Prevention Of Chronic Rejection Research Articles

Low Dose IL-2 Therapy Induces T Regulatory Cell Derived Circulatory Exosomes Containing PDL1 and CD73 and Abrogates Development of Chronic Cardiac Allograft Rejection

<h3>Purpose</h3> Aim of the study is to determine the effect of low dose interleukin-2 (IL-2) therapy on the kinetics of induction of circulating exosomes and their immunological properties resulting in prevention of chronic rejection following allogeneic murine cardiac transplantation (HTx). <h3>Methods</h3> BALB/c (H2^d) to C57BL/6 (H2^b) HTx was performed heterotopically in the abdominal cavity. Costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2) was administered to induce tolerance. IL-2 (2000IU/day) was administered from day 14 post-transplant for 3 weeks. Sera were collected on days 7-100 for analysis of exosome induction and characterization of cardiac self-antigens (SAgs) (Myosin, Vimentin) by western blot. Foxp3, PDL1, CD73 in exosomes were detected using aldehyde bead - based flow cytometry. Antibodies (Abs) to donor MHC and SAgs were detected using immunofluorescence and ELISA respectively. <h3>Results</h3> BALB/c to C57BL/6 HTx acutely rejected (AR) with median survival time of 8 days. Costimulatory blockade prevented AR but developed chronic rejection with mean of 42 days circulating exosomes contained significantly higher levels of Myosin and Vimentin in the exosomes day 15 (p<0.05) and day 30 (p<0.01). Abs to MHC (32±13.8% vs 2±1.1; p<0.001) was detected on day 30, whereas Abs to Myosin (1114.21±443.4 ng/ml; p<0.0001) and Vimentin (519±241 ng/ml; p<0.0001) were detected on day45 during chronic rejection with fibrosis (57.1±20.7 vs 2.3±1.7%). Low dose IL-2 significantly prolonged the survival, 115 vs 42 (p<0.001) and did not induce exosomes with cardiac SAgs or developed Abs to MHC or SAgs. Low dose IL-2 not only abrogated chronic rejection but also induced splenic CD4+CD25+ Foxp3 T regulatory (Treg) cells in the allograft by day 45. Flow cytometry of circulatory exosomes isolated in day 45 also demonstrated increased Foxp3+ PDL1+ (3.3 fold) and FoxP3+ CD73+ (3 fold) compared to exosomes isolated on day 45 from chronic rejecting animals. <h3>Conclusion</h3> Our results demonstrate that low dose IL-2 abrogated the development of chronic rejection following HTx and induced circulating Treg cells derived exosomes with PDL1 and CD73 along with splenic Tregs and graft infiltrating FoxP3 cells.

Risks and Benefits of Surveillance Transbronchial Biopsy after Lung Transplantation: A Single Center Experience

Acute rejection (including minimal grade A1), is a potential risk factor associated with chronic rejection. Although surveillance transbronchial biopsies (TBBs) are routinely offered to lung recipients in most centres, a favourable effect on survival and prevention of chronic rejection has not been clearly demonstrated. In this study we present the results of our surveillance program lasting for 4 years.

Prevention of chronic rejection after lung transplantation.

Long-term survival after lung transplantation (LTx) is limited by chronic rejection (CR). Therapeutic strategies for CR have been largely unsuccessful, making prevention of CR an important and challenging therapeutic approach. In the current review, we will discuss current clinical evidence regarding prevention of CR after LTx.

Organ Preservation : Milestones and Basic Principles

Current shortage in organ donors led to the expansion of criteria for organ donation placing organ preservation as one cornerstone for successful transplant, graft function and survival. The historical work of Belzer and Collins paved the way for key descriptions of physiopathology of cell ischemia and protection (cytokines roles, oxidative stress, energy shift to lactic acidosis and perfusion pressure changes). Good preservation means immediate recovery of function and prevention of chronic rejection. Two cooling approaches are available: static (SCS: simple cold storage) suitable for all organs, and dynamic (HMP: hypothermic machines perfusion) designed for kidneys and liver. A thorough discussion of historically manufactured and widely sold preservation solutions e.g. EuroCollins, UW solution (Viaspan®) as well as current used solutions e.g. Custodiol® and the new Celsior is available in this review. Obviously, every single organ exhibits different tolerance to warm and cold ischemia depending on its nature and demands after transplant. Future perspectives of organ preservation may be hidden in hibernators which may hold the enigmas of perfect human organ preservation.

