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The accurate measurement of anti-HLA alloantibodies in transplant candidates is required for determining the degree of sensitization and for the listing of unacceptable antigens for organ allocation. Both the configuration of the HLA molecules coated on the beads and the nature of detection antibodies may impede assessment of the presence and strength of anti-HLA IgG- with the Luminex single-antigen-bead assay. Sera antibodies of the end-stage renal disease patients were compared using LIFECODES (LC) and LABScreen (LS) beadsets monitored with polyclonal-Fab (IgHPolyFab) and monoclonal-IgG (FcMonoIgG) second antibodies. Positive results at mean fluorescence intensity (MFI) > 500 (at serum dilution 1/10) were used to calculate panel reactive antibody (cPRA) levels. LS-beadsets are coated with monomeric variants in addition to intact HLA antigens with or without peptides, while LC-beadsets are devoid of monomeric variants and with lesser levels of peptide-free heterodimers. Consequently, IgG antibodies against both classes of HLA were reactive to more antigens with LS than with LC-beadsets. For both classes, MFIs were also frequently higher with LS than with LC. For HLA-I, MFIs were higher with IgHPolyFab than with FcMonoIgG with the exception of sera with MFIs > 5000 where they were comparable. For HLA-II, the reverse occurred, with significantly higher levels with FcMonoIgG regardless of the beadsets. The intraindividual variability observed between beadsets with two detection antibodies elucidates that antigens found as acceptable with one beadset may end up unacceptable with the other beadsets, with the possibility of denying potentially compatible transplants to candidates.

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5-11.4 years, while three required reinstitution of IS after 5-8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Preexisting serum antibodies have long been associated with graft loss in transplant recipients. While most studies have focused on HLA-specific antibodies, the contribution of non-HLA-reactive antibodies has been largely overlooked. We have recently characterized mAbs secreted by B cell clones derived from kidney allograft recipients with rejection that bind to apoptotic cells. Here, we assessed the presence of such antibodies in pretransplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan-Meier survival analysis revealed that patients with high pretransplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year posttransplant. Moreover, the association between pretransplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactive to apoptotic cells contribute to presensitization. Taking these antibodies into consideration alongside anti-HLA antibodies during candidate evaluation would likely improve the transplant risk assessment., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Antibody mediated rejection (AMR) is associated with a variety of graft-reactive antibodies following kidney transplant. To characterize these antibodies, we immortalized 107 B cell clones from a patient with AMR. In a previous study, we showed that six clones were reacting to multiple self-antigens as well as to HLA and MICA for two of them, thus displaying a pattern of polyreactivity. We show here that all six polyreactive clones also reacted to apoptotic but not viable cells. More generally we observed a nearly perfect overlap between polyreactivity and reactivity to apoptotic cells. Functionally, polyreactive antibodies can activate complement, resulting in the deposition of C3d and C4d at the surface of target cells. Testing the serum of 88 kidney transplant recipients revealed a significantly higher IgG reactivity to apoptotic cells in AMR patients than in patients with stable graft function. Moreover, total IgG purified from AMR patients had increased complement activating properties compared to IgG from non-AMR patients. Overall, our studies show the development of polyreactive antibodies cross-reactive to apoptotic cells during AMR. Further studies are now warranted to determine their contribution to the detection of C4d in graft biopsies as well as their role in the pathophysiology of AMR., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background and Objectives: Chronic rejection leads to kidney allograft failure and develops in many kidney transplant recipients. One cause of chronic rejection, chronic antibody mediated rejection (CAMR), is attributed to alloantibodies. Maintenance immunosuppression including prednisone, mycophenolate mofetil (MMF) and calcineurin inhibitors may limit alloantibody production in some patients, but many maintain or develop alloantibody production, leading to CAMR. Therefore, no efficacious therapy to treat CAMR is presently available to prevent the progression of CAMR to kidney allograft failure., Design, Setting, Participants, and Measurements: We performed a retrospective review of 31 subjects with CAMR, of which 14 received Rituximab and 17 subjects did not. Response to Rituximab was defined as decline or stabilization of serum creatinine for at least one year. Data reviewed included demographic, clinical, allograft, post-transplant, and pathological variables. Pathological variables in the diagnostic allograft biopsy were scored according to Banff criteria., Results: The median survival time (MST) for allografts in the control group was 439 days, and for the Rituximab treated group was 685 days. The Rituximab group was dichotomous with 8 subjects showing a medial survival time of 1180 days, and 6 subjects having a median survival time of 431 days. The MST for the responders was statistically significant from the non-responders and controls. No pathological parameter distinguished any subset of subjects., Conclusions: These data show that Rituximab followed by standard maintenance immunosuppression shows a therapeutic effect in the treatment of CAMR, which is confined to a subset of treated subjects, not identifiable a priori., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antibody rejection is often accompanied by nondonor HLA specific antibodies (NDSA) and self-reactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B-cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self-antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B-cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

