RevIeWS. Sensitized renal transplant recipients: current protocols and future directions. James Gloor and Mark D. Stegall

RevIeWS Sensitized renal transplant recipients: current protocols and future directions James Gloor and Mark D. Stegall Abstract The identification of suitable donor kidneys for transplant candidates with high levels of circulating antibodies against human leukocyte antigen (HLA) is a major challenge and results in prolonged waiting times for transplantation. Technological advances in antibody characterization have permitted a more comprehensive assessment of anti-hla antibody activity, as well as providing new insights into the clinical effect of HLA antibody class and specificity. Protocols have been developed that enable successful transplantation in patients with donor-specific antibodies (anti-hla antibodies reactive against their donors). These protocols provide satisfactory early to intermediate-term allograft survival, and constitute an important advance in transplantation. Nevertheless, acute antibody-mediated rejection (AMR) remains a significant challenge, occurring in 20 50% of antibody-incompatible kidney transplantations. Although therapy directed toward lowering donor-specific antibody activity seems to be successful in reversing acute AMR, this condition still has an important negative impact on allograft survival. In addition, subclinical AMR seems to complicate a substantial proportion of positive-crossmatch transplantations even in the absence of allograft dysfunction, and may result in chronic histological abnormalities and shortened allograft function. New interventions for preventing acute AMR, such as anti-c5 antibody-mediated complement blockade and proteasome inhibitor-mediated plasma cell depletion, are promising therapeutic avenues currently under investigation. Gloor, J. & Stegall, M. D. Nat. Rev. Nephrol. 6, 297 306 (2010); published online 16 March 2010; doi:10.1038/nrneph.2010.34 Introduction a major challenge in renal transplantation today is the identification of acceptable kidney donors for individuals with renal failure. although the number of kidney transplantations performed annually in the us has consistently increased over the past decade, the number of people currently awaiting transplantation far exceeds the number of available donors (a situation that also exists internationally). adding to the complexity of this situation, the number of transplant candidates who are allosensitized to human leukocyte antigen (Hla) antigens has increased over the same period. 1 anti Hla antibodies develop after exposure to Hla antigens, typically following a blood transfusion, pregnancy, or a previous transplantation. 2 Historically, donor specific antibodies (Dsas) anti Hla antibodies with reactivity against a potential donor have constituted a contraindication for proceeding to transplantation, and testing for these antibodies has been an important component of the pretransplantation evaluation. Finding a compatible donor is often difficult and can take a long time for patients who, despite being otherwise acceptable candidates for kidney transplantation, have developed a broad range of anti Hla antibodies. For many highly allosensitized transplant candidates, an acceptable donor is never identified, and the patient Competing interests The authors declare no competing interests. remains on dialysis indefinitely. in an attempt to ameliorate this situation, innovative protocols have been developed that permit positive crossmatch transplantation that is, transplantation in patients with anti Hla antibodies directed against their donors. 3 7 these proto cols aim to acutely lower Dsa activity to below the level that causes immediate renal allograft injury, and to maintain this reduced level during the first weeks to months after transplantation. During this time, the renal allograft seems to develop a degree of relative resistance to antibodymediated injury, a condition referred to as accommodation, which is thought to be due in part to the upregulation of protective genes. 8,9 Despite requiring additional therapy beyond that needed for conventional transplantations, positive crossmatch kidney transplantations have demonstrated satisfactory short term to inter mediate term allograft survival in multiple investigations. 10 12 in this review, we discuss various requirements for successful positive crossmatch kidney transplantation. such requirements include the comprehensive characterization of Dsas (both before and after transplantation), protocols that lower Dsa activity, and prompt diagnosis and treatment of antibody mediated rejection (amr). Promising new approaches for preventing antibodymediated allograft injury, including complement blockade and the depletion of antibody secreting plasma cells from bone marrow using proteasome inhibition, will also be described. Department of Nephrology and Internal Medicine (J. gloor), Department of Surgery, Division of Transplant Surgery (M. D. Stegall), Mayo Clinic, 200 1 st Street SW, Rochester, MN 55905, USA. Correspondence to: J. Gloor gloor.james@mayo.edu nature reviews nephrology volume 6 may 2010 297

