Effect of Immunosuppressive Drugs on Humoral Allosensitization After Kidney Transplant

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Effect of Immunosuppressive Drugs on Humoral Allosensitization after Kidney Transplant

† † Olivier Thaunat,* Alice Koenig,* Claire Leibler, and Philippe Grimbert

*Service de Transplantation, Néphrologie et Immunologie Clinique, Hôpital Edouard Herriot, Hospices Civils de Lyon, INSERM UMR1111, Université de Lyon, Lyon, France; and †Service de Néphrologie et Transplantation, Hôpital Henri Mondor, Centre de référence maladie rare Syndrome Néphrotique Idiopathique, Institut Francilien de recherche en Néphrologie et Transplantation, INSERM U955, Université Paris Est Créteil, Assistance Publique-Hôpitaux de Paris, Creteil, France

ABSTRACT The negative effect of donor-specific antibodies on the success of solid transplant is Fc segments, resulting in the formation now clearly established. However, the lack of effective treatment to prevent the of ordered antibody hexamers with ex- development of antibody-mediated lesions deepens the need for clinicians to focus quisite ability to recruit and activate on primary prevention of de novo humoral allosensitization. Among the factors C1q.12 The probability that a sufficient associated with the risk of developing de novo donor–specific antibodies, therapeu- number of IgGs binds close enough to tic immunosuppression is the most obvious parameter in which improvement is form such hexameric complexes is possible. Beyond compliance and the overall depth of immunosuppression, it is strongly influenced by antibody titer. likely that the nature of the drugs is also crucial. Here, we provide an overview of In line with this idea is the fact that the molecular effect of the various immunosuppressive drugs on B cell biology. the ability of DSAs to bind C1q or Clinical data related to the effect of these drugs on de novo humoral allosensitiza- C3d in a solid-phase assay correlates tion are also examined, providing a platform from which clinicians can optimize with their MFI.11,13 Another clue that immunosuppression for prevention of de novo donor–specific antibody generation DSA titer is an important factor in at the individual level. their ability to activate complement is in the study by Yell et al.14 which shows J Am Soc Nephrol 27: ccc–ccc, 2016. doi: 10.1681/ASN.2015070781 that most C1q binding DSAs can be converted into non–C1q binding DSAs when diluted. However, DSAs with sim- Antibody-mediated rejection (AMR) is HLA molecules represent the most ilar MFI do not always activate the com- widely recognized as the leading cause of documented targets for DSAs, it is clear plement,11 which indicates that, beyond late transplant failure and accounts for that DSAs can also be directed against the quantity, some qualitative features of approximately two thirds of renal allo- other kinds of molecular targets, includ- antibodies also affect their ability to ac- graft losses.1,2 In 2003, AMR was added ing polymorphic minor histocompati- tivate the complement. It is well known to the Banff classification for inter- bility antigens and after a breakdown of pretation of renal allograft biopsy,3 B cell tolerance,8 nonpolymorphic auto- sparking intense interest in the trans- antigens.9 DSAs are massive proteins A.K. and C.L. contributed equally to this work. plant community. Clinical studies over that are largely sequestrated in the circu- the last 10 years have established that lation.10 Binding of circulating DSAs to Published online ahead of print. Publication date antibodies generated de novo post- directly accessible graft endothelial cells available at www.jasn.org. transplantation against donor-specific can trigger the activation of a classic Correspondence: Dr. Olivier Thaunat, Service de antigens (DSAs) are strongly associated complement pathway, a central process Transplantation, Néphrologie et Immunologie Clinique, Hôpital Edouard Herriot, 5 Place d’Ar- and may be an important cause of in the pathophysiology of acute AMR sonval, 69003 Lyon, France, or Dr. Philippe Grimbert, allograft loss.1,4,5 (i.e., AMR with acute graft dysfunction).11 Service de Néphrologie et de Transplantation, Hôpital Experimental studies have shed light Complement activation by antibodies Henri Mondor Hospital, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Creteil, France. Email: 6,7 on the natural history of AMR. The depends on the recruitment and activa- [email protected] or philippe.grimbert@hmn. sequence starts with the generation of tion of the component C1q. After anti- aphp.fr antibodies directed against the graft. Al- gen binding on cells, IgGs establish Copyright © 2016 by the American Society of though highly polymorphic mismatched specific noncovalent interactions between Nephrology