Role of Macrophage Subsets in Chronic Allograft Rejection and Novel Strategies to Target Them.

chronic rejection or transplant vasculopathy is a major problem in the clinic hindering long-term transplant survival. Chronic rejection primarily targets the graft vasculature, but the exact mechanism underlying the slow and progressive vasculature damage in transplanted organs remains poorly defined. In the present study, we addressed the role of macrophages in chronic allograft rejection in a mouse heart transplant model. We found that treatment of B6 recipients with a single dose of CTLA-4Ig prolonged the survival of Balb/c heart allografts(MST=35d) as compared to untreated controls (MST=8 days). Histologically, the CTLA-4Ig treated grafts showed signs of extensive chronic rejection characterized by heavy leukocyte infiltration and prominent neointima formation. Phenotypically, macrophages were a key infiltrating cell type in the vascular lesions in the grafts. Further studies revealed a M2 (F4/80+CD206+) phenotype for grafting infiltrating macrophages. An increased number of M2 macrophages were also detected in the spleen of recipient mice that experienced chronic rejection. We polarized macrophages in vitro to M1 and M2 subsets and systemically screened for differences and specific pathways that could be targeted for inhibition of M1 and M2 macrophages. We showed that when macrophages were polarized to a M2 phenotype, they preferentially expressedP2x7 receptor, a member of P2x family receptors that sense metabolites. We also found that oxidized ATP (P2x7R inhibitor)could effectively inhibit M2 induction in vitro. Importantly, in the heart transplant model, CTLA-4Ig plus oxidized ATP induced long-term heart allograft survival(MST>100 days), which was associated with markedly reduced neointima generation in the grafts, and prevention of chronic rejection was associated with significant inhibition of M2 macrophages in situ. Our studies identified a previously unknown mechanism of transplant vasculopathy and pinpointed as a molecular target in the inhibition of infiltrating M2 macrophages and chronic rejection.

Functional characterization of late outgrowth endothelial progenitor cells in patients with end‐stage renal failure

Renal transplantation is potentially curative in renal failure, but long-term efficacy is limited by untreatable chronic rejection. Endothelial damage contributes to chronic rejection and is potentially repairable by circulating endothelial progenitor cells (EPC). The frequency and function of EPC are variably influenced by end-stage renal failure (ESRF). Here, we isolated and functionally characterized the late outgrowth EPC (LO-EPC) from ESRF patients to investigate their potential for endothelial repair. Patients with ESRF generated more LO-EPC colonies than healthy controls and had higher plasma levels of IL-1rα, IL-16, IL-6, MIF, VEGF, Prolactin, and PLGF. Patients' LO-EPC displayed normal endothelial cell morphology, increased secretion of PLGF, MCP-1, and IL-1β, and normal network formation in vitro and in vivo. They demonstrated decreased adhesion to extracellular matrix. Integrin gene profiles and protein expression were comparable in patients and healthy volunteers. In some patients, mesenchymal stem cells (MSC) were co-isolated and could be differentiated into adipocytes and osteocytes in vitro. This is the first study to characterize LO-EPC from patients with ESRF. Their behavior in vitro reflects the presence of elevated trophic factors; their ability to proliferate in vitro and angiogenic function makes them candidates for prevention of chronic rejection. Their impaired adhesion and the presence of MSC are areas for potential therapeutic intervention.

B-lymphocyte homeostasis and BLyS-directed immunotherapy in transplantation

Current strategies for immunotherapy after transplantation are primarily T-lymphocyte directed and effectively abrogate acute rejection. However, the reality of chronic allograft rejection attests to the fact that transplantation tolerance remains an elusive goal. Donor-specific antibodies are considered the primary cause of chronic rejection. When naive, alloreactive B-cells encounter alloantigen and are activated, a resilient "sensitized" state, characterized by the presence of high-affinity antibody, is established. Here, we will delineate findings that support transient B-lymphocyte depletion therapy at the time of transplantation to preempt sensitization by eliminating alloreactive specificities from the recipient B-cell pool (ie, "repertoire remodeling"). Recent advances in our understanding of B-lymphocyte homeostasis provide novel targets for immunomodulation in transplantation. Specifically, the tumor necrosis factor-related cytokine BLyS is the dominant survival factor for "tolerance-susceptible" transitional and "preimmune" mature follicular B-cells. The transitional phenotype is the intermediate through which all newly formed B-cells pass before maturing into the follicular subset, which is responsible for mounting an alloantigen-specific antibody response. Systemic BLyS levels dictate the stringency of negative selection during peripheral B-cell repertoire development. Thus, targeting BLyS will likely provide an opportunity for repertoire-directed therapy to eliminate alloreactive B-cell specificities in transplant recipients, a requirement for the achievement of humoral tolerance and prevention of chronic rejection. In this review, the fundamentals of preimmune B-cell selection, homeostasis, and activation will be described. Furthermore, new and current B-lymphocyte-directed therapy for antibody-mediated rejection and the highly sensitized state will be discussed. Overall, our objective is to propose a rational approach for induction of humoral transplantation tolerance by remodeling the primary B-cell repertoire of the allograft recipient.