Key points The introduction of human leukocyte antigen (HLA) antibody characterization based on interactions between recipient serum and purified HLA antigens bound to solid-phase substrates has improved detection and quantification of donor-specific antibodies (DSAs) DSA removal through immunoadsorption or plasma exchange, and DSA inactivation using high-dose intravenous immunoglobulins enable successful positive-crossmatch kidney transplantation with good short- to intermediateterm outcomes Positive-crossmatch transplants are associated with an increased incidence of acute antibody-mediated rejection, which, although reversible, has a negative effect on long-term allograft survival Antibody-mediated rejection can occur subclinically and in time results in chronic injury to the renal microvasculature, transplant glomerulopathy, interstitial fibrosis, and tubular atrophy New interventions aimed at the prevention of DSA-mediated allograft injury using complement blockade, or the inhibition of DSA synthesis using proteasome inhibitor-mediated plasma cell depletion, are promising Antibody characterization the importance of anti Hla antibodies in the outcome of kidney transplantation has been recognized for over 40 years. 13 early reports of immediate allograft loss occurring within minutes of allograft reper fusion prompted the term hyperacute rejection. 14 the landmark investigation reported by Patel and terasaki in 1969 established that hyperacute rejection resulted from allograft injury caused by preformed antibodies directed against donor antigens. 15 Following this observa tion, testing transplant candidates for these antibodies became an essential component of pretransplantation evaluation. Cell based crossmatch assays were developed in which recipient sera were incubated with donor lymphocytes in the presence of complement, and cell lysis was interpreted as being an indication of Dsa activity. 16 Histocompatibility laboratories modified the testing procedure to suit their needs, and no single standard protocol was universally adopted. 17,18 some laboratories preferred crossmatch assays with high sensitivity so that lower levels of Dsas could be detected, at the expense of eliminating some donor recipient combinations that could have been successfully transplanted. other laboratories chose to use less sensitive assays with a goal of maximizing the number of transplantations performed, recognizing that as some transplantations would be performed in which low level Dsas were present there would be an increased risk of rejection. Despite the variability in testing techniques, complement dependent cyto toxicity crossmatching reduced the incidence of hyperacute rejection effectively. nevertheless, early rejection episodes still occurred, prompting efforts to develop assays with greater sensitivity than that seen when complement dependent cytotoxicity was used as an end point. 19 assays in which donor lymphocytes were incubated with recipient sera, exposed to anti human globulin labeled with fluorochrome, and then analyzed by flow cytometry were recognized as a more sensitive method for detecting Dsas. 19 the increased sensitivity of antibody assays, which may partly explain the increased number of sensitized patients awaiting transplantation, was, however, associated with decreased specificity, and some non Hla antigens irrelevant to transplantation were found to be capable of producing a positive crossmatch. 20 Furthermore, cell based crossmatch tests were also limited by their inability to clearly identify the antigen producing the positive reaction, as target cells expressed multiple different antigens on their surface. the identification of individual anti Hla antibodies was limited to studying patterns of positive reactions seen in panels of Hla typed lymphocytes subjected to crossmatch testing. Histocompatibility laboratories found that this approach was ineffective in broadly sensitized patients with a wide range of antibody specificities. in addition, assessing the relative importance of anti Hla class i and ii antibodies was complicated by the fact that none of the available cell targets for crossmatch testing expressed only class ii antigens. 18 the use of cell targets such as t lymphocytes, which express Hla class i antigens only, allowed Hla class i specific Dsas to be evaluated. nevertheless, because all nucleated cells express Hla class i antigens, no cellular targets specific for Hla class ii antigens were clinically available. reviewing the transplant literature up to the year 2000 reveals many reports of small series of transplantations with important differences in histocompatibility testing protocols from center to center, and markedly different conclusions drawn regarding the utility of differ ent crossmatch tests. 21 26 as the determination of Dsa specificity was severely limited by the use of cell based tests, donor selection focused on optimiza tion of Hla matching, with the assumption that the closer the match, the lower the likelihood of Dsa presence. the identification of acceptable donors for highly sensitized transplant candidates was dependent on the identification of a donor whose Hla phenotype most closely matched that of the patient. one of the most important developments occurring in transplantation over the past decade was the implementa tion of solid phase anti Hla antibody testing techniques. 27,28 in such tests, purified Hla antigens bound to solid surfaces such as flow beads or incubation wells are incubated with recipient sera and analyzed using flow cytometric or enzyme linked immunosorbent assay (elisa) techniques. Positive reactions are attributable to anti Hla antibodies as the testing platform contains only Hla antigen targets. naturally occurring substances can, however, interfere with interpretation of test results. 29 nevertheless, solidphase testing provides important advantages in sensitivity and specificity over cell based antibody screening techniques. 30 as these solid phase tests employ individual Hla antigens, the reactivity of individual anti Hla antibodies can be determined, which allows recognition of donor specificity. 31 33 in addition to their high degree of specificity, solid phase assays seem to be able to provide quantification of Dsa activity, and investigations have shown that solid phase test positivity correlates with both cell based crossmatch results and with transplant survival. 34,35 298 MAY 2010 volume 6 www.nature.com/nrneph