An idiopathic capillary leak syndrome ('engraftment syndrome') often occurs in recipients of hematopoietic cells, manifested clinically by transient azotemia and sometimes fever and fluid retention. Here, we report the renal pathology in 10 recipients of combined bone marrow and kidney allografts. Nine developed graft dysfunction on day 10-16 and renal biopsies showed marked acute tubular injury, with interstitial edema, hemorrhage and capillary congestion, with little or no interstitial infiltrate (≤10%) and marked glomerular and peritubular capillary (PTC) endothelial injury and loss by electron microscopy. Two had transient arterial endothelial inflammation; and 2 had C4d deposition. The cells in capillaries were primarily CD68(+) MPO(+) mononuclear cells and CD3(+) CD8(+) T cells, the latter with a high proliferative index (Ki67(+) ). B cells (CD20(+) ) and CD4(+) T cells were not detectable, and NK cells were rare. XY FISH showed that CD45(+) cells in PTCs were of recipient origin. Optimal treatment remains to be defined; two recovered without additional therapy, six were treated with anti-rejection regimens. Except for one patient, who later developed thrombotic microangiopathy and one with acute humoral rejection, all fully recovered within 2-4 weeks. Graft endothelium is the primary target of this process, attributable to as yet obscure mechanisms, arising during leukocyte recovery., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Specific tolerance after combined kidney and bone marrow transplantation for multiple myeloma with end-stage renal disease through mixed lymphohematopoietic chimerism has been achieved, as evidenced by prolonged normal renal function without ongoing immunosuppression., Methods: To achieve potent antimyeloma responses and induce tolerance for the renal allograft, seven patients (median age: 48 years [range: 34-55 years]) with multiple myeloma and end-stage renal disease underwent a combined human leukocyte antigen-matched kidney and bone marrow transplant with lead follow-up time of more than 12 years. Preparative therapy for the transplant consisted of high-dose cyclophosphamide, equine antithymocyte globulin and pretransplant thymic irradiation. Cyclosporine as the sole posttransplant immunosuppressive therapy was tapered and discontinued as early as day 73 posttransplant., Results: All seven patients achieved mixed chimerism. One patient developed acute graft-versus-host disease and two chronic graft-versus-host disease. Five of seven patients are alive, four with no evidence of myeloma from 4 to 12.1 years posttransplant. Three patients have normal or near-normal renal function without needing systemic immunosuppression. Two patients with normal renal function off immunosuppression were returned to immunosuppressive therapy without evidence of rejection because of the occurrence of chronic graft-versus-host disease., Conclusions: These long-term follow-up data show that sustained renal allograft tolerance and prolonged antimyeloma responses are achievable after human leukocyte antigen-matched kidney and bone marrow transplantation and the induction of mixed lymphohematopoietic chimerism.