J Am Soc Nephrol 27: ccc–ccc, 2016 ISSN : 1046-6673/2707-ccc 1 BRIEF REVIEW www.jasn.org that IgG4 is much less effective than INCIDENCE AND RISK FACTORS it is fundamental to take into account IgG1 and IgG3 in triggering the comple- FOR SENSITIZATION drug exposure evaluated by a pretrans- ment cascade. Strikingly, a recent study plant pharmagenomics analysis and reported that DSA subclasses identify The prevalence of de novo donor–specific post–transplant pharmacokinetic moni- distinct phenotypes of kidney allograft antigens (dnDSAs) is generally between toring. Indeed, the incidence of sensitiza- AMR. In this study, IgG3 DSAs were as- 5% and 10% at 1 year postrenal trans- tion is dramatically increased in patients sociated with a shorter rejection time, plantation and slowly increases there- who are nonadherent.2,19 Moreover, strict increased microcirculation injury, and after to 20% at 5 years.18–20 However, these monitoring of DSAs should be performed more C4d capillary deposition; in con- percentages should be interpreted with when clinical situations, such as infectious trast, IgG4 correlated with later allograft caution. During the last 10 years, assays episodes, BK virus viremia,21 diarrhea, or injury.15 for the detection of anti-HLA antibodies cancer,22 lead to a reduction of immuno- In contrast to acute AMR, activation have evolved from complement-dependent suppression. It is also noteworthy that of the classic complement pathway does cytotoxicity to the highly sensitive death with a functional allograft is cur- not seem to be critical for the develop- microbead–based Luminex assay. There- rently an important cause of graft loss. All ment of chronic AMR lesions. The first fore, it is possible that DSAs identified in recommendations regarding antirejection evidence supporting this concept was in the post-transplant period could, in fact, therapy must, therefore, take into account the study by Colvin and coworkers,16 be preformed DSAs that were not previously the related risk of overimmunosuppression which transplanted immunodeficient detected by the complement–dependent cy- and the benefit-to-risk ratio of all RAG knockout mice with allogenic totoxicity assay. Furthermore, a negative therapeutic decisions.28 Finally, defining heart. Colvin and coworkers16 observed pretransplant serum does not completely the best drug combination for primary that a passive transfer of noncomple- rule out the possibility that DSAs might be prevention requires an understanding of ment binding DSAs was sufficient expressed in the context of B cell memory B cell biology and the molecular mecha- enough to promote allograft vasculop- response. Indeed, using a novel HLA B Cell nisms by which each drug interferes with athy. Innate immune cells, including Enzyme-Linked ImmunoSpot Assay, dnDSA generation. neutrophils, macrophages, and NK cells, Lúcia et al.21 showed that, in 30% of pa- can bind to Fc fragments of antibodies tients, memory B cells could be detected and release lytic enzymes (a mechanism in highly sensitized patients in the ab- IMMUNE MECHANISMS called antibody–dependent, cell–mediated sence of corresponding anti–HLA anti- UNDERLYING DSA GENERATION cytotoxicity), which mediate smoldering bodies in the circulation. endothelial cell damages. In turn, chronic Risk factors for sensitization fall into DSAs are high-affinity IgGs directed vascular inflammation promotes the two categories: either they increase graft against protein antigen. These charac- progressive development of typical vascu- immunogenicity or they increase the teristics imply that DSA generation lar lesions (i.e., transplant glomerulopathy, recipient immune system’s ability to re- results from a thymo–dependent B cell allograft vasculopathy, and lamination spond to allogeneic epitopes. Graft immu- response. This type of humoral response, of the peritubular capillary basement nogenicity depends on both the quantity which develops within secondary lym- membrane). of allogeneic epitopes expressed by the phoid organs (spleen or lymph nodes), Current treatment protocols for graft (i.e., the number of mismatched involves two distinct events that are sep- AMR use permutations of a multi- amino acid polymorphisms)22 and their arated both spatially and temporally29 pronged approach that include DSA nature.23,24 It is also possible that graft (Figure 1). B cell activation is initiated removal with plasmapheresis, B or inflammation (either preexisting in in the B cell area of secondary lymphoid plasma cell depletion with anti-CD20 extended criteria donors or secondary to organs by the binding of cognate antigen and proteasome inhibitors (PIs), respec- ischemia-reperfusion injuries) could to surface B cell receptors (BCRs). This tively, and the blockade of antibody– increase immunogenicity.25 The ability step is strengthened with the help of