Humoral Antibodies in Organ Transplantation: Angels or Demons?

Hidalgo et al. (1) found that the development of de novo donor-specific antibodies was associated both with specific histological lesions in kidney transplant biopsies done late in the posttransplant course and a poor prognosis. These authors suggested that a careful assessment of de novo donor-specific antibodies should be performed in connection with all late kidney transplant biopsies. Studies aimed to investigate the role of donor-specific antibodies in organ transplantation are welcome as they may help to better understand the pathogenesis of chronic humoral rejection. However, one wonders whether such a costly monitoring of donor-specific antibodies will have a beneficial impact in clinical practice. In my opinion, caution is needed to interpret the role of humoral antibodies in this situation. Few doubts exist that chronic allograft rejection is caused, at least in part, by circulating antibodies, but it is still unclear if the anti-donor antibodies we can detect in the circulation are the same as those causing graft injury. It is conceivable that are antibodies aimed at inducing ‘accommodation’ (2). Experimental evidence from animal studies indicates that anti-HLA antibodies participate in allograft ‘accommodation’ (3). Expressing antiapoptotic genes can also induce allograft accommodation (4). As pointed out by Chang and Platt (2), it is conceivable that antibodies favoring the resistance of graft to immune injury cannot be absorbed by an allograft damaged by a previous immunological attack. As a consequence, the level of those antibodies increases in the circulation. Because we cannot determine whether circulating antidonor antibodies are harmful or protective, we should be cautious in making therapeutic decisions based on their measurement. If antidonor antibodies actually have a detrimental effect on graft outcome, removal therapies should be started when allo-antibody is first detected to prevent chronic rejection and prolong renal allograft survival. At present, however, there is not convincing evidence that therapies based on apheresis, B-cell removal or proteasome inhibition are of any substantial benefit in the treatment or prevention of chronic rejection. On the other hand, if circulating protective allo-antibodies are also operating in human allografts, these therapies might even be harmful, through removal of potentially protective allo-antibodies. Because a reinforcement of immunosuppression may be accompanied by serious or even life-threatening side effects, it is perhaps safer (and wiser) to keep the intensity of immunosuppression unchanged until further studies are able to elucidate the true nature and the heterogeneity (harmful or protective) of circulating antibodies.

Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule in cardiac allografts and significance thereof: experiment with rats

To investigate the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in allograft with chronic rejection (CR) after heart transplantation. Seventy-two SD rats receiving transplantation of the heats of 71 Wistar rats were divided into 4 equal groups: Group A, undergoing heterotopic cervical heart transplantation (transplanted with donor's heart subcutaneously at the neck) and receiving none intervention, and with the transplanted hearts taken out 3 d after transplantation to the end of CR; Group B, injected intravenously with the splenocytes (SPCs) of the donors on day 0, injected with cyclophomide (CP) on d2, transplanted with the donor hearts on d15, with the transplanted hearts taken out 15 - 120 days after transplantation; Group C, transplanted with the donor's heart and injected intraperitoneally with cyclosporine A (Cs A) 10 mg/kg every other day for 8 - 10 times, and with the transplanted hearts taken out 60 d after transplantation to the end of CR; and Group D, injected intravenously with the SPCs of the donors on day 0, injected with CP on d2, transplanted with the donor hearts on d15, with the transplanted hearts taken out 150 - 420 days after transplantation. Immunohistochemical method was used to detect the expression of ICAM-1 and VCAM-1 in the allografts. The cardiac allograft survival time of Group B was the longest. Less ICAM-1 and VCAM-1 expression and lymphocyte infiltration were observed in SPC and CP-pretreated group. Reversely, protein expression levels of ICAM-1 and VCAM-1 were both high and significant lymphocyte infiltration was seen in Groups B and D. There was no significant difference in the expression of ICAM-1 and VCAM-1 between Groups A and C (P > 0.05). The expression levels of ICAM-1 and VCAM-1 are associated with the occurrence and development of rejection and have positive correlation with the severity of graft rejection. Determination of ICAM-1 and VCAM-1 can predict the function of allograft and provide evidence for early diagnosis and prevention of CR.

