The increasing clinical importance of alloantibodies in kidney transplantation.

Immunological Investigations, 2014; 43(8): 775–789 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.910016

The increasing clinical importance of alloantibodies in kidney transplantation Oleh Pankewycz,1 Karim Soliman,2 and Mark R. Laftavi2 1

Immunol Invest Downloaded from informahealthcare.com by Selcuk Universitesi on 01/13/15 For personal use only.

Department of Medicine, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York, USA and 2 Department of Surgery, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York, USA Historically, cellular rather than humoral immunity has gathered the most attention in kidney transplantation. As the specter of cellular acute rejection and early graft loss has faded due to the availability of highly effective immunosuppressive therapy, scientific and clinical studies now focus on improving long-term graft survival. It is increasingly appreciated that alloantibodies directed against HLA and non-HLA antigens are key factors in determining graft longevity. Significant efforts are now being made to better understand the critical impact that B cells and alloantibodies make on organ allocation and graft survival. Future therapies directed specific for the humoral alloresponse will undoubtedly lead to improved outcomes after kidney transplantation. This review will cover some of the advances in the understanding and management of the continuum of humoral immunity in renal transplantation in the pre, peri and post-transplant periods. Keywords Alloantibodies, donor specific antibodies, humoral rejection, kidney transplantation

INTRODUCTION Over the past 50 years, the objective of immunosuppressive therapy for kidney transplantation has been preventing T cell mediated acute rejection. The outcome of these efforts is that modern medications are highly effective and relatively less toxic. Now, early cellular rejection rates are typically less than ten percent and the one year renal allograft survival rate approaches ninety percent (Matas et al., 2014). Despite this early success, long-term renal allograft survival rates have not shown equivalent improvements. The causes of chronic renal allograft loss that occur over the course of years are difficult to establish. Historically, many factors have been implicated in this process including: calcineurin toxicity, normal aging, infectious diseases, systemic illnesses such as hypertension and diabetes, consequences from ischemia reperfusion injury and subclinical rejection (Gago et al., 2012; Renders & Heemann 2012). Recently, the importance of humoral immunity to renal allograft function and survival has become increasingly appreciated (Coelho et al., 2013). A major technological advance was the development of the single antigen bead (SAB) assay in which recombinant HLA Correspondence: Oleh Pankewycz, Department of Medicine, Division of Nephrology, State University of New York at Buffalo, Eire County Medical Center, 462 Grider Street, Buffalo, NY 14215, USA. E-mail: [email protected]

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(Human Leukocyte Antigen) peptides are expressed on microbeads used to detect anti-HLA alloantibodies. The SAB assay easily allows for identification, enumeration and characterization of alloantibodies with exquisite specificity. This new tool altered the previously held T cell centric model of renal allograft damage to include a significant contribution from B cells (Colvin et al., 2010). Pre-transplant period The first renal transplantation was successful because the donor and recipient were identical twins. Sharing identical MHC (Major Histocompatibility Complex) regions, identical twins have little to no risk of having preformed alloantibodies to each other’s HLA antigens. When renal transplantation was offered to less than ideally MHC matched patients, the danger of preformed anti-donor alloantibodies became apparent after certain patients developed acute humoral rejection within hours to days. A major advance in transplantation occurred in 1968 when Terasaki and colleagues developed a serological test to detect preformed alloantibodies, the microcytotoxicity or complement dependent cytotoxicity crossmatch test (CDCC) (Mittal et al., 1968). Since that time, newer technologies improved the sensitivity of alloantibody detection at the cost of an ability to discern their pathogenic subset (Minucci et al., 2011). The CDCC is a functional test that assesses the pathogenic potential of preformed anti-donor antibodies (Figure 1). Prior to transplant, a final CDCC is performed to determine donor and recipient compatibility. A donor’s lymphocytes are mixed with recipient serum for 30 min followed by the addition of rabbit complement. The presence of a complement fixing anti-donor IgM or IgG alloantibody is confirmed if there is greater than 20% cell death determined by vital dye exclusion. Transplanting a kidney in the presence of a positive CDCC leads to hyperacute or acute rejection which results in rapid graft destruction and organ loss. Although the CDCC is highly specific for the risk of antibody mediated rejection (AMR), it is relatively insensitive. Thus, a negative CDCC does not guarantee the absence of hyperacute or acute humoral rejection. The CDCC assay was also instrumental in developing the traditional tissue typing system for the serologically defined Class I (A and B) and eventually Class II (DR) HLA antigens. Today, final crossmatches are performed by a more sensitive flow cytometry assay, a flow crossmatch (FXM) (Figure 1). To perform a FXM, donor cells are mixed with recipient’s serum. Binding of any preformed anti-donor IgG alloantibodies to donor cells is detected by the addition of a fluorescein labeled anti-human IgG. The cells are then analyzed in a flow cytometer by comparing their florescence signal before and after incubation with recipient serum. Typically, a positive FXM results in an increase in florescence intensity expressed as a ‘‘channel shift’’ of over 40 channels. However, FXM standards vary amongst individual laboratories. Labeled anti-T cell or anti-B cell antibodies can be added to differentiate between anti-Class I and Class II alloantibodies, respectively. False positive FXM results result from binding of non-HLA antibodies and non-specific immunoglobulin binding to donor cells. Unlike the CDCC assay, the FXM test does not distinguish between cytotoxic and non-pathogenic alloantibodies. Thus, the risk of a positive FXM to