Five patients with end-stage kidney disease received combined kidney and bone marrow transplants from HLA haploidentical donors following nonmyeloablative conditioning to induce renal allograft tolerance. Immunosuppressive therapy was successfully discontinued in four patients with subsequent follow-up of 3 to more than 6 years. This allograft acceptance was accompanied by specific T-cell unresponsiveness to donor antigens. However, two of these four patients showed evidence of de novo antibodies reactive to donor antigens between 1 and 2 years posttransplant. These humoral responses were characterized by the presence of donor HLA-specific antibodies in the serum with or without the deposition of the complement molecule C4d in the graft. Immunofluorescence staining, ELISA assays and antibody profiling using protein microarrays demonstrated the co-development of auto- and alloantibodies in these two patients. These responses were preceded by elevated serum BAFF levels and coincided with B-cell reconstitution as revealed by a high frequency of transitional B cells in the periphery. To date, these B cell responses have not been associated with evidence of humoral rejection and their clinical significance is still unclear. Overall, our findings showed the development of B-cell allo- and autoimmunity in patients with T-cell tolerance to the donor graft.

Numerous studies have demonstrated an association of posttransplant HLA antibodies with decreased long-term graft survival. The presence of C4d deposition in these cases supports the hypothesis that antibody and complement deposition are involved in the pathogenesis of graft failure. Development of HLA antibodies may predate the clinical manifestation of chronic rejection (CR). However, frequency of donor-specific antibody is low when all patients are screened regardless of their graft function, and it may be more valuable to look for antibody only in patients with mild dysfunction. Effective treatment for CR has not been identified, although increased immunosuppression has been shown to decrease antibody levels and stabilize graft function. Many patients have been identified with good graft function despite the presence of circulating donor-specific HLA antibody. Additional studies focusing on the mechanism behind the apparent protection from the detrimental effects of antibody in such patients are needed.

We sought evidence for non-MHC antibody-mediated rejection in renal allografts by a systematic study of rejected HLA-identical sibling renal allografts. Among 162 recipients of HLA-identical, ABO-compatible sibling donor kidneys transplanted at the Massachusetts General Hospital from 1964 to 2005, we identified 15 grafts that were lost from rejection and two additional grafts with reversible acute rejection, which provided 30 samples for study. All samples were stained for C4d by immunofluorescence in frozen tissue (n = 7) or by immunohistochemistry in paraffin embedded tissues (n = 10). We found that two of 17 grafts had positive C4d staining of peritubular capillaries. Histology revealed acute antibody-mediated rejection in one and acute cellular rejection type 1 in the other. Both grafts were matched at HLA-A, B, and C loci and had a nonreactive mixed lymphocyte response. Genotyping and serological analysis were not available. Compared with a published series, C4d+ irreversible rejection was more common in HLA nonidentical than HLA-identical grafts (75% vs 6.7%, respectively, P < .002). We conclude that antibody-mediated rejection, presumably due to non-MHC antigens other than ABO-blood groups does occur, but infrequently. This may account for some of the HLA antibody negative cases that develop antibody-mediated rejection.

In a list exchange (LE), the intended recipient in an incompatible pair receives priority on the deceased donor waitlist (DD-waitlist) after the paired incompatible donor donates a kidney to a DD-waitlist candidate. A nondirected donor's (ND-D) kidney is usually transplanted directly to a DD-waitlist candidate. These two established practices would help even more transplant candidates if they were integrated with kidney paired donation (KPD). We consider a scenario in which the donor of an LE intended recipient (LE-IR) donates to a compatible KPD intended recipient (KPD-IR), and the KPD donor (KPD-D) donates to the waitlist (an LE-chain). We consider a similar scenario in which an ND-D donates to a KPD-IR and the KPD-D donates to the DD-waitlist (an ND-chain). Using data derived from the New England Program for Kidney Exchange (NEPKE) and from OPTN/SRTR recipient-donor distributions, simulations are presented to evaluate the potential impact of chain exchanges coordinated with KPD. LE donors (LE-D) and ND-D who are ABO-O result in the highest number of additional transplants, while results for ABO-A and B donors are similar to each other. We recommend that both LE and ND donations be utilized through chain exchanges.