in- mediated effector functions with of recipients’ immune systems to respond nate immune effectors. Macrophages of intravenous Igs (IVIgs) or terminal to allogeneic epitopes is reflected in their the subcapsular sinus capture lymph- complement pathway inhibitors.7 The pretransplant immunization status (i.e., borne antigens,30 and the binding of 3-year graft survival rate with these pro- responses to previous challenges), acute these antigens to the surface Ig of the tocols is ,50%.17 Given the detrimental cellular rejection episodes, and younger cognate B cells contributes to the estab- effect of DSAs on transplant survival and age, all of which have been consistently lishment of the immunologic synapse the lack of effective treatment to prevent associated with increased risk of sensitiza- between the B cells and the subcapsular the development of antibody-mediated tion.19,26,27 Among the factors associated sinus macrophages30 (Figure 1, top lesions, the priority of clinicians in with post-transplant sensitization, thera- panel). BCR crosslinking leads to the charge of recipients of solid organs peutic immunosuppression represents the phosphorylation of immunoreceptor should be primary prevention of DSA most obvious modifiable risk factor. To tyrosine–based activation motifs by the generation. optimize immunosuppressive strategies, Src family kinase Lyn. This initiates the

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formation of the signalosome, an assembly of intracellular signaling molecules that, together with adaptor molecules, are re- sponsible for the coordinated regulation of downstream signaling pathways, including calcineurin and mammalian tar- get of rapamycin (mTOR) pathways, and ultimately, modify the gene expression. Importantly, signaling BCR microclus- ters also associate with molecular motor dynein, which allows for the coordinated movement of antigen into the intracyto- plasmic endosomal compartment of the B cell.29,31 Internalized antigen is processed and subsequently exposed on the B cell surface in association with MHC molecules.32 B cells that have received this first activation signal migrate toward the B zone-T zone boundary,33 where they pair with a particular subset of cognate CD4+ T cells called T follicular helper (Tfh) cells34 (Figure 1, bottom panel). This provides the second activation signal for B cells through cell surface costimulatory ligand interactions (especially CD40L) and directional cytokine production (in particular, IL-21).34 The activated B cells enter the germinal center, where they proliferate. Their pro- genies go through repeated cycles of random somatic hypermutation of Ig variable regions driven by the enzyme activation–induced cytidine deaminase followed by selection of progressively higher–affinity mutants. Differentiation of some of these high-affinity mutants into plasma cells or memory B cells leads to the increased affinity of serum antibodies and the high affinity of the recall response.35 Interestingly, recent evidence has shown that plasma cells and memory B cells generated by the germinal center reaction require additional helper signals from innate immune effectors, including eosinophils and basophils, to extend their lifespan in postgerminal center niches (summarized in ref. 36). Recent data suggest that B cell functions are not restricted to the deleterious role of secreting dnDSAs.37 In the inflammatory context, some B cell sub- Figure 1. Key molecular steps of B cell activation and sites of action of immunosuppressive sets have shown regulatory properties drugs. B cell activation is a two-step process. (Top panel) Binding of the cognate antigen to the that rely on either secretion of anti- surface Ig of B cells serves as the first signal of activation. BCR signaling drives the internalization inflammatory cytokines (IL-10) and/ of bound antigens (donor’s HLA molecules) to endosomal compartments, where they are or contact-dependent mechanisms. The

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fine equilibrium between these two roles onward and exceed baseline levels after 6 dnDSAs, and AMR was not specifically of B cells should also be taken into ac- months.42 Moreover, the composition of addressed).49,50 In a more recent trial, count when discussing immunosup- B cell subsets is altered compared with Brokhof et al.51 reported in a cohort of pressive strategies.38 that found pretransplant; the levels of 114 moderately sensitized recipients of Determining the actions of each im- naïve and transitional B cells increase, deceased donor renal transplants that in- munosuppressive drug on B cell biology whereas the absolute number of mem- duction with ATG is associated with a and their consequent effect on dnDSA ory B cells decreases significantly.42 In- reduction in the occurrence of dnDSAs