Alloantibodies and kidney transplant

Figure 1. Methods of detecting alloantibodies. (A) Complement dependent cytotoxicity assay detects lytic antibodies by the addition of a vital dye to measure cell death; (B) Flow crossmatch detects alloantibodies by measuring an increase in florescence ‘‘channel shift’’; (C) Single antigen bead assay detects alloantibodies by measuring the mean florescence intensity of HLA-coated bead. Red bars denote positive results above the threshold of 1,500 MFI units.

transplantation is relatively less than a positive CDCC but still represents a significant barrier to transplantation (Couzi et al., 2011; Kimball et al., 2011). Recently, two highly sensitive assays have been developed to pre-screen recipient serum for the number, specificity and binding strength of anti-HLA alloantibodies (Susal et al., 2013a). In the ELISA assay, purified HLA antigens isolated from well-defined human cell lines are fixed onto plastic wells. An individual’s serum is added to the wells followed by an enzyme-linked antihuman IgG antibody. Bound preformed alloantibodies are detected in a colorimeter after the addition of an appropriate substrate. The other screening assay is based on microbeads onto which recombinant variable regions a single HLA antigen (SAB) or several HLA antigens are fixed. Recipient serum is mixed with individual beads and binding of a preformed alloantibody is detected by flow cytometry after the addition of a fluorochrome labeled antihuman IgG. Results are reported as the mean florescence intensity (MFI) of individual beads (Figure 1). Typically, a MFI of greater than 1,000 or 1,500 is considered positive but this may vary between laboratories. Class I and II alloantibodies identified by SAB assay are significant risk factors for graft failure (Caro-Oleas et al., 2013; Dunn et al., 2011; Hirai et al., 2012; Mohan et al., 2012; Otten et al., 2012; Perez-Flores et al., 2013). Current organ allocation programs utilize the results of SAB assays to develop a