the introduction of solid phase anti Hla antibody testing techniques has had several important clinical ramifica tions. Firstly, the analysis of sera using tests capable of identifying individual anti Hla antibody speci ficities has made it possible to study the clinical impact of different classes of antibodies. the importance of anti Hla class ii antibodies, in terms of early acute rejection as well as long term chronic amr and transplant glomerulopathy, was not fully recognized until solid phase testing became available. 36 39 although studies had reported that anti Hla antibodies with specificity against class ii antigens had negative effects on outcome, these studies were limited by technical difficulties in antibody characterization using cell based crossmatch assays. 22,40,41 secondly, antibodies directed against histocompatibility antigens, such as Hla class i Cw and Hla class ii DQ, DP, and DQα antigens are now amenable to testing; the clinical relevance of these antibodies remains to be determined. Finally, the fact that specific anti Hla antibodies can now be identified has resulted in a profound change in the approach used to identify donors, particularly for transplant candidates who are highly sensitized. 42 45 instead of selecting acceptable donors on the basis of how closely their tissue typing matches the recipient s, selection is now made on the basis of the identification and avoidance of Hla antigens against which the recipient is sensitized. as a result, the number of potential donors for a highly sensitized transplant candidate is broadened to include anyone whose Hla phenotype does not include any of these unacceptable mismatches. this new approach to organ allocation has had several important clinical implications. one implication is that antibody compatible allografts can now be preferentially allocated to highly sensitized transplant candidates, improving access to transplantation. this method shortens the waiting time for highly sensitized deceased donor kidney transplantation in highly sensitized candidates. 42 in addition, by identifying organs with no unacceptable Hla antigens (negative virtual crossmatch ), transplant programs can proceed with a high level of certainty that the results of the final cellbased crossmatch testing will be acceptable, potentially shortening the time between organ procurement and implantation. 43 this approach has proven particularly beneficial in cardiac and lung transplantation. 42,44,45 Desensitization protocols Preconditioning regimens currently in use for positivecrossmatch kidney transplantation are administered with an immediate goal of preventing antibody mediated allograft injury, which can occur in two settings. Firstly, acute exposure of the renal allograft to high levels of Dsas results in preformed antibody mediated injury, termed hyperacute rejection, which occurs within minutes to hours after reperfusion at the time of transplantation. secondly, following exposure to donor antigens against which the transplant recipient is sensitized, memory B lymphocytes in bone marrow, spleen, and lymph nodes undergo an anamnestic reaction resulting in the formation of antibody secreting cells that produce high levels of Dsas. 46 this reaction typically occurs within days of transplantation, and results in acute humoral rejection characterized by acute tissue injury, activation of endothelial cells and deposition of complement degradation products in peritubular and glomerular capillaries, which often leads to allograft loss. 47,48 Positive crossmatch kidney transplantation therefore requires both pretransplantation and post transplantation interventions that have separate but complementary aims. Protocols for positive crossmatch kidney transplantation have been based on one of two distinct interventions: either high dose intravenous immunoglobulins or plasma exchange typically supplemented with lower doses of intravenous immunoglobulins. in addition to these interventions, pretransplantation induction using anti CD20 antibody therapy has been used on the basis of the rationale that depletion of B lymphocytes may reduce Dsa production. 49 high-dose intravenous immunoglobulin High dose intravenous immunoglobulin has been used for the desensitization of highly allosensitized transplant candidates for nearly 20 years. 5,6,12,50 53 although the exact mechanism of action has not been clearly delineated, an increasing number of investigations have demonstrated an important immunomodulatory effect of pooled igg products administered in high doses (typically >1 g/kg body weight). 