production represents the first step in terestingly, in organ transplantation, the and AMR compared with basiliximab. defining practical clinical immunosup- composition of the B cell compartment However, it should be noted that, in pressive strategies. is repeatedly identified as an important this study, (1) the difference became sta- determinant for graft outcome.43,44 tistically different only at 3 years, (2) Heidt et al.42 suggested that lymphocyte only a few patients assigned to basilixi- EFFECT OF INDUCTION THERAPY depletion with alemtuzumab may skew mab had a follow-up at 36 months, and ON DNDSA GENERATION the B cell compartment toward a proto- (3) the patients who received ATG also lerogenic profile. This hypothesis has received more plasmapheresis and IVIg Rabbit antithymocyte globulin (ATG) isa been further strengthened by another than the patients who received rituximab. mixture of polyclonal IgGs recovered work that showed that a significant Finally, if ATG should be considered from the sera of rabbits immunized expansion of regulatory–type B cells (de- as the first–line induction therapy in with human thymocytes. The binding fined as CD19+CD5+CD1dhigh) is associ- previously sensitized patients, no data of antibodies to recipient’sTlympho- ated with superior graft function and that are available concerning a potential cytes results in complement– and/or this pattern is more common after alem- benefit of this induction strategy in non- antibody–dependent, cell–mediated cy- tuzumab induction.45 However, al- sensitized recipients. totoxicity and an interaction with T cell though this hypothesis is interesting, it surface antigens, possibly resulting in has been challenged by recent conflicting apoptosis or anergy (Figure 1). Interest- reports from two independent groups EFFECT OF MAINTENANCE ingly, because certain epitopes are shared showing that alemtuzumab–induced IMMUNOSUPPRESSION ON between T and B cells and because pedi- B cell depletion/reconstitution is as- DNDSA GENERATION atric thymi also contain CD20+ B cells sociated with a higher incidence of and CD138+ plasma cells, ATG contain AMR compared with induction with Corticosteroids IgGs directed against these latter sub- ATG.46,47 Corticosteroids are the oldest immune sets.39 As a result, in vitro studies have Basiliximab is a chimeric mouse- response–modifying drugs. Their mech- shown that ATG induces apoptosis of human nondepleting mAb targeting anism of action is diverse and includes naive, activated B cells and bone marrow the a-chain (CD25) of the IL-2 receptor interference with intracellular transcrip- resident plasma cells at clinically rele- (Figure 1). Basiliximab was developed tion factors and signaling pathways of vant concentrations (1–100 ng/ml).39 for induction therapy on the basis of several surface receptors, including the However, the importance of ATG– the concept that it would block IL-2 T cell antigen receptor52 and down- induced plasma and/or B cell depletion binding, thereby inhibiting T cell prolif- stream kinases.53 in vivo has not been clearly shown.40 eration. Interestingly, activated B cells High doses of corticosteroids (.1 An alternative to ATG as a depleting also express CD25,48 and IL-2 has a cru- mg/kg), used for induction therapy in agent for induction is alemtuzumab, a cial role in driving the differentiation of transplantation, also induce lymphocyte humanized mAb directed against CD52, activated B cells toward plasma cells in apoptosis (including B cells)46,54 and can which is a protein widely distributed on vitro.48 prevent in vitro differentiation of B cells the surface of lymphocytes and mono- In practice, ATG should be preferred into plasma cells.55 High concentrations cytes (but with an unknown function). in sensitized patients without DSAs, of corticosteroids are also likely to inter- Alemtuzumab induces rapid and pro- because two randomized clinical trials fere with Fc g–receptor signaling, in- found B (and T) lymphocyte depletion41 showed beneficial short–term effects on cluding the inhibitory Fc g–RIIb, which (Figure 1). A recent study showed that, acute rejection, graft function, and acts as a key negative modulator of B cell after this initial depletion, B cells repo- survival (however, the relationship be- function and controls bone marrow pulate the peripheral blood from 6 weeks tween induction immunosuppression, plasma cell apoptosis.56 The effect of a

processed. Selected peptides are then loaded onto recipient MHC class 2 molecules and presented on the B cell surface. (Bottom panel) B cells interact with cognate Tfh cells that provide the second costimulation signal for the completion of B cell activation. Therapies with proven efﬁciency are shown as red lines. Putative mechanisms of action are dashed red lines with question marks. TCR, T cell receptor.