Effect of a Novel Inducible Nitric Oxide Synthase Inhibitor in Prevention of Rat Chronic Aortic Rejections

Cytotoxic nitric oxide (NO) is produced during ischemia-reperfusion injury and acute and chronic rejection in allografts by expression of inducible (i) NO synthase (NOS). Therefore, continuous inhibition of iNOS may prevent early graft dysfunction and immune injury (rejection) and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity that works by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n > or = 6) were involved in this study. FR260330, tacrolimus, or both were administered orally for 14 or 90 days, according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. Both low and high doses of FR260330- or tacrolimus-treated grafts showed significantly decreased intima/(intima+media) ratios at day 90 compared with placebo controls. Combination therapy of low-dose FR260330 with low-dose tacrolimus produced a significant decrease of intima/(intima+media) ratios with intact endothelium compared with placebo controls. Anti-alpha-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. A selective inhibitor of NOS, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose FR260330 with tacrolimus produces significant protection of immune injury and may serve to improve long-term graft survival and function.

THE EFFECT OF A NOVEL INDUCIBLE NITRIC OXIDE SYNTHASE INHIBITOR IN PREVENTION OF CHRONIC AORTIC REJECTION IN RATS

P1219 Background: Cytotoxic nitric oxide (NO) is produced during ischemia/reperfusion (I/R) injury, acute and chronic rejection in the allograft by expression of inducible NO synthase (iNOS). Therefore, continuous inhibition of iNOS may prevent early graft disfunction and immune injury (rejection), and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. Methods: Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n ≥ 6) were involved in this study. FR260330 and/or tacrolimus were administered orally for 14 or 90 days according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. Results: Both low and high doses of FR260330 or tacrolimus treated grafts showed significantly decreased intimal/(intimal + media) ratios at days 90 compared to placebo controls. Combination therapy of low-dose of FR260330 with low-dose of tacrolimus produced significant decrease of intimal/(intimal + media) ratios with intact endothelium to compare with placebo controls. Anti-α-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. Conclusion: A selective inhibitor of nitric oxide synthase, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose of FR260330 with tacrolimus produces significant protection of immnue injury and may serve to improve long-term graft survival and function.

