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Proteasome inhibitor-based therapy for antibody-mediated rejection

2012; Elsevier BV; Volume: 81; Issue: 11 Linguagem: Inglês

10.1038/ki.2011.502

1523-1755

R. C. Walsh, Rita R. Alloway, Alin Girnita, E. Steve Woodle,

Cytomegalovirus and herpesvirus research

The development of donor-specific anti-human leukocyte antigen antibodies (DSAs) following renal transplantation significantly reduces long-term renal graft function and survival. The traditional therapies for antibody-mediated rejection (AMR) have provided inconsistent results and transient effects that may be due to a failure to deplete mature antibody-producing plasma cells. Proteasome inhibition (PI) is a novel AMR therapy that deletes plasma cells. Initial reports of PI-based AMR treatment in refractory rejection demonstrated the ability of bortezomib to deplete plasma cells producing DSA, reduce DSA levels, provide histological improvement or resolution, and improve renal allograft function. These results have subsequently been confirmed in a multicenter collaborative study. PI has also been shown to provide effective primary AMR therapy in case reports. Recent studies have demonstrated that PI therapy results in differential responses in early and late post-transplant AMR. Additional randomized studies are evaluating the role of PI in transplant induction, acute AMR, and chronic rejection in renal transplantation. An important theoretical advantage of PI-based regimens is derived from several potential strategies for achievement of synergy. The development of donor-specific anti-human leukocyte antigen antibodies (DSAs) following renal transplantation significantly reduces long-term renal graft function and survival. The traditional therapies for antibody-mediated rejection (AMR) have provided inconsistent results and transient effects that may be due to a failure to deplete mature antibody-producing plasma cells. Proteasome inhibition (PI) is a novel AMR therapy that deletes plasma cells. Initial reports of PI-based AMR treatment in refractory rejection demonstrated the ability of bortezomib to deplete plasma cells producing DSA, reduce DSA levels, provide histological improvement or resolution, and improve renal allograft function. These results have subsequently been confirmed in a multicenter collaborative study. PI has also been shown to provide effective primary AMR therapy in case reports. Recent studies have demonstrated that PI therapy results in differential responses in early and late post-transplant AMR. Additional randomized studies are evaluating the role of PI in transplant induction, acute AMR, and chronic rejection in renal transplantation. An important theoretical advantage of PI-based regimens is derived from several potential strategies for achievement of synergy. The development of anti-human leukocyte antigen (HLA) antibodies (Abs) specific for the renal allograft is associated with diminished allograft survival, regardless of whether the donor-specific anti-HLA antibody (DSA) is de novo or anamnestic in origin.1.Lee P.C. Zhu L. Terasaki P.I. et al.HLA-specific antibodies developed in the first year posttransplant are predictive of chronic rejection and renal graft loss.Transplantation. 2009; 88: 568-574Crossref PubMed Scopus (140) Google Scholar, 2.Terasaki P.I. Ozawa M. Castro R. Four-year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival.Am J Transplant. 2007; 7: 408-415Crossref PubMed Scopus (281) Google Scholar, 3.Mao Q. Terasaki P.I. Cai J. et al.Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study.Am J Transplant. 2007; 7: 864-871Crossref PubMed Scopus (185) Google Scholar However, prompt and complete DSA elimination may improve allograft survival.4.Everly M.J. Everly J.J. Arend L.J. et al.Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss.Am J Transplant. 2009; 9: 1063-1071Crossref PubMed Scopus (173) Google Scholar,5.Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2010; 89: 277-284Crossref PubMed Scopus (153) Google Scholar There are currently no immunosuppressive agents approved by the Food and Drug Administration for antibody-mediated rejection (AMR) treatment. Historically, AMR has been treated with a variety of approaches, including intravenous immunoglobulin (IVIg), therapeutic plasma exchange, rabbit anti-thymocyte globulin, and rituximab. These approaches, however, do not deplete the source of Ab production—the mature plasma cell.6.Ramos E.J. Pollinger H.S. Stegall M.D. et al.The effect of desensitization protocols on human splenic B-cell populations in vivo.Am J Transplant. 