4 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BRIEF REVIEW standard maintenance dose of cortico- because calcineurin inhibitors (CNIs), in engagement of the BCR activates PI3K, steroid therapy on human B cells was contrast with genetic deletion, do not which beyond activation of NF-kB– studied in a seminal in vitro study by completely suppress calcineurin activity dependent transcription, also initiates a Cupps et al.57 in the mid-1980s. Early in lymphocytes.64 In fact, when Heidt distinct signaling pathway involving events in B cell activation, such as et al.65 tested the effect of CNIs in T and the Akt and mTOR serine/threonine anti-IgM–induced B cell activation and B cell cocultures, they observed that, kinases.70 proliferation, are profoundly suppressed whereas cyclosporin A (CsA) and tacroli- Surprisingly little is known about the by corticosteroids.57 To be effective, the mus were both capable of inhibiting Ig functions of the mTOR complexes in B corticosteroids have to be present in production when B cells were cultured cells. Homozygous knockin mice for the culture during the first 24 hours, with T cells that were not preactivated, hypomorphic mTOR showed decreased suggesting a drug-induced modulation they failed to do so when preactivated T ability to produce antibodies.69 To deter- of an early event.57 cells were used to stimulate B cells. These mine whether these effects were caused Over the last decades, several attempts results indicate that CNIs affect the hu- by a direct mTOR blockade in the B cells have been made to develop steroid- moral immune response of patients with or only reflected T cell deficiency, the sparing strategies to avoid their important transplants by interfering with T helper same group examined the humoral re- deleterious side effects. No increased risk signals and not by targeting B cells di- sponses in mTOR conditional B cell in sensitization has been reported in rectly.65 This hypothesis is supported by knockout mice.71 Mice with mTOR de- patients without corticosteroids in a pro- the study by Struijk et al.,66 which showed leted in their B cell lineage produced spective single–center study on kidney that patients with renal transplants who fewer splenic germinal centers and also, transplantation58 and two retrospective were stable on prednisolone plus CsA exhibited a decrease in switched high– studies on liver transplantation.59,60 failed to develop antibodies against affinity antibody responses in contrast These encouraging results are also sup- T cell–dependent model antigens but re- to their wild-type littermates.71 The ported by the conclusions in the work by sponded normally to T cell–independent fact that mTOR inhibitors are able to Delgado et al.,61 which randomized 37 antigens. prevent the in vitro production of Ig recipients of kidney transplants with Some studies have reported that the use with equal efficiency when human B ATG induction, tacrolimus, and myco- of CsA is associated with an increased risk cells are cultured with preactivated or phenolic acid (MPA) for corticosteroid of dnDSAs compared with tacrolimus, not preactivated T cells further shows withdrawal 1 week post-transplantation. 59,67 butthisdifferenceisnotalways that mTOR inhibitors, in contrast with Delgado et al.61 did not observe any dif- found.5 The effect of the nature of CNIs CNIs, have a direct effect on B cells.65,72 ference in dnDSA incidence at 5 years. (CsA or tacrolimus) on dnDSA occur- This concept is also validated by the re- However, because early steroid with- rence is made difficult to assess because sults of a recent study that compared the drawal is associated with a higher in- of the specific pharmacokinetic features effects of CNI and mTOR inhibitors on cidence of subclinical rejection and of each molecule (including variance of B cells from healthy volunteers. Only chronic graft injury in recipients with oral bioavailability, action on entero- sirolimus was able to inhibit the prolifer- high immunologic risk, such as hepatic circulation of MPA observed ation of B cells and their differentiation blacks,62 a steroid-sparing strategy with CsA, and genetic polymorphisms of into plasma cells. Interestingly, the effect should only be considered in selected re- tacrolimus), which are likely to modify of sirolimus seemed more