A NEW SYNTHETIC IMMUNOMODULATOR, KRP-203, ABROGATES CHRONIC REJECTION IN A RAT HEART TRANSPLANTATION MODEL

O126 Aims: Chronic rejection is a major cause of graft failure, limiting long-term survival after organ transplantation. KRP-203, a novel sphingosine-1-phosphate receptor agonist, has been recently synthesized by Kyorin Pharmaceutical Co., LTD. (Tokyo, Japan). This study was designed to investigate the potency of KRP-203 for the allograft survival and prevention of chronic rejection in a rat heart transplantation model. Methods: Heterotopic heart transplantation was performed in minor histocompatibility complex (mHC)-disparate rat combination of LEW (RT1l) to F344 (RT1lvl). The recipients were divided into 3 oral treatment groups: 1) vehicle (VH, distilled water, n=8), 2) KRP-203 0.1 mg/kg/day (KRP0.1, n=5), 3) KRP-203 1 mg/kg/day (KRP1, n=8). The heart allografts were followed until the day of rejection or for 100 days after transplantation. The percentage of vascular occlusion (%VO) by neointimal formation in the coronary arteries of allografts was calculated as follows: %VO = {area of intima / (area of intima + vascular lumen)} x 100. Perivascular inflammatory cell infiltration (PI) including macrophage and T-cell infiltration (MI and TI, respectively), and myocardial fibrosis (MF) were evaluated using scoring systems as follows: PI, MI and TI: Grade (G)-0, no infiltration; G-1, focal infiltration; G-2, moderate infiltration; G-3, diffuse infiltration; MF: G-0, no fibrosis; G-1, focal fibrosis; G-2, moderate fibrosis; G-3, diffuse fibrosis. The expression of endotherin-1 (ET-1) in the coronary arteries of allografts was also evaluated by immunohistochemical examination. The peripheral blood samples obtained from the recipients were examined for total WBC counts and WBC subpopulation. The homing of peripheral lymphocytes into secondary lymphoid organ was also analyzed by flow cytometry. Results: In the VH group, 6 of 8 heart allografts were rejected at days 21, 24, 53, 74, 79 and 87 after transplantation, respectively. In contrast, all allografts survived without rejection for 100 days after transplantation in both KRP-203 groups. Quantitative analyses were shown as follows: %VO (VH, 54.1±4.6; KRP0.1, 29.8±4.1*; KRP1 24.7±3.4*), PI (VH, 2.5±0.1; KRP0.1, 1.3±0.1*; KRP1, 1.3±0.1*), MI (VH, 2.7±0.2; KRP0.1, 1.6±0.2*; KRP1, 1.1±0.1*), TI (VH, 1.9±0.2; KRP0.1, 1.3±0.2*; KRP1, 1.2±0.1*), MF (VH, 2.3±0.1; KRP0.1, 1.4±0.1*; KRP1 1.3±0.1*), *P<0.05 vs. VH group. While the marked expression of endothelin-1 (ET-1) was observed in the neointimal lesion of coronary arteries in the VH group, ET-1 expression was suppressed in the KRP-203 groups, suggesting that KRP-203 abrogates transplant arteriosclerosis. In KRP-203-treated rats, total peripheral WBC counts decreased by 50 to 60 %, compared to VH-treated rats (VH, 7.08±1.11; KRP0.1, 4.08±0.49*; KRP1, 3.76±0.58* (x103/μl); P<0.05 vs. VH group). The WBC subpopulation analysis revealed that the number of peripheral lymphocytes remarkably decreased by the treatment with KRP-203 (VH, 4.98±0.16; KRP0.1, 1.08±0.16*; KRP1, 1.02±0.18 (x103/μl); *P<0.05 vs. VH group). Flow cytometric analysis demonstrated that treatment with KRP-203 enhanced lymphocyte homing into peripheral lymph nodes such as mesenteric, axillar and submandibular lymph nodes. Conclusions: KRP-203 prolonged long-term allograft survival and prevented chronic rejection in a rat heart transplantation model. These findings suggest that KRP-203 has a therapeutic potential in patients with organ transplantation.

132 CONSECUTIVE PEDIATRIC LIVER TRANSPLANTATIONS WITHOUT HOSPITAL MORTALITY. LESSONS LEARNED AND OUTLOOK TO THE FUTURE.

O161* Aims: The present study analyses our last 132 consecutive pediatric liver transplantations in which we did not experience any in-hospital death, while applying all available techniques including re-transplantations to rule out the most likely explanation for this encouraging improvement in outcome and to find future perspectives of improvements in pediatric liver transplantation. Methods: From April 2001 until December 2003, 18 whole organs (14%), 17 reduced-size (13%), 53 split organs (42%) (46 ex situ, 7 in situ) and 44 organs from living donors (33%) were transplanted into 113 patients. 112 were primary liver transplantations, 18 first re-transplantations, one third and one fourth liver transplantation. Of the 132 liver transplantations, 26 were high-urgent (20%). The outcome of these 132 consecutive pediatric liver transplantations was retrospectively analysed. Results: After 132 consecutive pediatric liver transplantations no patient died within 6 months after transplantation (100% patient survival <6 months) and overall three recipients (2%) died during further follow-up. The three months and the actual graft survivals are 92% and 86%, respectively. 16 children (12%) had to undergo re-transplantation. The causes were chronic rejection (3,8%), primary non function (3,8%), primary poor function (1,5%) and arterial thrombosis (3%). Biliary complication rate was 6%; arterial complications occurred in 8,3%; intestinal perforation was observed in 3% and in 5% postoperative bleeding required re-operation. Portal vein complication rate was 2%. Conclusions: These very good immediate results are a sign that liver transplantation has reached the gate to a new era where survival of the patient is considered the rule. Analysing our complete series of pediatric liver transplantation after starting our program in 1989, we have experienced that the year of transplantation turns out to be the most important prognostic factor of patient and graft survival in a multivariate analysis. Recurrence of disease, side effects of immunosuppression, diagnosis and prevention of chronic rejection and quality of life are some of the topics that will dominate our concerns in the years to come.