2007; 7: 402-407Crossref PubMed Scopus (123) Google Scholar This limitation may contribute to the suboptimal and unreliable results observed with non-plasma cell-depleting agents. Given the need for reliable and durable elimination of anti-HLA alloantibodies, considerable efforts are being focused on developing new antihumoral therapies. Recent reports have described the use of the proteasome inhibitor bortezomib (Millennium Pharmaceuticals, Cambridge, MA) in treating AMR. The utility of bortezomib was first demonstrated in the treatment of refractory AMR,7.Everly M.J. Everly J.J. Susskind B. et al.Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.Transplantation. 2008; 86: 1754-1761Crossref PubMed Scopus (330) Google Scholar and has subsequently been shown to provide effective primary therapy for AMR in case reports.5.Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2010; 89: 277-284Crossref PubMed Scopus (153) Google Scholar The purpose of this review is to discuss the role of proteasome inhibition (PI) in AMR treatment. The 26S proteasome is a large multimeric enzymatic structure present in the cytosol of all eukaryotic cells. The proteasome structurally resembles a cylinder and consists of a 20S core with 19S regulatory subunits capping each end (Figure 1). The 20S core consists of four stacked heptameric rings, with two beta rings surrounded by two alpha rings. Residing inside of the cylinder, the proteolytic activity of the proteasome is protected from the cytosol. Three distinct proteolytic activities exist within the beta ring: chymotryptic-like, tryptic-like, and postglutamyl (that is, caspase-like) hydrolyzing activity.8.DeMartino G.N. Slaughter C.A. The proteasome, a novel protease regulated by multiple mechanisms.J Biol Chem. 1999; 274: 22123-22126Crossref PubMed Scopus (490) Google Scholar,9.Adams J. The proteasome: structure, function, and role in the cell.Cancer Treat Rev. 2003; 29: 3-9Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar Each alpha ring provides a restricted opening, which limits the entry of proteins destined for proteolysis. Proteasomal function provides cellular homeostasis via the selective degradation of misfolded proteins, cell-cycle regulatory proteins, transcription factors, and inhibitory molecules.8.DeMartino G.N. Slaughter C.A. The proteasome, a novel protease regulated by multiple mechanisms.J Biol Chem. 1999; 274: 22123-22126Crossref PubMed Scopus (490) Google Scholar,9.Adams J. The proteasome: structure, function, and role in the cell.Cancer Treat Rev. 2003; 29: 3-9Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar Bortezomib interrupts this homeostasis through reversible binding of the β5-subunit, the site of chymotryptic-like proteolytic activity, within the 20S core of the 26S proteasome.9.Adams J. The proteasome: structure, function, and role in the cell.Cancer Treat Rev. 2003; 29: 3-9Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar PI results in dysregulation of numerous cellular processes, including antigen processing and mitosis regulation (Figure 2). The ubiquitin–proteasome system is the major intracellular mechanism for production of peptide fragments of suitable length for presentation on major histocompatibility complex (MHC) class I molecules.10.Goldberg A.L. Cascio P. Saric T. et al.The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.Mol Immunol. 2002; 39: 147-164Crossref PubMed Scopus (275) Google Scholar,11.Kloetzel P.M. Ossendorp F. Proteasome and peptidase function in MHC-class-I-mediated antigen presentation.Curr Opin Immunol. 2004; 16: 76-81Crossref PubMed Scopus (339) Google Scholar This process is facilitated by the 26S proteasome; however, in inflammatory states, differing β-subunits with differing enzymatic activities are produced that provide an alternative array of peptides for antigen presentation. Cytokine stimulation via interferon-γ enhances immunoproteasome activity as well as upregulates cell surface MHC class I expression. Through PI, the degree of antigen processing and presentation on MHC class I molecules can be inhibited.10.Goldberg A.L. Cascio P. Saric T. et al.The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.Mol Immunol. 2002; 39: 147-164Crossref PubMed Scopus (275) Google Scholar,11.Kloetzel P.M. Ossendorp F. Proteasome and peptidase function in MHC-class-I-mediated antigen presentation.Curr Opin Immunol. 2004; 16: 76-81Crossref PubMed Scopus (339) Google Scholar There are four major physiological effects of proteasome inhibitor therapy that are thought to be primarily responsible for its immunomodulatory effects and likely its effectiveness in AMR. These include: (1) inhibition of nuclear factor-kappa B (NF-κB) activity, (2) inhibition of proliferation and induction of apoptosis via cell cycle arrest, (3) induction of apoptosis via ER stress, and (4) inhibition of Class I MHC expression via reduction in endogenous peptide production. Cell cycle progression is mediated through a precisely choreographed series of enzymatic events via cyclins. Throughout mitosis, the synthesis and subsequent degradation of cyclins is required for cell cycle progression at multiple checkpoints. The ubiquitin–proteasome system maintains appropriate levels of these key cyclins.12.Pagano M. Tam S.W. Theodoras A.M. et al.Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.Science. 1995; 269: 682-685Crossref PubMed Scopus (1734) Google Scholar, 13.Muratani M. Tansey W.P. How the ubiquitin-proteasome system controls transcription.Nat Rev Mol Cell Biol. 2003; 4: 1-10Crossref Scopus (674) Google Scholar, 14.Yew P.R. Ubiquitin-mediated proteolysis of vertebrate G1- and S-phase regulators.J Cell Physiol. 2001; 187: 1-10Crossref PubMed Scopus (84) Google Scholar Interruption of this precise series of events (for example, viaPI) results in cell cycle arrest and apoptosis. Protein synthesis and folding are managed within the endoplasmic reticulum (ER) and are vital for the maintenance of normal cellular homeostasis. When a critical threshold of unfolded or misfolded proteins accumulates within the ER, a number of protective responses are generated that comprise the unfolded protein response (UPR). The UPR is characterized by three major processes: (1) reduction in synthesis of nascent proteins, (2) upregulation of ER chaperones and foldases, and (3) expression of proteins that constitute the ER-associated degradation pathway and facilitate protein trafficking from the ER to the proteasome.15.Kim R. Emi M. Tanabe K. et al.Role of the unfolded protein response in cell death.Apoptosis. 2006; 11: 5-13Crossref PubMed Scopus (396) Google Scholar,16.Obeng E.A. Carlson L.M. Gutman D.M. et al.Proteasome inhbitors induce a terminal unfolded protein response in multiple myeloma cells.Blood. 2006; 107: 4907-4916Crossref PubMed Scopus (862) Google Scholar PI potently induces an UPR due to the accumulation of high levels of misfolded proteins that have been targeted for proteasomal digestion. If the accumulation of misfolded proteins is not controlled, a terminal UPR results, which consists of mitochondrially and caspase-mediated cellular apoptosis.15.Kim R. Emi M. Tanabe K. et al.Role of the unfolded protein response in cell death.Apoptosis. 2006; 11: 5-13Crossref PubMed Scopus (396) Google Scholar PI also effectively inhibits production of pro-inflammatory cytokines. NF-κB-mediated transcription of pro-inflammatory cytokines is regulated through the binding of inhibitor nuclear factor-kappa B (IκB) to NF-κB. IκB is downregulated by ubiquitination and subsequent proteasomal degradation. Therefore, PI results in IκB stability and a resulting diminishment in production of pro-inflammatory cytokines such as interleukin (IL)-2, IL-6, IL-10, IL-13, interferon-γ, and tumor necrosis factor-α.13.Muratani M. Tansey W.P. How the ubiquitin-proteasome system controls transcription.Nat Rev Mol Cell Biol. 2003; 4: 1-10Crossref Scopus (674) Google Scholar,17.Mattingly L.H. Gault R.A. Murphy W.J. Use of systemic proteasome inhibition as an immune-modulating agent in disease.Endocr Metab Immune Disord Drug Targets. 2007; 7: 29-34Crossref PubMed Scopus (29) Google Scholar Reduction in the pro-inflammatory cytokines by PI has also been demonstrated in anti-CD3-stimulated T cells.18.Luo H. Wu Y. Qi S. et al.A proteasome inhibitor effectively prevents mouse heart allograft rejection.Transplantation. 2001; 72: 196-202Crossref PubMed Scopus (63) Google Scholar Reduction in the inflammatory cytokine milieu improves overall inflammation and allograft damage, ultimately strengthening the role of PI in immunologically mediated damage post transplantation. Proteasome inhibitors have been evaluated in animal models of humorally mediated autoimmune disease. These studies have provided an enhanced understanding of the effects of systemic proteasome inhibitor therapy. A disease that closely mimics systemic lupus erythematosus develops spontaneously in NZB/W F1 mice.19.Neubert K. Meister S. Moser K. et al.The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis.Nat Med. 2008; 14: 748-755Crossref PubMed Scopus (480) Google Scholar Autoantibodies to double-stranded DNA mediate glomerulonephritis and play a critical role in disease progression in this model. Ultimately, these double-stranded DNA autoantibodies and the associated glomerulonephritis result in significant mortality in this model. Neubert and colleagues utilized this animal model to assess the response to PI with bortezomib and elucidate the mechanism by which disease amelioration is induced. Flow cytometry analysis of CD138+ short- and long-lived plasma cells from spleen and bone marrow demonstrated a bortezomib-mediated decrease in both cell populations, with an overall 95% reduction in the bone marrow plasma cell population.19.Neubert K. Meister S. Moser K. et al.The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis.Nat Med. 2008; 