pronounced on cipients with low immunologic risk. drug exposure. Finally, beyond the nature the CD19+CD27+ memory subset.73 of the CNI, compliance19 and trough lev- Several clinical studies have reported Calcineurin Inhibitors els59 have been established as the most successful conversion to an mTOR in- It has been known since the mid-1990s important risk factors for sensitization of hibitor–based regimen.74–77 Although that, similar towhat happens in T cells, the patients on CNI. several of them showed a significant im- engagement of BCR activates NFAT provement in renal function after conver- through a calcineurin-dependent pathway mTOR Inhibitors sion to a CNI-free regimen, long-term in B lymphocytes (Figure 1). The impor- Immunosuppressive drugs targeting the data on the humoral response are limited. tance of this pathway for T cell–dependent mTOR signaling pathway have been de- In the ZEUS Trial,74 recipients of kidney humoral responses has been studied in veloped to avoid or reduce CNI-associated transplants on CsA-based immunosup- mice, in which calcineurin was deleted toxicity. Sirolimus and everolimus pression were randomized at 4 months specifically in B cells.63 These mice had engage FK binding proteins, forming a post-transplantation to be either main- larger germinal centers, reduced plasma complex that inhibits mTOR. This in- tained on the same regimen or converted cell development, and lower antigen– hibition prevents the translation of to everolimus-based immunosuppres- specific antibody titers, indicating a block mRNA-encoding proteins needed to en- sion. In a retrospective analysis of a subset in plasma cell differentiation.63 Although ter the cell cycle68 (thus reducing T cell of patients, Liefeldt et al.78 reported that interesting, these experimental results proliferation) and cytokine produc- 23% of those receiving everolimus had cannot be directly translated into patients, tion40,69 (Figure 1). Interestingly, developed dnDSAs compared with only

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11% of those who were receiving CsA. influences the B cell fate. This mechanism which is crucial for the expansion of This difference was, however, only ob- could account for the recent observation the rare antigen–specific clones and served when steroid withdrawal was al- that the frequency and absolute number therefore, the development of humoral lowed. In contrast, the 4-year report of of CD19+CD24hiCD38hi B cells (a subset response. Azathioprine and mycophe- the CONCEPT Trial79 did not find signif- that has previously been associated with nolate mofetil (MPA) both block cell di- icant difference in dnDSA incidence be- operational tolerance43,44) are increased vision by inhibiting DNA synthesis tween patients switched to sirolimus at 3 in patients on belatacept compared with through two distinct mechanisms (Fig- months and the CsA controls (12% versus controls on CNIs.90 ure 1). Azathioprine is a prodrug for 21%; P=0.24). In line with these last reas- Most of the clinical data where belata- 6-mercaptopurine; its metabolites block suring results, a case-control study evalu- cept was used come from two multina- purine synthesis enzymes and impede ating the effect of late conversion from tional phase 3 studies: the BENEFIT Trial later steps of DNA synthesis by being in- CNI to everolimus (median of 22 months and the BENEFIT-EXT Trial. The BENEFIT corporated into newly synthetized DNA. post-transplantation) also fails to show Trial studied patients who received a In contrast, MPA only acts as a selective increased dnDSA incidence.80 Finally, kidney from a living donor or a standard inhibitor of inosine-59-monophosphate the question of whether mTOR inhibitors criteria deceased donor. The BENEFIT- dehydrogenase, a key enzyme in the de favor dnDSA emergence is still controver- EXT Trial, however, studied patients who novo synthesis of purine. Therefore, sial. Available data suggest that conversion receivedakidneyfromanextendedcriteria MPA does not incorporate false purine to mTOR inhibitors after complete CNI deceased donor. In both studies, patients analogs into DNA. withdrawal is a potential risk factor, espe- received corticosteroids and basiliximab Adding MPA to primary human B cially if it is made before the end of the first for induction therapy intraoperatively