54,55 Historically, this immunomodulatory effect has been attributed to the interaction between anti idiotypic antibodies and Dsas. 56 59 the importance of the complement inhibitory effect of intravenous immuno globulin through the blockade of C3, resulting in decreased formation of the potent anaphylatoxin C5a and the C5b C9 membrane attack complex, has since been recognized. 60,61 a second mechanism whereby high dose intra venous immunoglobulin may have an immuno modulatory role involves its interaction with the neonatal Fc receptor, Fcrn, an Hla class i like molecule with a β2 microglobulin subunit expressed on endothelial cells. this molecule serves to protect igg molecules from lysosomal catabolism following pinocytosis. in high doses, exogenous intravenous immunoglobulin saturates the Fcrn, reducing the availability of this ligand to protect injurious endogenous anti Hla antibodies, which results in increased catabolism of Dsas. the Fc portion of the immunoglobulin molecule itself is also capable of immuno modulatory effects via the induction of the inhibitory receptor FcγriiB. 62 over the past few years, the anti inflammatory activity of intravenous immunoglobulin has been linked to a population of sialylated Fc fragments present in pooled preparations of igg. these fragments interact with the C type lectin receptor DC sign (dendritic cell specific intercellular adhesion molecule 3 grabbing nonintegrin) expressed on dendritic cells, resulting in enhanced expression of FcγriiB molecule and release of soluble anti inflammatory cytokines from sialylated Fc activated regulatory cells. 63 Finally, high dose intravenous immunoglobulin has been shown to induce apoptosis of B lymphocytes. 64 nature reviews nephrology volume 6 may 2010 299

an open label, single center study published in 2008 showed that high dose intravenous immunoglobulin administered in conjunction with the anti CD20 antibody rituximab improved transplantation rates of highly allosensitized living and deceased donor kidney transplant candidates. 12 in this study, 20 patients received a preconditioning regimen of 2 g/kg body weight intravenous immuno globulin (given twice over the preconditioning period) and two infusions of rituximab. Panel reactive antibody levels decreased significantly following treatment, and 16 patients were successfully transplanted within 4 months of treatment. importantly, the six patients who received a deceased donor transplant had been on the waiting list for a mean of 144 months before receiving the preconditioning treatment, without a crossmatch compatible donor being identified. although transplanta tion was achieved, 13 of 16 recipients had persistently positive crossmatches at the time of transplantation, which indicates that although the preconditioning regimen allowed transplantation to take place, it was not effective in producing a negative crossmatch. nevertheless, no hyperacute rejections occurred. although the amr rate after transplantation was 31%, 1 year allograft survival was 94%, leading the authors to conclude that intravenous immunoglobulin and anti CD20 antibody induction is an effective desensitizing regimen for patients awaiting living or deceased donor transplants. in another investigation of positive crossmatch kidney transplantation using high dose intravenous immunoglobulin, lefaucheur et al. reported a series of 18 transplantations. 65 recipients were characterized as having negative complement dependent cytotoxicity crossmatches at baseline, with Dsas identified using single antigen flow bead testing. Preconditioning regimens consisted of high dose intravenous immunoglobulin, and immunosuppression treatment consisted of anti thymocyte globulin induction, tacrolimus, mycophenolate mofetil, and corticosteroids. acute amr occurred in five of 18 patients (28%) and was associated with worse graft survival over 8 years follow up (63% in patients diagnosed with amr versus 85% in those without amr; P <0.0001). another study of positive crossmatch kidney transplantation following intravenous immunoglobulin preconditioning in a series of eight patients reported that acute amr incidence was 13% and allograft survival was 88% over a mean follow up of 15 months. 51 importantly, although this group had positive cell based crossmatch tests before transplantation, only three of the eight patients had demonstrable Dsas on solid phase testing. plasma exchange in contrast to preconditioning regimens involving highdose intravenous immunoglobulin, protocols based on plasma exchange aim to physically remove Dsas before transplantation. with this approach, transplant candidates undergo a series of plasma exchange treatments, often supplemented with low dose intravenous immuno globulin before transplantation. transplantation is performed once Dsa activity has been reduced to an acceptable level. 3,4,7,10,11 During the first days to weeks after transplantation, many protocols incorporate further plasma exchange with or without intravenous immunoglobulin therapy directed towards maintaining low levels of Dsas. the rationale for plasma exchange as a preconditioning treatment for positive crossmatch kidney trans plantation was based on reports showing that this therapy was effective in the treatment of acute amr. 66 early reports of successful positive crossmatch kidney transplantation demonstrated that hyperacute rejection was avoided but that acute amr occurred in many recipients within the first several days to weeks after transplantation. 