14: 748-755Crossref PubMed Scopus (480) Google Scholar Importantly, when compared to treatment with dexamethasone or cyclophosphamide, bortezomib demonstrated a greater reduction in the total number of splenic- and bone marrow-derived plasma cells. However, only bortezomib significantly reduced serum levels of anti-double-stranded DNA immunoglobulin G levels, which may have resulted from the induction of a terminal UPR in plasma cells, as evidenced by the induction of the protein-folding chaperone BiP. Clinically, reduction in plasma cells and anti-double-stranded DNA Abs result in amelioration of glomerulonephritis and prolonged survival. Pathological examination of bortezomib-treated animals demonstrated an absence of glomerulitis, vasculitis, and Ab deposition that was present in control animals. Proteinuria, a marker of renal dysfunction, also significantly improved with bortezomib treatment. Notably, infectious complications were not apparent as mice treated with bortezomib survived for more than 10 months without signs of infection. Given the evidence presented, bortezomib appears to possess considerable potential as a plasma cell–depleting agent. Vanderlugt et al.20.Vanderlugt C.L. Rahbe S.M. Elliott P.J. et al.Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519.J Autoimmun. 2000; 14: 205-211Crossref PubMed Scopus (79) Google Scholar evaluated the effects of PI in a murine model (SJL/J mouse) of relapsing experimental autoimmune encephalomyelitis (an experimental model of multiple sclerosis induced through immunization with myelin protein eptitopes). Mice treated with the proteasome inhibitor PS-519 showed improvement in paralysis scores, incidence of clinical relapse decreased, delay type hypersensitivity reaction, and spinal cord histology. Palombella et al.21.Palombella V.J. Conner E.M. Fuseler J.W. et al.Role of the proteasome and NK-κB in streptococcal cell wall-induced polyarthritis.Proc Natl Acad Sci USA. 1998; 95: 15671-15676Crossref PubMed Scopus (269) Google Scholar studied the effects of PI in a Lewis rat polyarthritis model induced by intraperitoneal injection of group A Streptococcal cell wall peptidoglycan and polysaccharide. This polyarthritis model is characterized by NF-κB-mediated upregulation of cell adhesion molecules and proinflammatory cytokines with histology that closely mimics rheumatoid arthritis. Utilizing several methodologies to assess disease severity, Palombella et al.21.Palombella V.J. Conner E.M. Fuseler J.W. et al.Role of the proteasome and NK-κB in streptococcal cell wall-induced polyarthritis.Proc Natl Acad Sci USA. 1998; 95: 15671-15676Crossref PubMed Scopus (269) Google Scholar demonstrated improvement in clinical manifestations of polyarthritis (assessed by the total arthritis index) in bortezomib-treated animals. Additionally, average hind paw volume, an objective measure of polyarthritis, was also markedly reduced in bortezomib-treated animals. Finally, histology of hind paw joints at necropsy demonstrated a reduction in cellular infiltrates as well as an attenuation of the degradation of articular cartilage and erosion of subchondral bone. Three studies have examined potential roles for proteasomes and PIs in transplant rejection models. Luo et al.18.Luo H. Wu Y. Qi S. et al.A proteasome inhibitor effectively prevents mouse heart allograft rejection.Transplantation. 2001; 72: 196-202Crossref PubMed Scopus (63) Google Scholar evaluated the PI dipeptide boronic acid (DPBA) and found it to suppress T-cell proliferation and IL-2, IL-6, IL-10, IL-13, and γ-interferon production in vitro in response to anti-CD3 monoclonal Ab. Short-term administration of DPBA (16 days) was found to prolong murine heart allograft survival for up to 35 days compared with 7 days in control mice. This group of investigators also examined DPBA in a murine islet allograft model.22.Wu Y. Han B. Luo H. et al.Dipeptide boronic acid, a novel proteasome inhibitor, prevents islet-allograft rejection.Transplantation. 2004; 78: 360-366Crossref PubMed Scopus (16) Google Scholar In this experience, the authors first demonstrated that DPBA suppressed mixed lymphocyte reactions and cytotoxic T-cell generation in vitro. In murine islet transplant recipients, a 17-day DPBA course provided 50% islet allograft survival at 60 days, whereas control mice demonstrated islet allograft rejection at 7 days. No effect of DPBA was found on islet function following glucose challenge. In a more recent study, administration of bortezomib on day 20 following MHC-mismatched heart transplantation prolonged cardiac allograft survival to 31.7 days compared with 6.3 days in untreated controls.23.Ashton-Chess J. Mai H.L. Jovanovic V. et al.Immunoproteasome beta subunit 10 is increased in chronic antibody mediated rejection.Kidney Int. 2010; 77: 880-890Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar The authors also found lower levels of anti-MHC class I and II Abs at 7 days following transplantation. In a chronic AMR rat cardiac transplant model, administration of bortezomib beginning at 60 or 80 days following transplantation reduced anti-donor MHC class I and II Abs. Histological improvements were also observed with bortezomib administration, including reduction in C4d expression, interstitial fibrosis, and vasculopathy.23.Ashton-Chess J. Mai H.L. Jovanovic V. et al.Immunoproteasome beta subunit 10 is increased in chronic antibody mediated rejection.Kidney Int. 2010; 77: 880-890Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Recent clinical experiences have provided evidence for the ability of proteasome inhibitor-based regimens to reverse AMR. In the first report of bortezomib in renal transplantation, we treated a series of patients with refractory mixed acute rejection, defined as biopsy-proven rejection meeting the Banff criteria for both AMR and acute cellular rejection.7.Everly M.J. Everly J.J. Susskind B. et al.Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.Transplantation. 2008; 86: 1754-1761Crossref PubMed Scopus (330) Google Scholar In this report, six patients treated with refractory mixed acute rejection were treated for eight rejection episodes. Each rejection episode was treated with one cycle of bortezomib (4 doses of 1.3mg/m2). Two patients received an additional bortezomib cycle for recurrent rejection. In all cases, these mixed acute rejection episodes had previously failed multiple therapies, including plasmapheresis, rituximab, rabbit anti-thymocyte globulin, and IVIg. Immunodominant DSA (iDSA), which is defined as the highest-level DSA at the time of rejection diagnosis, was used as the marker of AMR treatment efficacy. Bortezomib therapy reduced iDSA levels by more than 50% in all cases.7.Everly M.J. Everly J.J. Susskind B. et al.Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.Transplantation. 2008; 86: 1754-1761Crossref PubMed Scopus (330) Google Scholar This includes one case where bortezomib was administered alone with plasmapheresis and resulted in >70% reduction in the iDSA and a >90% reduction in other DSA specificities. Interestingly, the majority of iDSA specificities were MHC class II specificities, and half of all the rejection episodes had an HLA-DQ specificity for the iDSA. Previously, it has been suggested that MHC class II Abs are generally more refractory to treatment.24.Zachary A.A. Montgomery R.A. Leffell M.S. Factors associated with and predictive of persistence of donor-specific antibody after treatment with plasmapheresis and intravenous immunoglobulin.Hum Immunol. 2005; 66: 364-370Crossref PubMed Scopus (84) Google Scholar Renal function improved or remained stable in the majority of rejection episodes. Also notably, renal allograft biopsies showed that all patients experience resolution or improvement in acute cellular rejection, including one patient with a Banff grade IIA acute cellular rejection, in which a severe endotheliitis completely resolved with bortezomib treatment.7.Everly M.J. Everly J.J. Susskind B. et al.Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.Transplantation. 2008; 86: 1754-1761Crossref PubMed Scopus (330) Google Scholar In this initial experience, the toxicity profile of bortezomib was reasonable, as only two patients were reported to experience adverse events.7.Everly M.J. Everly J.J. Susskind B. et al.Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.Transplantation. 2008; 86: 1754-1761Crossref PubMed Scopus (330) Google Scholar One patient experienced transient thrombocytopenia and diarrhea (which resolved with anti-diarrheal therapy). Another patient experienced febrile neutropenia (without infection), which resulted in holding of the final dose of bortezomib. Neutropenia was attributed to the additive effects of multiple immunosuppressants and anti-infective agents in this patient. Opportunistic infection and malignancy were not reported for any patients. This case series demonstrates the ability of bortezomib to provide a marked reduction in DSA levels, resolution of allograft biopsy histology, and improvement in allograft function. A subsequent report described the use of bortezomib in combination with daily plasmapheresis and IVIg in two positive cross-match kidney recipients with AMR.25.Perry D.K. Burns J.M. Pollinger H.S. et al.Proteasome inhibition causes apoptosis of normal human plasma cells preventing alloantibody production.Am J Transplant. 