and cells stimulated in vitro with IL-21, anti- year and/or associated with steroid with- MPA and tapering doses of the steroid CD40, and anti-IgM or CpG-B inhibits drawal. postoperatively. Results from these trials B cell proliferation and the generation of indicate that belatacept may have a para- antibody–producing plasma cells.93 Belatacept doxical effect on T versus B cell–mediated Similar findings have been reported in Belatacept is a human fusion protein alloimmune response. The BENEFIT the T/B coculture model.65,72 As expec- combining the extracellular portion of Trial revealed surprisingly low rates (5%– ted, however, the drug has no effect on cytotoxic T lymphocyte–associated anti- 6%)ofdnDSAformationat3years, terminally differentiated plasma cells,93 gen 4 (CTLA4), which has been mutated whereas the incidence of acute T cell– suggesting that it can only prevent (but to confer greater binding avidity to mediated rejection was markedly in- not cure) AMR. CD80 and CD86, and the constant re- creased.91 Schwarz et al.92 performed a Some clinical studies suggest that MPA gion fragment of human IgG1. The retrospective patient match analysis of 14 might perform better than azathioprine CTLA4 binds surface costimulatory patients on belatacept originally enrolled in preventing dnDSA formation. For ligands (CD80 and CD86) of antigen- in the phase 2 multicenter trial. Fifty-six instance, two retrospective studies in kidney presenting cells, thus preventing their patients treated with CsA were matched transplantation5,26 have reported that interaction with CD28 and thereby, according to age at transplantation, first azathioprine is more frequently used blocking T cell activation81 (Figure 1). transplant/retransplant, and donor type. than MPA among patients who develop The efficiency of belatacept in prevent- None of the patients treated with belatacept dnDSAs. However, this difference, which ing dnDSA generation is generally attrib- had DSAs$10 years post-transplantation was not observed in liver transplanta- uted to its effect on Tfh cells.82 B cells are, compared with 38.5% of tested subjects tion,59 could also be explained by the dos- indeed, known to lack CD28, which has treated with CsA (P=0.05).92 Although age (or the nature) of other concomitant expression that is specifically repressed by extremely promising, these results need medications. In a study designed to deter- the B cell master regulator gene Pax-5.83 to be confirmed through additional ex- mine the effect of immunosuppression However, several experimental studies perimentation. It should also be remem- on both naïve and memory humoral have shown the role of reverse signaling bered that part of the effect could be responses, Struijk et al.66 reported that of CD80/CD86 in CTLA4-mediated coin- related to the fact that, in contrast with all recipients of kidneys on prednisone hibition.84 CTLA4-Ig binds to CD80/ other immunosuppressive drugs, belata- combined with either CsA, MPA, or CD86 molecules on dendritic cells and cept is given intravenously monthly in an everolimus all generated significantly promotes the induction of the immuno- outpatient clinic, which could limit fewer antibodies during primary humoral modulatory enzyme indoleamine 2,3 noncompliance, a major risk factor for responses compared with healthy con- dioxygenase85,86 and the tolerogenic mol- dnDSA generation.2,19 trols. In contrast, when patients were ecule HLA-G.87 Experimental data sug- tested for memory responses against the gest that reverse signaling of CD80/ Purine Inhibitors tetanus toxin, only patients on prednisone CD86 also exists in B cells.88,89 It is, there- After signal 1 (BCR engagement) and plusMPAshowedimpairedsecondary fore, tempting to speculate that, when be- signal 2 (provided bycognate Tfh cells), B humoral responses, suggesting that MPA latacept binds to CD80/CD86, it directly cells undergo massive proliferation, could be of special interest in preventing