3,4 Despite this finding, however, early allograft loss was rare, and the feasibility of the approach was established. Positive crossmatch kidney transplantation using plasma exchange and low dose intravenous immunoglobulin has since been implemented in other transplantation centers. thielke et al. reported that a negative crossmatch was successfully achieved in 51 of 57 positive crossmatch patients treated with anti thymocyte and anti CD20 antibody induction therapy in addition to plasma exchange and low dose intra venous immunoglobulin. the rate of allograft survival was 93% at 1 year and 81% at 2 years. 10 similar to previous reports, 3,4,7,10,11 amr was diagnosed in 22 of 51 patients (43%). using a similar protocol, magee and colleagues reported a series of 28 positive crossmatch transplantations in which allograft survival rate was 90% at 1 year and 80% 2 years. in this series, amr was diagnosed in 12 of 28 patients (42%). 11 in another series of 41 positive crossmatch transplant recipients treated with a preconditioning regimen of plasma exchange supplemented with intravenous immuno globulin, 1 year and 5 year allograft survival rates were 89.9% and 69.4%, respectively, and five patients developed amr (12%). importantly, this investigation compared 8 year allograft survival in the treatment group with that in a control group of 41 patients with negative crossmatch (matched for sex, age, race, retransplantation and year of transplantation), and found that allograft loss was signifi cantly higher in the positive crossmatch group. 67 in an analysis of 119 positive crossmatch kidney transplanta tions, our group found that increasing baseline Dsa levels, determined either with cell based crossmatch tests or solid phase assays, was associated with an increased risk of amr. 35 acute amr, which complicated over 50% of transplanta tions performed in patients with the highest baseline levels of Dsas, also occurred in 30% of transplantations performed in patients with low baseline Dsa levels. allograft survival was also related to baseline Dsa level. the 3 year allograft survival rate ranged from 70% in patients with high baseline Dsa levels (complement dependent cytotoxicity assay positive crossmatch) to 94% in patients with lower baseline Dsa levels (flow cytometric positive crossmatch). Challenges of desensitization protocols although both high dose intravenous immuno globulin and plasma exchange with or without intravenous immuno globulin effectively achieve an acceptable level 300 MAY 2010 volume 6 www.nature.com/nrneph

Table 1 Acute AMR incidence* and its effect on 1-year and 2-year allograft survival Study number of patients AMr incidence (%) 1-year death-censored allograft survival (%) 2-year death-censored allograft survival (%) Lefaucheur et al. (2008) 65 43 35 89 89 Thielke et al. (2009) 10 51 32 93 81 Magee et al. (2008) 11 28 39 92 89 Gloor et al. (2010) 35 119 41 89 89 Haririan et al. (2009) 67 41 12 90 85 vo et al. (2008) 12 16 30 94 Not reported *After positive-crossmatch kidney transplantation. Abbreviation: AMR, antibody-mediated rejection. of Dsas in many positive crossmatch candidates, these approaches do have disadvantages. Firstly, both interventions increase resource utilization, in terms of preconditioning (plasma exchange, intravenous immuno globulin, and Dsa characterization), as well as therapy for acute amr. Disadvantages of high dose intravenous immunoglobulin include an association with thrombotic complica tions and the difficulty of administering large fluid volumes to anuric dialysis patients awaiting transplantation. 68,69 in a comparison of positive crossmatch transplant candidates undergoing preconditioning treatments, our group found that neither high dose intravenous immunoglobulin nor plasma exchange supple mented with intravenous immunoglobulin were effective in achieving a negative crossmatch in patients with high baseline Dsa levels, despite both approaches being effective in candidates with low Dsas. 70 notably, high dose intravenous immunoglobulin was administered as one dose in our investigation, whereas other protocols using this agent incorporate multiple doses administered over several weeks to months. 5,6 High dose intravenous immunoglobulin seems to be more effective than plasma exchange in achieving transplantation in highly sensitized candidates awaiting deceased donor transplantation. Dsa levels rapidly rebound to pretreatment levels upon discontinuing plasma exchange supplemented with intra venous immuno globulin therapy, making deceased donor transplantation impractical if this approach is used. 71,72 in a multicenter, randomized, placebo controlled trial, Jordan et al. evaluated serial infusions of high dose intravenous immunoglobulin in a cohort of highly sensitized candidates awaiting deceased donor transplantation. 52 they found that the rate of transplanta tion was higher in the group treated with intravenous immunoglobulin than in the placebo group, although the incidence of amr was greater in patients treated with intravenous immunoglobulin. Despite the increase in amr, however, the 2 year allograft survival was similar in both groups, which led the authors to conclude that periodic infusions of high doses of intravenous immuno globulin are an effective approach to desensitization before transplantation for highly sensitized candidates. Antibody-mediated rejection although the low incidence of hyperacute rejection and the favorable renal allograft survival rates observed in recent reports of positive crossmatch transplantations are encouraging, acute amr remains a common occurrence, complicating 20 50% of positive crossmatch transplants regardless of the method used for pretransplanta tion conditioning (table 1). 