2009; 9: 201-209Crossref PubMed Scopus (265) Google Scholar One patient was highly sensitized pre-transplant and exhibited a flow cross-match channel shift >300. This patient received a perioperative plasmapheresis and IVIg protocol, with AMR developing during treatment with this protocol. The second patient was less sensitized initially, and therefore did not receive the perioperative plasmapheresis and IVIg protocol. Both patients received bone marrow biopsies at the time of AMR and 1 week following treatment to assess therapeutic response to AMR treatment with bortezomib. Both patients had numerous alloantibody specificities at the time of AMR, both donor specific and third-party anti-HLA Abs.25.Perry D.K. Burns J.M. Pollinger H.S. et al.Proteasome inhibition causes apoptosis of normal human plasma cells preventing alloantibody production.Am J Transplant. 2009; 9: 201-209Crossref PubMed Scopus (265) Google Scholar Ab levels were measured, by single antigen anti-HLA beads, at the time of rejection and 1 year following bortezomib treatment. In both patients, the total number of allospecificities and levels of remaining Abs were reduced in response to treatment with bortezomib. Bone marrow aspirates in these two patients showed that the percentage of plasma cells in bone marrow declined in response to bortezomib therapy. One year post transplant, both patients maintain normal renal function and transplant glomerulopathy was absent on protocol biopsies. Notably, total immunoglobulin levels were normal in both patients despite the absence of DSA-producing plasma cells. Bone marrow biopsies done during AMR treatment in these patients provide a unique perspective on the effects of bortezomib on bone marrow–resident plasma cells.25.Perry D.K. Burns J.M. Pollinger H.S. et al.Proteasome inhibition causes apoptosis of normal human plasma cells preventing alloantibody production.Am J Transplant. 2009; 9: 201-209Crossref PubMed Scopus (265) Google Scholar An ELISpot assay for the detection of antitetanus Ab demonstrated a greater than 50% reduction in Ab production in bone marrow–derived plasma cells treated with bortezomib. More recently, PI with bortezomib has been evaluated as the primary therapy for AMR in a series of two patients.5.Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2010; 89: 277-284Crossref PubMed Scopus (153) Google Scholar Both patients were treated with the combination therapy, which consisted of bortezomib, rituximab, and plasmapheresis. Bortezomib (1.3mg/m2) was given on treatment days 1, 4, 8, and 11. Patients received plasmapheresis before each bortezomib dose and every other day for 3 sessions beginning 72h after the final bortezomib dose. Rituximab (375mg/m2) was administered on treatment day 1 following bortezomib and plasmapheresis. A representation of the histological and immunological improvement seen with bortezomib-based therapy is depicted in Figure 3. The first patient developed AMR on post-transplant day 13 following his third kidney transplant.5.Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2010; 89: 277-284Crossref PubMed Scopus (153) Google Scholar Although DSAs were absent before the transplant, an acute rise in creatinine level on post-transplant day 13 prompted a renal allograft biopsy, which demonstrated C4d immunostaining of the peritubular and glomerular capillaries (Figure 3a and b). High levels of DSA were detected on single HLA-antigen bead testing by Luminex. Bortezomib-based treatment was initiated on post-transplant day 14. Following treatment, DSA was undetectable (Figure 3) and repeat renal allograft biopsy on post-transplant day 28 demonstrated a reduction in glomerular and peritubular capillary C4d staining (Figure 3c and d). Serum creatinine levels returned to pre-rejection baseline and proteinuria was not observed. The second patient to receive bortezomib-based primary therapy for AMR was a 41-year-old woman who received a one-haplotype-matched living donor kidney transplant from her son.5.Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2010; 89: 277-284Crossref PubMed Scopus (153) Google Scholar DSA was absent before transplant, but a low-level DSA was detected through surveillance on post-transplant day 7. DSA continued to rise, and on post-transplant day 13 a renal allograft biopsy demonstrated faint peritubular capillary and strong glomerular capillary C4d staining. DSA was also markedly increased, and treatment was initiated on post-transplant day 15. Plasmapheresis was done before the first two bortezomib doses, but otherwise held to reduce risk of bleeding complications from recent surgical procedures. Treatment response included DSA elimination, return of serum creatinine levels to baseline, and resolution of proteinuria. Approximately 2 months following initial treatment, the patient developed recurrent DSA elevation without renal dysfunction, which was treated with a second course of bortezomib-based therapy. Again, DSA was decreased below level of detection and serum creatinine level remained stable. Interestingly, the iDSA of the first rejection episode (anti-HLA B7) was abs