Background: In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients., Methods: Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4)., Results: All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR., Conclusion: ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+ cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.

The 2 principal factors implicated in late kidney allograft failure are chronic rejection (also called chronic allograft nephropathy) and death of the patient with a functioning graft (mainly from cardiovascular causes). Despite lifelong immunosuppression of the recipient, immunological responses remain the leading factor in the pathogenesis of chronic rejection and both cellular and humoral immune mechanisms have been shown to play important roles. In this review, we highlight the relevance of humoral mechanisms of rejection to the pathogenesis of late allograft loss. Non immunological factors, such as donor organ quality, initial ischemic injury, calcineurin inhibitor (CNI) toxicity, hypertension, and hyperlipidemia, also contribute to progressive chronic allograft injury, but will not be reviewed in detail here. Possible strategies to stabilize or improve allograft function in patients with already established "chronic rejection/chronic allograft nephropathy" (CR/CAN) are the addition of mycophenolate mofetil (or sirolimus) with or without a reduction of cyclosporine dosage, or conversion from cyclosporine to tacrolimus. However, prospective randomized clinical trials are needed to test the efficacy of these strategies. A major current challenge for transplant physicians is to develop regimens that prevent CR/CAN, since, once established, the process typically progresses inexorably to renal allograft loss in most recipients. Evidence is now accumulating that new immunosuppressive regimens must control not only T cell but also B cell responses (i.e. limit antidonor antibody production) in order to prevent CR/CAN and improve long-term allograft survival.

The use of interferon-alpha (IFN) in hepatitis C (HCV)-infected renal recipients has been associated with acute rejection and graft loss. We reviewed our recent experience in HCV (+) renal recipients treated with antiviral therapy for biopsy proven chronic hepatitis C. Twelve HCV (+) recipients who recently received antiviral therapy were analyzed. Post-treatment sera were tested for donor-specific human leukocyte antigen (HLA) antibodies (DSA). Within 6 months of initiating antiviral therapy, two of 12 patients (17%) developed acute rejection, which was characterized as acute humoral rejection (de novo DSA in serum and C4d deposits in peritubular capillaries). Both progressed to graft failure. Nine of the remaining 10 patients tested did not have DSA. The use of IFN was associated with severe acute humoral rejection (C4d +, DSA +). The recognition of IFN-associated acute humoral rejection in this series may explain the high rate of graft loss reported previously in renal recipients receiving IFN.

Background: 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'., Methods: 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., Results: 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., Conclusion: 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.

Background: Acute rejection (AR) associated with de novo production of donor-specific antibodies (DSA) is a clinicopathological entity that carries a poor prognosis (acute humoral rejection, AHR). The aim of this study was to determine the incidence and clinical characteristics of AHR in renal allograft recipients, and to further analyze the antibodies involved., Methods: During a 4-year period, 232 renal transplants (Tx) were performed at our institution. Assays for DSA included T and B cell cytotoxic and/or flow cytometric cross-matches and cytotoxic antibody screens (PRA). C4d complement staining was performed on frozen biopsy tissue., Results: A total of 81 patients (35%) suffered at least one episode of AR within the first 3 months: 51 had steroid-insensitive AR whereas the remaining 30 had steroid-sensitive AR. No DSA were found in patients with steroid-sensitive AR. In contrast, circulating DSA were found in 19/51 patients (37%) with steroid-insensitive AR, and widespread C4d deposits in peritubular capillaries were present in 18 of these 19 (95%). In at least three cases, antibodies were against donor HLA class II antigens. DSA were not found in the remaining 32 patients but C4d staining was positive in 2 of 32. The DSA/C4d positive (n=18) and DSA/C4d negative (n=30) groups differed in pre-Tx PRA levels, percentage of re-Tx patients, refractoriness to antilymphocyte therapy, and outcome. Plasmapheresis and tacrolimus-mycophenolate mofetil rescue reversed rejection in 9 of 10 recipients with refractory AHR., Conclusion: More than one-third of the patients with steroid-insensitive AR had evidence of AHR, often resistant to antilymphocyte therapy. Most cases (95%) with DSA at the time of rejection had widespread C4d deposits in peritubular capillaries, suggesting a pathogenic role of the circulating alloantibody. Combined DSA testing and C4d staining provides a useful approach for the early diagnosis of AHR, a condition that often necessitates a more intensive therapeutic rescue regimen.