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‘‘virtual’’ crossmatching organ sharing system that predicts unacceptable donor/recipient pairs based on the presence of preformed alloantibodies (Susal et al., 2013). This system allows for a more rapid allocation of organs with fewer rejections and improved outcomes (Bray et al., 2006). However, solid phase assays do not determine the pathogenicity of alloantibodies, are not fully standardized amongst laboratories and are subject to false positive results (Gombos et al. 2013; Grenzi et al. 2013; Reed et al. 2013; Tait et al. 2013). Therefore, assessing the actual risk of rejection in individual patients from solid phase assay results is challenging. Overall, the clinical impact of an alloantibody detected in positive sold phase assays appears less significant than if identified by CDCC or FXM (Couzi et al., 2011). A recent innovation in SAB technology has been the ability to evaluate whether alloantibodies bind complement. Preliminary results from clinical trials predicting post-transplant outcomes based on the results of pre-transplant complement binding SAB assays are promising and lend support for additional study (Loupy et al., 2013). Avoiding preformed alloantibodies based on solid phase assays is now the foundation of modern organ allocation (Tait et al., 2013). Based on solid phase assays, patients waiting for transplantation are assigned a calculated panel reactive antibody (cPRA) score. The cPRA reflects percentage of the donor population that expresses the HLA antigens against which the recipient has preformed alloantibodies. In addition, cPRA indirectly estimates post-transplant immunological risk. Patients with a cPRA greater than 20–30% are considered sensitized to HLA antigens and are felt to be at higher risk for rejection compared to unsensitized individuals with a 0% cPRA. The cPRA values can fluctuate over time as new anti-HLA antibody specificities are uncovered and others disappear. Peri-transplant period During the early post-transplant period, renal allografts are at risk of acute antibody mediated (AMR) or humoral (AHR) rejection. Should the transplant be performed in the face of a positive final CDCC, the result may be ‘‘hyperacute’’ AMR. In hyperacute AMR, preformed alloantibodies bind to HLA antigens expressed on the donor kidney’s endothelium and activate complement. This results in complement mediated direct cellular toxicity, activation of the coagulation cascade, vascular thrombosis and an influx of immune cells including macrophages and NK cells all of which leads to rapid destruction of the allograft (Hidalgo et al., 2010). Given a negative final FXM, hyperacute AMR a rare event in today’s transplantation practice. In sensitized patients, having a negative CDCC or FXM at the time of transplantation does not exclude developing an accelerated humoral rejection (AHR). In cases of AHR, renal allograft function suddenly declines or ceases within days after transplantation. AHR occurs after a donor specific antibody (DSA) rapidly develops presumably due to a B memory cell response. Risk factors for developing AHR include having a pre-existing anti-donor alloantibody that escaped detection by FXM or having a prior ‘‘historic’’ alloantibody that was absent at the time of transplant.



Due to the increasing shortage of kidneys suitable for transplantation, the previously held truism that preformed alloantibodies preclude successful transplantation has been reassessed. The new view is that assays to detect preexisting DSAs are measures of relative rather than absolute immunological risk after transplantation. As mentioned, the greatest risk for renal allograft rejection and failure is a positive CDCC due to either Class I or Class II antidonor HLA antibodies. A positive FXM prohibits transplantation in cases of deceased donation but is considered a less daunting barrier to living donor transplantation (Bentall et al., 2013). Pioneering work done at several leading transplant centers including the Mayo Clinic, UCLA and Johns Hopkins University explored a variety of preconditioning or ‘‘desensitization’’ strategies to eliminate alloantibodies in order to successfully transplant sensitized patients (Gloor et al., 2003; Jordan et al., 2012; Montgomery & Zachary, 2004; Reinsmoen et al., 2012). Various combinations of preemptive therapies and protocols to remove or lower the titers of CDCC or FXM identified alloantibodies have been evaluated (Gloor et al., 2003; Jordan et al., 2012; Montgomery et al., 2012). Preconditioning regimens included splenectomy, high or low doses of IVIG infusions, plasmapheresis, anti-CD20 monoclonal antibody (rituximab) and, most recently, the use of a cellular proteosome toxin (bortezomib) (Ejaz et al., 2013; Everly et al., 2012; Vo et al., 2010). Clinical trials indicated that high titers of CDCC detected alloantibodies, greater than 1:32 dilutions, pose a significant threat to rejection post-transplant with poor outcomes and so are generally avoided (Montgomery, 2010). Splenectomy has fallen out of favor as current desensitization protocols use an aggressive combination of pre-transplant plasmapheresis followed by infusions of IVIG (200 mg/kg/session) and one or more doses of rituximab. Immunosuppressive therapy with tacrolimus and mycophenolic acid are usually initiated prior to transplant. Frequent monitoring of alloantibody titers is imperative and proceeding with surgery when titers are deemed acceptable per center protocol. Following transplantation, continued therapy with rabbit anti-thymocyte globulin (rATG) combined with plasmapheresis and IVIG infusions are usually required. Despite these efforts, vigorous AMR occurs in upwards of 60% of desensitized patients requiring additional therapy (Gloor et al., 2010; Lefaucheur et al., 2008). Overall clinical outcomes in desensitized patients are significantly inferior when compared to unsensitized recipients (Bentall et al., 2013). However, patient outcomes following desensitization and transplantation are significantly better compared to those who never receive a kidney transplant (Montgomery et al., 2012). As with CDCC assays, it is difficult to desensitize a recipient who has a strongly positive FXM assay of greater than 300 channel shifts (Bentall et al., 2013). For strongly FXM positive patients, a desensitization protocol of IVIG plus rituximab lowered the intensity of the FXM allowing for successful living and deceased donor transplantation (Gloor et al., 2010; Vo et al., 2010). For weaker FXM, desensitization protocols include pre-transplant high dose IVIG or plasmapheresis combined with lower doses of IVIG without the routine addition of rituximab. Transplantation proceeds when the FXM becomes negative or falls to an acceptable range determined by the individual transplant center. Even after acceptable FXM desensitization, AMR occurs