NEW INSIGHTS INTO ICOS-B7h PATHWAY IN ALLOIMMUNE RESPONSE

O456* Aims: Recent data indicate that the new T cell costimulatory pathway ICOS-B7h plays an important role in alloimmune responses in vivo. Although its role has been investigated in acute allograft rejection, relatively little is known of its functions in the process of chronic rejection. In this study, we investigated the role of ICOS-B7h pathway in development of chronic rejection of vascularized cardiac allografts. Methods: We utilized a MHC class II disparate cardiac transplantation model (Bm12 into C57BL/6). In this model, Bm12 cardiac allografts survive long-term but spontaneously develop moderately severe chronic vasculopathy, cellular infiltration and fibrosis by 6-8 weeks post-transplant. Results: We treated mice with a blocking anti-B7h mAb (HK5.3) either in the initiation phase (n=6, 0.25mg, every other day, 0-20 days after transplantation) or in the progression phase (n=6, 21-41 days) of chronic rejection. At 6 weeks, histopathological examination of grafts was performed by a blinded observer and scored in terms of vasculopathy (score 0-4), fibrosis (grade 0-3) and cellular infiltration (grade 0-3). Early blockade of ICOS-B7h pathway didn’t show a protective effect on chronic rejection (vasculopathy; 3.5±0.4, fibrosis; 2.2±0.3, cellular infiltration; 2.3±0.3: mean±SEM) compared to untreated controls (n=6, vasculopathy; 3.6±0.1, fibrosis; 2.7±0.2, cellular infiltration; 2.7±0.2). In contrast, delayed blockade of B7h significantly inhibited the development of chronic vasculopathy (1.8±0.4 vs. control, P=0.043), fibrosis (1.5±0.2, P=0.004) and cellular infiltration (1.7±0.3, P=0.03). To address the underlying mechanisms, we performed quantative real-time PCR analysis. There was significant decrease in local intragraft expression of cytokines, such as TNF-α, IFN-γ and IL-4, and several key chemokines, such as RANTES and IP-10, as well as adhesion molecules such as E-selectin in recipients with delayed blockade of B7h compared to controls. Moreover, there was significantly less infiltration of CD4 and CD8 T cells (p=0.007 and p=0.042 vs. control). Thus, delayed blockade of ICOS-B7h pathway downregulated local immune activation, thereby resulting in the less infiltration of alloreactive cells into cardiac grafts. Furthermore, to rule out the possibility that early blockade simply was not sufficient enough to display the protective effect on chronic rejection in this model, we treated mice with mAb in the entire period (n=6, 0-41 days, every other day). Interestingly, these mice demonstrated severe chronic rejection (vasculopathy; 3.3±0.4, fibrosis; 2.7±0.2, cellular infiltration; 2.8±0.2). Taken together, ICOS pathway may be critical in the progression phase of chronic rejection. In addition, its blockade in the initiation phase may have a detrimental effect on the prevention of chronic rejection. Conclusions: These data highlight the importance of ICOS-B7h costimulatory pathway in the pathogenesis of progression of chronic allograft rejection and provide the rationale to explore novel blockade strategy to treat this important clinical problem.

Prevention of Chronic Rejection with Immunoregulatory Cells Induced by Intrathymic Immune Modulation with Class I Allopeptides

Intrathymic immune modulation with RT1.Aa allopeptides in the PVG.R8-to-PVG.1 U rat strain combination leads to long-term survival of cardiac allografts. This regimen, however, does not induce transplantation tolerance, since most long-surviving allografts undergo chronic rejection. We investigated recipients with chronic rejection for donor-specific immune nonresponsiveness and immunoregulatory cells as possible mechanisms responsible for long-term graft survival. There was a significant reduction in the proliferative response of T cells from long-term allograft recipients to donor alloantigens as compared with that of naïve T cells. Adoptive transfer of splenocytes from intrathymically manipulated primary long-term graft survivors into minimally irradiated secondary hosts resulted in indefinite survival of > 80% of allografts, providing evidence for immunoregulatory cells. Secondary recipients had total absence of donor-reactive cellular and humoral responses. Immunoregulation was also transferable from secondary to tertiary graft recipients. More importantly, there was a significant reduction in the incidence of chronic rejection in secondary hosts (> 85%) and complete prevention of acute and chronic rejection in tertiary hosts. This study demonstrates that intrathymic immunomodulation with class I allopeptides results in the generation of immunoregulatory cells that do not block chronic rejection in primary hosts where they develop, but prevent both acute and chronic allograft rejection when adoptively transferred into secondary and tertiary recipients.