The pathogenesis of chronic renal allograft rejection (CR) remains obscure. The hypothesis that a subset of CR is mediated by antidonor antibody was tested by determining whether C4d is deposited in peritubular capillaries (PTC) and whether it correlates with circulating antidonor antibodies. All cases (from January 1, 1990, to July 31, 1999) that met histologic criteria for CR and had frozen tissue (28 biopsies, 10 nephrectomies) were included. Controls were renal allograft biopsies with chronic cyclosporine toxicity (n = 21) or nonspecific interstitial fibrosis (n = 10), and native kidneys with end-stage renal disease (n = 10) or chronic interstitial fibrosis (n = 5). Frozen sections were stained by two-color immunofluorescence for C4d, type IV collagen and Ulex europaeus agglutinin I. Antidonor HLA antibody was sought by panel-reactive antibody analysis and/or donor cross matching in sera within 7 wk of biopsy. Overall, 23 of 38 CR cases (61%) had PTC staining for C4d, compared with 1 of 46 (2%) of controls (P < 0.001). C4d in PTC was localized at the interface of endothelium and basement membrane. Most of the C4d-positive CR tested had antidonor HLA antibody (15 of 17; 88%); none of the C4d-negative CR tested (0 of 8) had antidonor antibody (P < 0.0002). The histology of C4d-positive CR was similar to C4d-negative CR, and 1-yr graft survival rates were 62% and 25%, respectively (P = 0.05). Since August 1998, five of six C4d-positive CR cases have been treated with mycophenolate mofetil +/- tacrolimus with a 100% 1-yr graft survival, versus 40% before August 1998 (P < 0.03). These data support the hypothesis that a substantial fraction of CR is mediated by antibody (immunologically active). C4d can be used to separate this group of CR from the nonspecific category of chronic allograft nephropathy and may have the potential to guide successful therapeutic intervention.

Between February 1963 and December 2000, 1,627 kidney transplants were performed at the Massachusetts General Hospital. The majority (62%) were from cadaveric donors, although in recent years (1996-2000) 52% have been allografts from living donors, with an increase in living unrelated donors. The introduction of CsA and OKT3 in 1984 was associated with a significant improvement in actuarial renal allograft survival, although a persistent late attrition of allografts continues beyond the first year after transplantation. As reported in other centers, current actuarial survival for living unrelated allografts is superior to that of cadaveric allografts, and is quite similar to that observed in recipients of non-HLA identical living-related transplants. Our preliminary laparoscopic donor nephrectomy experience is encouraging as excellent allograft survival and function has been observed, with minimal morbidity associated with the procedure and a low rate of conversion to open nephrectomy. Recent changes in immunosuppressive protocols have resulted in lower early acute rejection rates, however the incidence of delayed graft function remains unchanged in cadaveric renal transplantation. The role of humoral immunity in allograft rejection has been progressively clarified and new approaches to control donor specific alloantibody production have been shown to be effective. Current clinical studies are ongoing to determine the optimal type and dose of calcineurin inhibitors beyond the first year after transplantation and to study whether avoidance of steroids is safe and feasible. Finally, an innovative tolerance induction protocol using the mixed chimerism approach has been successfully accomplished in selected patients with end-stage renal disease secondary to multiple myeloma. These encouraging observations emphasize that major changes from current immunosuppressive regimens are likely to occur over the next few years as more approaches to tolerance induction are explored clinically.