10 12,35,65,67 in addition, acute amr is increasingly recognized as a risk factor for poor graft outcome after transplantation. in a study comparing long term outcomes of a series of 41 positive crossmatch transplants with those of a matched group of conventional transplants, Haririan et al. found that acute amr was a strong predictor of inferior graft survival. 67 similarly, lefaucheur et al. observed that in the absence of acute amr, 8 year allograft survival was similar in patients with and without Dsas at transplantation, whereas among transplant recipients who developed acute amr, those with Dsas had markedly reduced graft survival compared with those without Dsas. 65 the graft loss associated with acute amr was not limited to the immediate time period in which the rejection occurred; 1 year allograft survival was reduced to 60% in patients diagnosed as having acute amr, compared with 89% in the absence of amr, and another 15% of patients with amr lost function over the following 7 years. in a series of positive crossmatch transplant biopsies performed 1 year after transplantation, our group reported that the incidence of transplant glomerulopathy a manifestation of chronic amr was 44% in patients with previous acute amr, compared with 12% in patients without previous acute amr. 73 thus, the early successful reversal of acute amr with recovery of allograft function might not indicate that the rejection process has been halted. Historically, amr has often been considered to be an event that complicates antibody incompatible transplantations. amr is, however, increasingly being recognized as a process, that begins with acute histological lesions and progresses to chronic changes. 74 initially, acute histological injury, characterized by endothelial cell swelling, detachment from the basement membrane, and fragmentation, predominantly affects peritubular and glomerular capillaries. these changes are visible by electron microscopy before their detection by light microscopy, and may occur early in the post transplantation transplant period. 75 77 with time, chronic injury to the allograft microvasculature results in remodeling of the peritubular and glomerular capillary basement membrane, producing the characteristic multilayering or lamellation pattern indicative of transplant glomerulo pathy. 78 the nature reviews nephrology volume 6 may 2010 301

a Figure 1 The effect of antibody-mediated rejection on peritubular capillaries. a Normal peritubular capillaries (arrows). CD34 immunoperoxidase stain; original magnification 20. b Obliteration of peritubular capillaries (arrows) following chronic antibody-mediated rejection. CD34 immunoperoxidase stain; original magnification 20. Figure 2 Infiltration of peritubular capillaries by mononuclear cells in subclinical chronic antibody-mediated rejection. Arrow shows infiltrated capillary. Periodic acid Schiff stain; original magnification 40. b final result of this obliterative vasculopathy involving the allograft microcirculation is progressive interstitial fibrosis and tubular atrophy (Figure 1). 75 this evolution in our understanding of the nature of amr is reflected in the continuing refinement of the Banff classification of renal allograft pathology. in 2003, the Banff classification system originally proposed in 1997 was modified to include diagnostic criteria for acute amr, including evidence of acute tissue injury, detection of circulating Dsas, and deposition of the complement degradation product C4d in peritubular capillaries. 47,79 this classification was further modified in 2005 to incorporate criteria for chronic amr, characterized by peritubular and glomerular capillary basement membrane remodeling, including transplant glomerulopathy. 80 although many of the acute histological changes associ ated with acute amr are mediated through comple ment activation, complement independent mech anisms are increasingly being recognized to also be involved. the involvement of complement independent mechanisms has been most clearly demonstrated for anti Hla antibodies with class i specificity, in which interaction with Hla antigens on cell surfaces can stimulate intracellular signal transduction mechanisms, resulting in cell proliferation. 81 83 although often associated with allograft dysfunction, amr commonly occurs subclinically (Figure 2). in 2007, Haas et al. reported histological evidence of subclinical acute amr consisting of peritubular capillary monocytic infiltration in 10 of 83 positive crossmatch kidney transplant recipients who were biopsied during the first year after transplantation. 84 although the majority of this group never developed acute allograft dysfunction, an increase in transplant glomerulopathy, chronic inter stitial fibrosis and tubular atrophy were seen on subsequent biopsies. in 2009, the same group reported the incidence of subclinical amr to be 40% on protocol kidney biopsies performed 12 months after transplantation in a series of 50 positive crossmatch kidney transplant recipients. 