FTY720, an immunosuppressant that alters lymphocyte trafficking, abrogates chronic rejection in combination with cyclosporine A

Chronic rejection remains the leading cause of failure after transplantation (Tx). FTY720, a new immunosuppressant altering lymphocyte trafficking, is effective against acute rejection, but its activity against chronic rejection is not known. A valid model of chronic rejection was produced. Heart transplantation (HTx) was performed using fully mismatched RA (RT1p) and PVG (RT1c) rats. Administration of donor-specific blood transfusion 12 days before HTx prolongs graft survival, but features of chronic rejection including intimal hyperplasia and vascular obliteration (VO) develop with time only in allogeneic Tx. This is therefore a valid model of chronic rejection. VO was assessed on post-Tx day 90 in six groups differing according to the maintenance immunosuppressive regimen administered. group 1, donor-specific blood transfusion only and no other treatment; group 2, FTY720 (0.3 mg/kg/day orally) for 90 days; group 3, cyclosporine A (CsA) (1 mg/kg/day orally) for 90 days; group 4, combined administration of FTY720 and CsA for 90 days; group 5, transient administration of combined FTY720 and CsA for 7 days; and group 6, syngeneic HTx (RA to RA). Graft infiltrate, endothelial immunoglobulin (Ig) G deposition, and complement binding were also examined on post-Tx day 90. In control group 1, severe VO was observed, compared with syngeneic HTx (group 6). Monotherapy with FTY720 (group 2) or with CsA (group 3) significantly but partially reduced VO. On the contrary, combined administration of FTY720 and CsA (group 4) abrogated VO. A 1-week treatment with combined FTY720 and CsA (group 5) reduced VO but only partially. In group 1, arteriosclerosis was accompanied by graft infiltrate, endothelial IgG deposition, and complement binding. In groups 2, 3, and 5, graft infiltrating scores were partially decreased compared with group 1 but remained higher than in syngeneic controls; endothelial IgG deposition and complement binding were still present. In group 4, continuous administration of combined FTY720 and CsA reduced graft infiltrate to the level of syngeneic control and abrogated both endothelial IgG deposition and complement binding. Maintenance treatment with either FTY720 or CsA monotherapy partially prevents chronic rejection; short-term treatment with combined FTY720 and CsA reduces chronic rejection only partially; and continuous treatment with combined FTY720 and CsA abrogates chronic rejection, and this is accompanied by dramatic reduction of graft infiltrating cells, endothelial IgG deposition, and complement binding. Prevention of chronic rejection by maintenance treatment with FTY720 and CsA represents indirect evidence that normal lymphocyte trafficking and function are mandatory for development of chronic rejection.

Prevention of chronic rejection by pravastatin in a rat kidney transplant model.

Pravastatin when administered with cyclosporine (CsA) has been shown to ameliorate transplant vasculopathy in the clinical setting. Previously we showed that pravastatin prevents chronic rejection in rat cardiac and liver transplant models. Here we determine whether pravastatin prevents chronic rejection in a rat renal allograft model. Orthotopic renal transplantations were performed using Fisher 344 rats as donors and Lewis rats as recipients. Recipients were treated with low-dose CsA for 10 days to prevent acute rejection. Recipients were divided into three groups: CsA, CsA + pravastatin, and syngeneic. Renal function was assessed by serum creatinine level at day 130. Allografts were evaluated by histology and immunohistochemistry. Serum levels of alloantibodies were measured by flow cytometry. Intragraft cytokine mRNA expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction. Intragraft levels of the antiapoptotic Bag-1 gene were measured by Western blot. Unlike allografts from the pravastatin group, control allografts demonstrated glomerulosclerosis, vascular obliteration, tubular atrophy, and interstitial fibrosis. Serum creatinine levels and graft infiltration of T cells and macrophages in the pravastatin-treated animals were significantly lower. Intragraft cytokines showed a T helper 2 polarization and decreased transforming growth factor-beta in the pravastatin group. Intragraft expression of Bag-1 was increased in the pravastatin group. This study demonstrates the ability of pravastatin to inhibit chronic rejection in rat renal allografts. Pravastatin's pleiotropic effects of reducing intragraft inflammatory cytokines, inhibiting immune cell infiltration, and causing up-regulation of the antiapoptotic gene Bag-1 suggest that its ability to prevent transplant chronic rejection may be multifactorial.

Control of antidonor antibody production with tacrolimus and mycophenolate mofetil in renal allograft recipients with chronic rejection.