6 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2016 www.jasn.org BRIEF REVIEW the rise of DSA titers in presensitized re- the scope of this review, because DSAs are (Figure 1). In the context of AMR, PI cipients.66 present before drug administration). An- seems to reduce the titers of immunodo- other problem to determine their effect is minant DSAs by 50%, especially in early In Clinical Practice the fact that these drugs were mostly used AMR.102 A similar but transient reduc- A maintenance immunosuppression in combination with plasma exchanges tion rate has also been observed for strategy should be defined according to and/or IVIg.95 preformed anti–HLA antibodies in a the level of the pretransplant sensitiza- Rituximab is a chimeric depleting prospective trial of PI-based desensitization status. A conventional strategy using antibody directed against the B cell tion.103 Interestingly, Woodle et al.103 CNI plus MPA and avoiding minimiza- surface molecule CD20 that is not found also reported a low rate of dnDSA for- tion in the absence of specific clinical on mature plasma cells (Figure 1). Thus, mation (12.5%) in patients who were indications should be chosen in sensi- rituximab should not affect serum anti- desensitized. tized recipients. In recipients with low body titers directly. However, some immunologic risk, belatacept can now be studies have reported that prolonged B considered as a promising alternative cell depletion with rituximab (in associ- CONCLUSION strategy in association with MPA and ste- ation with IVIg) could lead to a decrease roids. Regarding the other CNI–sparing in DSA titers in patients diagnosed with The prevention of dnDSA generation is strategies, mTOR inhibitors with MPA chronic AMR.96,97 Arecentstudyalso critical to the success of modern renal should not be considered as the first found significantly less HLA antibody transplant practice. The depth of immu- choice in either highly or moderately rebound in patients treated with rituxi- nosuppression together with the nature of sensitized recipients. In recipients with mab compared with a control cohort de- the drugs used play an important role in low immunologic risk, the effect of sensitized and transplanted without the success of the transplant. The data both mTOR inhibitors (with or without rituximab.98 Compared with controls, provided by clinical trials regarding the low-dose CNI) as steroid-sparing strate- patients treated with rituximab had a effect of therapeutic immunosuppression gies has to be clearly defined by addi- significantly greater mean reduction in on the risk of developing dnDSAs are still tional analysis. DSA MFI but a similar rate of DSA per- limited, relatively recent, and considerably Maintenance immunosuppression sistence.98 Some patients sensitized to varied. This because of the rapid evolution should also be adapted to the results of HLA antigens do not have antibody pre- of methods used to detect anti-HLA clinical, pharmacologic, and immuno- sent in serum before transplanta- antibodies as well as the heterogeneity of logic monitoring. DSA monitoring tion.21,97 Such patients are at risk for monitoring and follow-up across centers. should be performed at least once be- an anamnestic response resulting Nevertheless, these data together with the tween 3 and 12 months after transplan- from a proinflammatory response to basic advances in our understanding of B tation. An evaluation of DSAs should also the trauma of transplant surgery. Elim- cellbiology providea platformfromwhich be performed when a change of immu- ination of HLA–specific memory B cells clinicians in charge of patients with trans- nosuppressive regimen is considered and with rituximab in these patients success- plants can now design immunosuppres- when nonadherence or graft rejection is fully abrogated anamnestic response.99 sive strategies tailored to the specific suspected.94 Detection of a significant Regarding prevention of dnDSA gener- immunologic risk of each recipient. level of dnDSAs should prompt verifica- ation with rituximab, a Japanese group tion of drug compliance, and pharmaco- has recently reported that dnDSA in- fi – kinetic analyses should be performed to cidenceissigni cantly lower in ABO DISCLOSURES ensure that drug exposure is within ther- incompatible renal transplantations None. apeutic ranges (in particular, in patients induced with rituximab.100 Incidence with therapeutic minimization). of chronic AMR was also significantly reduced in the group of ABO-incompati- REFERENCES ble recipients who received induction THE EFFECT OF B OR PLASMA therapy with rituximab or splenectomy 1. 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