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in approximately 30-40% of recipients requiring further plasmapheresis, IVIG and rituximab to treat resistant cases (Bentall et al., 2013; Gloor et al., 2010; Higgins et al., 2007; Vo et al., 2010). Today, solid phase assays are used to direct pre-transplant desensitization protocols (Hirai et al., 2012). As with CDCC and FXM, desensitization strategies with IVIG and rituximab have been more successful in patients with a low degree of sensitization (Djamali et al., 2013; Hirai et al., 2012; Marfo et al., 2012). In general, positive pretransplant DSAs detected by SAB alone predict poor patient outcomes even after desensitization with upwards of 30% experiencing acute rejection (Higgins et al., 2007; Yamanaga et al., 2013). However, a significant percentage of DSA positive patients do not reject and enjoy good results (Perez-Flores et al., 2013). Thus, the clinical significance of a pre-transplant DSA detected in a solid phase assay without a concomitantly positive FXM or CDCC remains uncertain (Gupta et al., 2008; Susal et al., 2011). In the future, the intensity of the alloantibody signal or its ability to bind complement in a SAB assay may prove useful in discriminating pathogenic alloantibodies that require desensitization from those that are less harmful (Bartel et al., 2013; Loupy et al., 2013). Alloantibodies detected at MFIs in the range of 5,000 or greater are more likely to be cytotoxic and represent strong barriers to transplantation (Fidler et al., 2013; Lefaucheur et al., 2010). Patients with DSA MFIs between 1,500 and 4,000 are considered moderately sensitized and are readily desensitized with IVIG infusions prior to transplantation. Rejections in these circumstances are easier reversed with better long-term results. Post-transplant period As acute cellular rejection has become less of a threat to early allograft survival, transplant research now focuses on improving long-term graft survival. Recent technological advances such as protocol biopsies and SAB testing for de novo DSA (dnDSA) provide novel methods to monitor the immunological health of allografts. These innovations led to awareness that the therapeutic window of immunosuppressive therapy is less than previously suspected and that individualized drug therapy is a critical but unmet goal. In the absence of reliable biomarkers reflecting the health of the allograft, the use of surveillance allograft biopsies has become commonplace (Pankewycz et al., 2011). Protocol biopsies revealed that acute rejections occur in the absence of detectable changes in renal function. Such subclinical rejections represent an unsuspected state of inadequate immunosuppression that, if not treated, may result in chronic humoral immune mediated damage. A pivotal finding in this regard was that late kidney failure was frequently associated with dnDSA and microcirculatory inflammation indicative of chronic humoral rejection (Einecke et al., 2009; Gaston et al., 2010; Hidalgo et al., 2009). SAB assays may provide another means to track the adequacy of immunosuppressive therapy. Prospective studies monitoring SAB assays demonstrated that, in low risk patients, the yearly incidence of dnDSAs ranges from 2% to upwards of 10%) (Cooper et al., 2011; Devos et al., 2012; Dieplinger et al., 2014; Everly et al., 2013; Everly et al., 2014; Lachmann et al., 2009; Wiebe et al., 2012). Typically, dnDSAs appear after an episode of early acute rejection