In renal transplantation, chronic rejection is a major cause of late allograft loss. Recent studies indicate that a subset of chronic rejection is associated with anti-HLA donor specific antibodies (DSA) and complement C4d deposition in peritubular capillaries (PTC). Since rescue therapy with tacrolimus and mycophenolate mofetil has been found to limit antidonor B-cell responses in recipients with acute humoral rejection, we sought to determine whether a similar immunosuppressive regimen might be effective in patients with 'chronic humoral rejection'. Four renal allograft recipients with 'chronic humoral rejection' were prospectively identified. The diagnosis was based on: (1) progressive rise in serum creatinine over 12 months; (2) typical pathologic features by light microscopy (transplant arteriopathy and glomerulopathy); (3) widespread C4d deposits in PTC by immunofluorescence; (4) detection of 'de novo' DSA at the time of biopsy. Maintenance immunosuppression was CsA, prednisone and azathioprine (n=3) or prednisone and azathioprine (n=1). Rescue therapy with tacrolimus and mycophenolate mofetil was initiated in all patients, 12 hr after cyclosporine and azathioprine discontinuation. At diagnosis, the mean serum creatinine was 3.9 mg/dl (range: 3.3 to 5.4 mg/dl). DSA was an IgG directed against HLA class II (n=3) or class I (n=2), that is one patient had both anti-HLA class I and class II antibodies. Pretreatment antibody titers varied between 1:8 and 1:128. Rescue therapy was associated with a rapid and sustained decrease in antibody titers. In two patients, DSA became undetectable after 9 months and a repeat biopsy performed after 12 months revealed a decrease in C4d deposition in PTC. These results suggest that a decrease in DSA production can be induced in renal allograft recipients with 'chronic humoral rejection' by using an immunosuppressive regimen that combines tacrolimus and mycophenolate mofetil. Limitation of antidonor antibody synthesis may be important for the treatment or the prevention of chronic rejection in organ transplantation.

Chronic Rejection of Renal Transplants: New Clinical Insights

Chronic rejection is the most important cause for returning to dialysis after failure of a renal transplant. The term chronic allograft nephropathy refers to the progressive decline of renal function seen in some renal transplant recipients in association with alloantigen-dependent and alloantigen-independent factors. This review examines the role of factors related to allorecognition, injury, nephron dosing, and donor and recipient characteristics in the development of chronic allograft nephropathy. The clinical associations to chronic allograft nephropathy are presented in the context of pathogenetic mechanisms of renal damage and disease progression. As there is no therapy available at this time for established chronic allograft nephropathy, possible areas of intervention for the prevention of chronic rejection are discussed.

DISTINCT PATTERNS OF SOLUBLE HLA CLASS I AND FREE HEAVY CHAIN RELEASE IN LUNG TRANSPLANT PATIENTS DURING REJECTION VS CMV INFECTION

147 Background: HLA heavy chains (HC) are cleaved from the surface of activated cells by a metalloproteinase (MPase)-dependent pathway. Some of the cleaved 36kD HC can rapidly re-associate with β2m, while the rest remain β2m-free. Proteinase-independent pathways generate 39kD & 44kD sHLA/β2m. Significance: Because β2m-free HCs of donor origin may be easily processed and presented by host APC (i.e. the indirect pathway of allorecognition), it is important to determine when and where they are generated. Hypothesis: We hypothesized that donor sHLA/β2m and HC are released by MPase cleavage in transplant rejection and infection. Methods: The concentration and molecular weights of donor sHLA and β2m-associated or free forms of HLA HC in bronchoalveolar lavage (BAL) or sera from lung transplant recipients were determined by ELISA (n=50) and by immunoprecipitation with specific mAbs followed by Western blot (n=11). Results: Acute rejection (AR) events were correlated with a release of β2m-free HC and HLA/β2m into the lung epithelial lining fluid (sampled by BAL) and a 50% increase in systemic release of donor sHLA; serum β2m-free HC level was unchanged. In contrast, serum levels of both β2m-free HC and donor sHLA doubled during CMV events, but there was no significant increase of sHLA in the BAL. Western blots revealed proteolytically cleaved (36kD) sHLA/β2m and β2m free HC in the BAL fluid and donor sHLA in the serum. (Table)TableConclusions: The data support involvement of the MPase pathway of intragraft soluble HLA release during lung transplant AR and in AR + CMV, suggesting a possible avenue of intervention to limit the supply of donor free HC to the indirect pathway of allograft recognition and prevention of chronic rejection. CMV by itself had a negligible impact on the local generation of free HC, but a substantial impact on the generation of free HC (most likely of host origin) in the peripheral blood.