85 renal allograft function at the time of biopsies was not significantly different between patients with subclinical amr and those without. similar to Haas and colleagues, lerut et al. also found that monocytic infiltration and margination of peritubular capillaries seen on protocol biopsies performed 3 months after transplantation was predictive of chronic amr by 12 months after transplantation. 86 Finally, in the series reported by our group, transplant glomerulopathy developed by 36 months in 77% of positive crossmatch transplants complicated by acute amr compared with 55% of transplants without acute amr. 35 the findings of these investigations underscore the fact that active antibody mediated renal allograft injury occurs in a substantial proportion of positive crossmatch kidney transplants even in the absence of acute allograft dysfunction, and that over time this process results in chronic histological injury. evidence is accumulating that the renal allograft has a limited capacity to accommodate to long term exposure to Dsas. Treatment of antibody-mediated rejection one of the key advances that has permitted positivecrossmatch kidney transplantation is the characterization of the histological appearance of acute amr. 48,87 89 establishing the criteria for a diagnosis of acute amr improved our ability to assess the efficacy of treatment; the resistance of acute amr to therapy directed against acute cellular rejection has been recognized since the 1990s. 88 Currently, treatment for acute amr aims to lower the level of Dsa activity, in addition to attempting to decrease further Dsa production. 90 Both plasma 302 MAY 2010 volume 6 www.nature.com/nrneph

exchange and immunoadsorption have been shown to rapidly lower circulating levels of Dsas. 90,91 in a small, randomized, controlled trial that compared the efficacy of immunoadsorption with the efficacy of conventional therapy in a series of 10 patients with acute amr, the high rate of allograft loss in the control group prompted early termination of the study. 92 the investigators concluded that immunoadsorption was effective in reversing severe, acute amr. intravenous immuno globulin has also been used in the treatment of acute amr, using the same rationale as that proposed for its use in pretransplant conditioning. 93 in addition to lowering the level of circulating Dsa, therapies for acute amr also aim to inhibit further synthesis of antibodies. several interventions including antithymocyte antibody treatment, depletion of B lympho cytes using anti CD20 antibody, and splenectomy have been proposed to accomplish this aim. 94 99 studies showing efficacy of these interventions have been limited by small numbers, a lack of control patients, and the incorporation of the interventions into complex treatment programs without a standard protocol. anti CD20 antibody therapy, through its celldepleting effect on B lymphocytes, has the potential to interrupt the production of Dsa secreting plasma cells, and constitutes a rational target for therapy in the setting of acute amr. nevertheless, although a single dose of the chimeric, monoclonal anti CD20 antibody, rituximab, results in the effective depletion of CD20 + B lymphocytes from peripheral blood, CD27 + memory B cells are still demonstrable in spleen. 100 although anti CD20 antibody therapy might seem to be an appropriate intervention for the treatment of acute amr, the data supporting its use remain inconclusive. Future directions although current protocols effectively prevent hyperacute rejection, acute amr remains an important complica tion in positive crossmatch kidney transplantation and carries a substantial risk of allograft loss. in addition, individuals with high Dsa levels at baseline are often resistant to treatment and are unable to proceed to transplantation. 4,10,12 although positive crossmatch protocols have improved access to transplantation for highly sensitized candidates, further advances are clearly needed. two potentially important innovations that are currently being explored are the prevention of antibodymediated endothelial cell injury by complement blockade and the depletion of Dsa secreting plasma cells from the bone marrow using proteasome inhibition. Complement blockade High levels of Dsas activate complement, thereby leading to the formation of membrane attack complex that injures donor endothelium and results in the histo logical abnormalities diagnostic of acute amr. eculizumab is a humanized monoclonal antibody that binds to the complement factor C5 with high affinity, inhibiting its cleavage to C5a and C5b, and preventing the formation of the membrane attack complex. in a presensitized murine model of acute amr, C5 blockade in combination with conventional immunosuppression was shown to prevent antibody mediated allograft injury, resulting in prolonged graft survival. 101 in a case report, locke et al. describe the resolution of severe amr in a single patient following treatment with anti C5 antibody. 102 although the response to therapy was temporally related to the anti C5 antibody administra tion, the authors caution that the treatment was given in the context of multiple different interventions, making it diffi cult to determine the impact of the anti C5 antibody on outcome. this observation highlights how difficult it is to evaluate the efficacy of new agents without controlled trials. nevertheless, a study reported by Burns et al. found that increasing Dsa level (determined by flow cytometric crossmatch) correlated with an increasing risk of acute amr in a series of 70 positive crossmatch transplant recipients who had simultaneous measurement of Dsas and allograft biopsies performed during the first month after transplantation. 