(El Ters et al., 2013). However, dnDSAs can also be detected in apparently stable patients (Cooper et al., 2011; Terasaki & Cai, 2008; Wiebe et al., 2012). When detected prior to clinical rejection, dnDSA may be the result of previous or ongoing subclinical rejection (Wiebe et al., 2012). The time to onset of dnDSAs is variable depending on the patient population studied and the test parameters used to determine positivity. Typically, dnDSAs appear within the first two years after transplantation, however, late onset anti-Class II alloantibodies were commonly observed 2 to 4 years posttransplant (Cooper et al., 2011; Dieplinger et al., 2014; El Ters et al., 2013; Everly et al., 2013, 2014; Lee et al., 2009; Ntokou et al., 2011; Wiebe et al., 2012). Existing information now strongly supports the contention that the presence of an IgG dnDSA is a major risk factor for developing more frequent and severe rejections as well as worse graft function and survival (Cooper et al., 2011; Everly et al., 2013, 2014; Wiebe et al., 2012). In addition to acute rejection, several other risk factors are implicated in the development of dnDSAs. Patient noncompliance is a major cause of dnDSAs usually late anti-Class II alloantibodies. Estimates are that over 50% of patients develop dnDSAs due to a failure to abide by their drug regimen or clinic visit schedule resulting in inadequate or inconsistent immunosuppression (Sellares et al., 2012; Wiebe et al., 2012). Young recipients, recipients of kidneys from deceased donors, patients with non-DSA anti-HLA antibodies prior to surgery and those with Class II HLA mismatches are additional risk factors for developing dnDSA (Everly et al., 2013; Wiebe et al., 2012). De novo DSAs may occur in patients who have a historic positive crossmatch with their donor or a DSA that was incorrectly undetected at the time of surgery (Everly et al., 2013; Lefaucheur et al., 2008). When not prospectively monitored, circulating dnDSAs are often detected during an episode of rejection. In acute AMR, renal damage is mediated by DSA binding to HLA alloantigens along the allograft endothelium and activating complement (Taflin et al., 2011). That complement is a major cause of renal pathology in AMR is supported by observing C4d present along the capillary walls surrounding renal tubules accompanied by inflammation or capillaritis (Cornell, 2013; de Kort et al., 2013). Moreover, in cases of severe AMR with widespread inflammation and vasculitis an antibody that targets and inhibits complement component C5a, eculizumab, effectively halts tissue damage and blocks further renal injury (Barnett et al., 2013a; Locke et al., 2009). Generally, the diagnosis of AMR consists of having a DSA, capillaritis, C4d deposits and renal dysfunction (Mengel et al., 2012). However, the pathological manifestations of AMR may be quite variable and, at times, difficult to establish (Mengel et al., 2012; Rafiq et al., 2009; Sis et al., 2010). Confounding diagnostic efforts, the pathological criteria for AMR are constantly being revised as new information is generated (Haas et al., 2014). Moreover, cellular rejection may coexist with AMR (Dorje et al., 2013; Dunn et al., 2011; Lefaucheur et al., 2013; Matignon et al., 2012). A future molecular based approach may prove more decisive in diagnosing AMR () (Akalin & O’Connell, 2010; Halloran et al., 2013; Sellares et al., 2013). Regardless of the clinical circumstances, the therapy of destructive acute AMR is aimed at lowering the titer and eliminating DSAs (Djamali et al., 2014)

