 inhibited Flt3L‐differentiated DC‐mediated proliferation of allo‐CD4⁺ T cells and production of IFN‐γ and IL‐17. These data provide new evidence of the potential regulatory effects of galectin‐1 in alloimmune responses in a murine model of liver transplantation.     

De Novo Donor‐Specific HLA Antibodies Decrease Patient and Graft Survival in Liver Transplant Recipients

The role of de novo donor‐specific HLA antibodies (DSA) in liver transplantation remains unknown as most of the previous studies have only focused on preformed HLA antibodies. To understand the significance of de novo DSA, we designed a retrospective cohort study of 749 adult liver transplant recipients with pre‐ and posttransplant serum samples that were analyzed for DSA. We found that 8.1% of patients developed de novo DSA 1 year after transplant; almost all de novo DSAs were against HLA class II antigens, and the majority were against DQ antigens. In multivariable modeling, the use of cyclosporine (as opposed to tacrolimus) and low calcineurin inhibitor levels increased the risk of de novo DSA formation, while a calculated MELD score >15 at transplant and recipient age >60 years old reduced the risk. Multivariable analysis also demonstrated that patients with de novo DSA at 1‐year had significantly lower patient and graft survival. In conclusion, we demonstrate that de novo DSA development after liver transplantation is an independent risk factor for patient death and graft loss.     

Role of T Cell Recruitment and Chemokine‐Regulated Intra‐Graft T Cell Motility Patterns in Corneal Allograft Rejection

Keratoplasty is the primary treatment to cure blindness due to corneal opacification. However, immune‐mediated rejection remains the leading cause of keratoplasty failure. Here, we utilize an in vivo imaging approach to monitor, track, and characterize in real‐time the recruitment of GFP‐labeled allo‐specific activated (Bonzo) T cells during corneal allograft rejection. We show that the recruitment of effector T cells to the site of transplantation determined the fate of corneal allografts, and that local intra‐graft production of CCL5 and CXCL9/10 regulated motility patterns of effector T cells in situ, and correlated with allograft rejection. We also show that different motility patterns associate with distinct in vivo phenotypes (round, elongated, and ruffled) of graft‐infiltrating effector T cells with varying proportions during progression of rejection. The ruffled phenotype was characteristic of activated effectors T cells and predominated during ongoing rejection, which associated with significantly increased T cell dynamics within the allografts. Importantly, CCR5/CXCR3 blockade decreased the motility, size, and number of infiltrating T cells and significantly prolonged allograft survival. Our findings indicate that chemokines produced locally within corneal allografts play an important role in the in situ activation and dynamic behavior of infiltrating effector T cells, and may guide targeted interventions to promote graft survival.     

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4⁺CD25⁺Foxp3⁺ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4⁺CD25⁺Foxp3⁺ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG^?/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4⁺CD25⁺Foxp3⁺ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4⁺CD25⁺Foxp3⁺ iTregs were further able to induce endogenous naïve T cells to convert to CD4⁺CD25⁺Foxp3⁺ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro‐generated donor‐specific CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.     

A Novel,Blocking, Fc‐Silent Anti‐CD40 Monoclonal Antibody Prolongs Nonhuman Primate Renal Allograft Survival in the Absence of B Cell Depletion

CD40–CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti‐CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti‐CD40 antibody can still promote graft survival. The parent anti‐CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC‐mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc‐silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98–100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well‐tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc‐silent anti‐CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.     

The Impact of C4d Pattern and Donor‐Specific Antibody on Graft Survival in Recipients Requiring Indication Renal Allograft Biopsy

We examined the pattern of PTC C4d by immunohistochemistry and DSA in 297 kidney recipients with indication biopsies, and evaluated their predictive value for graft survival. Median biopsy time was 5.1 months posttransplant. Patients were followed for 17.9 ± 9.4 months postbiopsy. An 18.5% had focal and 15.2% had diffuse C4d, with comparable graft survival (adjusted graft failure HR: 2.3, p = 0.001; HR:1.9, p < 0.02, respectively). 31.3% were DSA+, 19.5% class I and 22.9% class II DSA. Only those with class II DSA had worse outcome (adjusted HR:2.5, p = 0.001 for class II only; HR:2.7, p < 0.001 for class I/II DSA). Among patients with <10%C4d, 23.9% had DSA, compared to 68.9% with diffuse staining. For patients biopsied in first‐year posttransplant presence of DSA, regardless of C4d positivity in biopsy, was a poor prognostic factor (adjusted graft failure HR: 4.2, p < 0.02 for C4d?/DSA+; HR:4.9, p = 0.001 for C4d+/DSA+), unlike those biopsied later. We have shown that focal C4d had similar impact on graft survival as diffuse pattern. During the first‐year posttransplant either class I or II DSA, and afterward only class II DSA were associated with worse graft survival. DSA was predictive of worse outcome regardless of C4d for patients biopsied in first year and only with C4d positivity afterward, supporting the importance of assessment of both DSA and C4d pattern in biopsy.     

Pancreas Allograft Biopsies with Positive C4d Staining and Anti‐Donor Antibodies Related to Worse Outcome for Patients

C4d+ antibody‐mediated rejection following pancreas transplantation has not been well characterized. Therefore, we assessed the outcomes of 27 pancreas transplantation patients (28 biopsies), with both C4d staining and donor‐specific antibodies (DSA) determined, from a cohort of 257 patients. The median follow‐up was 50 (interquartile range [IQR] 8–118) months. Patients were categorized into 3 groups: group 1, patients with minimal or no C4d staining and no DSA (n = 13); group 2, patients with either DSA present but no C4d, diffuse C4d+ and no DSA or focal C4d+ and DSA (n = 6); group 3, patients with diffuse C4d+ staining and DSA (n = 9). Active septal inflammation, acinar inflammation and acinar cell injury/necrosis were significantly more abundant in group 3 than in group 2 (respective p‐values: 0.009; 0.033; 0.025) and in group 1 (respective p‐values: 0.034; 0.009; 0.002). The overall uncensored pancreas graft survival rate for groups 1, 2 and 3 were 53.3%, 66.7% and 34.6%, respectively (p = 0.044). In conclusion, recipients of pancreas transplants with no C4d or DSA had excellent long‐term graft survival in comparison with patients with both C4d+ and DSA present. Hence, C4d should be used as an additional marker in combination with DSA in the evaluation of pancreas transplant biopsies.