103 in that investigation, 92% of biopsies obtained at the time that the donor B lymphocyte flow cytometric crossmatch channel shift exceeded 359 were diagnostic of acute amr. 103 this observa tion not only has potential importance in patient manage ment, but also serves to provide a comparison group for later investigations aimed at studying the efficacy of new interventions directed at preventing amr. the same investigators reported a series of 10 positive crossmatch kidney transplant recipients treated with anti C5 antibody, in which five patients developed high Dsa levels during the first weeks following transplantation. Despite having Dsa levels similar to those associated with amr in the earlier study, none of the five patients had abnormal biopsy findings on light or electron microscopy. 104,105 the authors concluded that C5 blockade effectively prevents complement mediated allograft injury in the first month after transplantation. proteasome inhibition in addition to physically removing or inhibiting of Dsas, decreasing Dsa production is a goal for positivecrossmatch transplant protocols. nevertheless, agents currently in use such as anti CD20 antibodies, antithymocyte antibodies, and intravenous immunoglobulin are ineffective in depleting antibody secreting plasma cells resident in the bone marrow, which has prompted investigators to consider other agents. 106 in the past decade, proteasome inhibition has been recognized as a potentially important intervention with applications for enabling transplantation in allo sensitized patients through its role in cell cycle regulation, cell cell interactions, and apoptosis. 107 the proteasome inhibitor bortezomib is effective in the treatment of multiple myeloma, a plasma cell malignancy. 108,109 in 2008, everly et al. reported that bortezomib use was associated with rapid improvement in allograft function and suppression of recurrent rejection over a 5 month period in six kidney transplant recipients with severe combined amr and acute cellular rejection. 110 nature reviews nephrology volume 6 may 2010 303

in an attempt to mechanistically study the impact of proteasome inhibition on plasma cells, Perry and colleagues cultured bone marrow derived CD138 + plasma cells using an enzyme linked immunosorbent spot (elispot) technique. 106,111 when cultured in the presence of Hla antigens, antibody secreting plasma cells were detectable in cultures obtained from allosensitized individuals, and anti Hla antibodies were detectable in the culture supernatant. 106 no significant decrease in plasma cells was observed when cultures were incubated with anti CD20 or antithymocyte antibody or intravenous immunoglobulin, but when the proteasome inhibitor bortezomib was added to the culture, a significant decrease in the number of plasma cells occurred, along with an increase in the number of plasma cells undergoing apoptosis and a decrease in anti Hla antibody detectable in the culture supernatant. 111 in an in vivo study of the effect of bortezomib on bone marrow plasma cells in highly allosensitized kidney transplant candidates, two of three patients treated with bortezomib had a significant decrease in CD138 + antibody secreting cells following treatment. 112 although the investigations described above are preliminary, they are indicators that interventions directed toward decreasing Dsa production may be feasible. Conclusions advances in anti Hla testing techniques, together with the ability to characterize the histological appearance of acute amr more accurately, have provided clinicians with the tools necessary to begin to address the important issue of transplantation in allosensitized patients with renal failure. Protocols involving either depletion or inactivation of Dsas are effective in preventing hyperacute rejection, but acute amr remains an important problem, complicating 20 50% of positive crossmatch transplantations. although therapies are available that are acutely effective in reversing amr, this condition carries an important negative prognostic effect. new interventions for preventing acute amr that are based on complement blockade and proteasome inhibition seem promising. Review criteria We searched the MeDLINe databases for papers in english published between 1980 and 2009 (inclusive), using the terms: kidney transplantation, crossmatch, HLA antibody, antibody-mediated rejection, plasmapheresis and intravenous immunoglobulin. The reference lists of retrieved articles were also searched to include relevant papers. 1. Annual Report 1998 2007 HHS/HRSA/HSB/ DOT (OPTN/SRTR, 2008). 2. Moore, S. B., Sterioff, S., Pierides, A. M., Watts, S. K. & Ruud, C. M. Transfusion-induced alloimmunization in patients awaiting renal allografts. Vox Sang. 47, 354 361 (1984). 3. Montgomery, R. A. et al. Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients. Transplantation 70, 887 895 (2000). 4. Schweitzer, e. J. et al. A high panel-reactive antibody rescue protocol for cross-match-positive live donor kidney transplants. 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