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AMRs occurring within the first year are often associated with preformed or recall anti-Class I and/or Class II dnDSAs, severe graft dysfunction, vasculitis and worse immediate graft survival (Dorje et al., 2013; Lee et al., 2009; Lefaucheur et al., 2013). Identified quickly, early AMRs have been successfully reversed using a combination of plasmapheresis, IVIG, rituximab and bortezomib (Djamali et al., 2014; Dorje et al., 2013; Fehr & Gaspert, 2012). The treatment of late onset dnDSAs, those occurring after the first posttransplant year, in the absence of severe renal dysfunction or significant inflammation remains challenging (Einecke et al., 2009; Fehr & Gaspert, 2012; Wiebe et al., 2012). Late AMRs are usually characterized by patient noncompliance with medical care, the presence of de novo anti-Class II DSAs, a slowly progressive increase in serum creatinine and poor long-term graft function (Dorje et al., 2013; Einecke et al., 2009; Hidalgo et al., 2009; Ntokou et al., 2011; Sellares et al., 2012; Wiebe et al., 2012). Renal biopsy findings show glomerular and vascular injury with or without the presence of mild inflammation and capillaritis (Einecke et al., 2009; Hill et al., 2011; Sis et al., 2007; Wiebe et al., 2012). In such cases, complement does not appear to be a critical mediator of renal damage. Rather, renal injury may be caused by a direct stimulatory effect of anti-HLA antibodies on vascular cells to produce collagen and fibrosis (Zhang & Reed, 2009). Late appearing anti-HLA antibodies resist eradication as their cells of origin may be long-lived plasma cells that reside in specialized bone marrow niches not readily accessible to current therapies (Gloor et al., 2008). Over the long term, late onset dnDSA, especially anti-Class II alloantibodies, lead to glomerular basement membrane duplication and sclerosis resulting in accelerated graft loss (Gloor et al., 2008; Wiebe et al., 2012; Yamanaga et al., 2013). Recently, an effort to eliminate dnDSA using combinations of plasmapheresis, bortezomib and IVIG has shown promise (Everly et al., 2012). There is now considerable agreement that the presence of any dnDSA is generally a poor prognostic indicator portending a more rapid loss of renal allograft function (Lachmann et al., 2009) (Everly et al., 2013, 2014). However, it is also clear that a significant percentage of patients with dnDSAs do not undergo accelerated graft failure (Dunn et al., 2011; Everly et al., 2014; Wiebe et al., 2012). Each patient’s clinical situation is unique and often presents a difficult decision regarding the risks and benefits of a potentially hazardous treatment modality. Ongoing research is now focused on predicting risk by defining particular biological properties of dnDSAs. The factors that determine the differences in pathogenicity of particular dnDSAs are undoubtedly complex and involve both the character of the antibody as well as its antigen target. As with pre-transplant alloantibodies, the antibody titer and circumstance in which it was found appear to be important factors. Prospective studies demonstrated that low titer dnDSAs found in the absence of graft dysfunction can be transitory. However, if found during an evaluation for graft dysfunction even low-titer dnDSA can be associated with AMR. In contrast, high titer dnDSA having a MFI of greater than 8,000 are highly correlated with AMR (Fidler et al., 2013). In the absence of AMR, the treatment of even high titer dnDSA identified by surveillance testing remains uncertain. However, the presence of a dnDSA even in the absence of renal dysfunction warrants a biopsy to investigate the possibility of subclinical AMR that predicts poorer