The effect of long-term pharmacologic immunosuppression (PI) on anti-Galalpha1-3Gal (alphaGal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured alpha Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in alpha Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of alphaGal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate alphaGal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce Gal Ab levels sufficiently to be of clinical value in xenotransplantation.

The distinction between acute humoral rejection (AHR) and acute cellular rejection (ACR) in renal allografts is therapeutically important, but pathologically difficult. Since AHR is probably mediated by antibodies to the donor endothelium that activate the classical complement pathway, it was hypothesized that peritubular capillary C4d deposition might distinguish this group. Renal biopsies (n = 16) from 10 patients with AHR who had acute graft dysfunction, neutrophils in peritubular capillaries, and a concurrent positive cross-match were stained for C4d by immunofluorescence. Control biopsies for comparison showed ACR (n = 14), cyclosporin A toxicity (n = 6), or no abnormality (n = 4). Peribiopsy sera were tested for anti-donor HLA antibody. C4d deposited prominently and diffusely in the peritubular capillaries in all AHR biopsies (16 of 16). IgM and/or C3 were also present in 19 and 44%, respectively. With two-color immunofluorescence, C4d was localized in basement membranes (type IV collagen+) and in the endothelium (Ulex europaeus agglutinin-I+). In ACR, no more than trace C4d was found in peritubular capillaries (P < 0.0001 versus AHR), and no patient had anti-donor HLA antibodies (0 of 8); 27% had neutrophils in peritubular capillaries. One of six biopsies with cyclosporin A toxicity had similar C4d deposits, and circulating anti-donor class I antibody was detected. Grafts with AHR were lost (40%) more often than those with ACR (0%; P < 0.02). C4d in peritubular capillary walls distinguishes AHR from ACR, is more specific and sensitive than traditional criteria, and is a potentially valuable adjunct in the diagnosis of graft dysfunction.

Background: Experimental and clinical evidence has demonstrated that the establishment of allogeneic chimerism after bone marrow transplantation may provide donor-specific tolerance for solid organ allografts., Methods: Based on the preliminary results of a clinical trial using nonmyeloablative preparative therapy for the induction of mixed lymphohematopoietic chimerism, we treated a 55-year-old woman with end stage renal disease secondary to multiple myeloma with a combined histocompatibility leukocyte antigen-matched bone marrow and renal transplant after conditioning with cyclophosphamide, antithymocyte globulin, and thymic irradiation., Results: The posttransplant course was notable for early normalization of renal function, the absence of acute graft-versus-host disease, and the establishment of mixed lymphohematopoietic chimerism. Cyclosporine, which was the only posttransplant immunosuppressive therapy, was tapered and discontinued on day +73 posttransplant. No rejection episodes occurred, and renal function remains normal on day + 170 posttransplant (14 weeks after discontinuing cyclosporine). Although there is presently no evidence of donor hematopoiesis, there is evidence of an ongoing antitumor response with a recent staging evaluation showing no measurable urine kappa light chains. The patient remains clinically well and is off all immunosuppressive therapy., Conclusion: This is the first report of the deliberate induction of mixed lymphohematopoietic chimerism after a nonmyeloablative preparative regimen to treat a hematological malignancy and to provide allotolerance for a solid organ transplant.