outcome (de Kort et al., 2013; Wiebe et al., 2012). Therefore, surveillance protocols for dnDSA remain investigational and their impact on long-term graft outcome remains under study (Cooper et al., 2011). As reviewed previously, dnDSAs found associated with AMR need rapid and aggressive therapy. The ability of DSA IgG1 and IgG3 isotypes to fix complement appears to be another important pathogenic feature (Everly et al., 2013, 2014; Freitas et al., 2013). IgM dnDSA appear quite commonly posttransplant occurring in 29% of patients. The time to development of IgM dnDSA was significantly shorter than for IgG dnDSA suggesting that alloantibody responses to grafts undergo class switching. When occurring in isolation, IgM dnDSAs were not associated with increased risk of rejection or worse graft survival (Everly et al., 2014). However, rejections in the presence of both IgG and IgM dnDSAs were more severe and frequently associated with vasculitis. The greatest risk for graft failure was observed in patients with persistent IgM and IgG3 dnDSAs. Studies such as these may lead to means by which high-risk dnDSAs can be identified and targeted in only select patients thereby resulting in improved outcomes while placing fewer patients at risk from therapy unnecessarily. Mechanisms and prevention of dnDSAs Given the gravity of developing a dnDSA and the therapeutic challenges posed by dnDSAs, the best approach is preventing their appearance altogether. One tactic to prevent dnDSAs is improved HLA matching. The development of molecular HLA typing technology now allows for precise genotyping of a broader range of alloantigens that were previously overlooked or unknown. With the development and widespread application of molecular HLA typing, the importance of Class II mismatches at the DR, DRw, DQ and DP regions is increasingly becoming evident (Devos et al., 2012; Everly et al., 2013, 2014; Freitas et al., 2013). Molecular typing allows for better matching and more effective organ sharing. It remains to be seen if these efforts will lead to the much anticipated improvements in long-term graft survival. It is also evident that our current immunosuppressive strategies are inadequate in preventing the formation of dnDSA. Although the immunosuppressive drugs may be effective, inadequate dosing either through noncompliance or planned drug minimization may underlie their apparent inability to prevent dnDSAs. Commonly used immunosuppressive agents such as the calcineurin inhibitors (CNI), mycophenolic acid (MPA) and steroids all have significant adverse effects that often limit their dosing intensity. Thus, maintaining an adequate level of immunotherapy using current medications that is necessary to prevent dnDSAs may not always be possible. Another successful strategy to prevent dnDSAs is to incorporate lymphocyte depleting agents such as antithymocyte globulin or anti-CD20 monoclonal antibodies for highly sensitized patients (Barnett et al., 2013b; Thibaudin et al., 1998). The discovery of costimulation as a critical mechanism of lymphocyte activation allowed for the development of completely innovative therapies (Kinnear et al., 2013). Inhibiting ‘‘second’’ signaling pathways critical for productive T and B cell interactions has the theoretical potential of preventing dnDSAs. One such costimulatory pathway inhibitor is a molecularly engineered soluble form of CTLA-4 (CTLA4-Ig), belatacept. Belatacept binds to

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surface proteins CD80 and CD86 found on antigen presenting cells including B cells and inhibit T cell activation. Early clinical trials with belatacept therapy showed promise with fewer patients developing dnDSA compared to cyclosporin treated patients (Pestana et al., 2012). Another potential target for costimulation blockade is the B cell costimulatory molecule CD40. Phase 2 clinical trials are in progress examining the therapeutic efficacy of an antiCD40 antibody. Yet other costimulatory inhibitors, e.g. an anti-CD28 antibody fragment, are being developed with the goal of preventing chronic humoral immune mediated graft injury. B cells require activation signals delivered by specific cytokines which may prove to be effective targets in preventing dnDSAs. One B cell growth factor, B cell activating factor (BAFF) provides critical early signals for the development of B2 and marginal zone B cells (Mackay & Schneider, 2009). In human kidney transplantation, elevated BAFF or BAFF-receptor levels correlated with rejection and graft dysfunction (Thibault-Espitia et al., 2012). However, an initial clinical trial of anti-BAFF therapy proved ineffective and was prematurely halted http://clinicaltrials.gov/ct2/show/NCT01025193. A B cell maturation factor, APRIL (a proliferation-inducing ligand) favors the establishment and survival of late plasmablasts and promotes long-lived antibody responses. Clinical trials of APRIL directed therapy have not yet been conducted. We studied the potential relevance of the B cell stimulating, growth and maturation promoting cytokine, interleukin 14 (IL14) in transplantation (Ambrus et al., 1993). IL14 transcript levels in human T cells increase in vitro following allostimulation. In transplant recipients, IL14 mRNA levels are increased in those undergoing rejection or infection compared to stable patients (Leca et al., 2008). These data, suggest that IL14 may prove to be a biomarker indicating graft health and may play a role in the generation of dnDSA after kidney transplantation. Further studies will be required to fully explore the role of B cell stimulating cytokines and the therapeutic benefits of targeting these factors.

ACKNOWLEDGEMENTS We gratefully acknowledge the critical reviews of this manuscript by Dr. Tom Shanahan and Lin Feng. We also wish to thank Dr. Tom Shanahan for providing figures used in this review.

DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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