Background: Acute renal allograft rejection associated with the development of donor-specific alloantibody (acute humoral rejection, AHR) typically carries a poor prognosis. The best treatment of this condition remains undefined., Methods: During a 14-month period, 73 renal transplants were performed. During the first postoperative month, five recipients (6.8%) with AHR were identified. The diagnosis was based on: (1) evidence of severe rejection, resistant to steroid and antilymphocyte therapy; (2) typical pathologic features; and (3) demonstration of donor-specific alloantibody (DSA) in recipient's serum at the time of rejection. Pretransplant donor-specific T- and B-cell cross-matches were negative., Results: Plasma exchange (PE, four to seven treatments per patient) significantly decreased circulating DSA to almost pretransplant levels in four of five patients, and improvement in renal function occurred in all patients. One patient had recurrent renal dysfunction in the setting of an increase in circulating DSA. A second series of five PE treatments decreased DSA and reversed the rejection episode. Rescue therapy with tacrolimus (initial mean dose: 0.14+/-0.32 mg/kg/day) and mycophenolate mofetil (2 g/day) was used in five of five and four of five patients, respectively. With a mean follow-up of 19.6+/-5.6 months, patient and allograft survival are 100%. Renal function remains excellent with a mean current serum creatinine of 1.2+/-0.3 mg/dl. (range: 0.9-1.8 mg/dl)., Conclusions: Our findings suggest that a therapeutic approach combining PE and tacrolimus-mycophenolate mofetil rescue has the potential to improve the outcome of AHR.

We report a case of accelerated acute rejection of a renal allograft from a presumed ABO histo-blood group A2 donor in an O recipient, in which all of the published criteria for compatibility had been met. Flow cytometric analysis of the A and H antigen expression on the kidney donor's erythrocytes suggested that this donor did not have an A2 phenotype, but rather another subgroup of A. Some of the reported cases of accelerated acute rejection of A2 renal allografts in O recipients may have resulted from misapplication of the results of standard lectin agglutination to the transplant setting. The current case suggests that a more sophisticated method of ABO phenotyping, such as erythrocyte flow cytometric analysis, may be necessary in the transplant setting.

Objective: To determine whether the appropriate use of scarce donor resources has been accomplished by renal retransplantation by reviewing the initial and long-term outcomes of second-renal transplant recipients at the Massachusetts General Hospital, Boston., Patients and Results: With a mean follow-up of nearly 5 years following transplantation, 54 (68%) of 80 second-transplant recipients had functioning allografts (allograft failure was defined by patient death or a return to dialysis). Rejection was the most common cause of failure (14 [54%] of 26 patients). The 1-, 3-, and 5-year actuarial allograft survival rates were 86%, 78%, and 69%, respectively, which were not significantly different from the survival rates of primary allografts at this center. These results support the continued approach of providing both cadaver-donor and living-donor renal allografts for recipients whose primary renal allograft has failed. The antiglobulin crossmatch may have contributed to the successful outcome by accurately determining compatibility and by averting early rejection failures., Conclusions: Health care policy reviewers should clearly distinguish the prospects for successful second renal transplants from the outcomes of extrarenal retransplantation. Moreover, because excellent second-renal allograft survival is attainable and comparable to primary-renal allograft survival and because the costs are comparable, restricting suitable patients to subsequent lifelong dialysis becomes unethical.

Thirty-eight sequentially placed liver and kidney allografts were evaluated with respect to patient and graft survival, and the influence of preformed lymphocytotoxic antibodies was analysed. The results suggest that the survival rate of combined liver and kidney transplantation is similar to the survival rate of liver transplantation alone. Sequentially placed kidney allografts may be protected from hyperacute rejection in the presence of donor specific lymphocytotoxic antibodies, but not in all instances. Both patient and kidney allograft survival was lower in positive crossmatch patients (33% and 17% respectively) than in negative crossmatch patients (78% and 75%). High levels of panel reactive antibodies (> 10%) also appeared to have a deleterious effect on survival, although the majority of the patients who failed also had a positive crossmatch. Although performed lymphocytotoxic antibodies are not an absolute contraindication to combined liver-kidney transplantation, they do appear to have a deleterious effect on long-term graft survival. However, more correlation with clinical parameters is needed.