Immunoproteasome beta subunit 10 is increased in chronic antibody-mediated rejection
2010; Elsevier BV; Volume: 77; Issue: 10 Linguagem: Inglês
10.1038/ki.2010.15
ISSN1523-1755
AutoresJoanna Ashton‐Chess, Hoa Le, Vojislav Jovanović, Karine Renaudin, Yohann Foucher, Magali Giral, Philippe Moreau, Emilie Dugast, Michael Mengel, Maud Racapé, Richard Danger, Claire Usal, Helga Smit, Marina Guillet, Wilfried Gwinner, Ludmilla Le Berre, Jacques Dantal, Jean‐Paul Soulillou, Sophie Brouard,
Tópico(s)Xenotransplantation and immune response
ResumoChronic active antibody-mediated rejection is a form of late rejection with a poor prognosis. To identify specific markers of this, we analyzed several microarray studies in the literature and performed mRNA profiling of 65 biopsies and 165 blood samples of a large cohort of renal transplant patients with precisely characterized pathologies. Immunoproteasome beta subunit 10 was found to be specifically increased in the graft and blood samples during chronic active antibody-mediated rejection and was also significantly increased in rat cardiac allografts undergoing acute rejection as well as chronic active antibody-mediated rejection. This syndrome is characterized by chronic transplant vasculopathy associated with diffuse C4d staining and circulating donor-specific antibodies. Using this animal model, we found that administration of the proteasome inhibitor, Bortezomib, delayed acute rejection and attenuated the humoral response in both the acute phase and established state of this syndrome in a dose-dependent manner. Following treatment with this reagent, donor-specific antibodies and C4d deposition were reduced. These studies highlight the role of the proteasome in chronic rejection and identify this molecule as a marker of this syndrome. Chronic active antibody-mediated rejection is a form of late rejection with a poor prognosis. To identify specific markers of this, we analyzed several microarray studies in the literature and performed mRNA profiling of 65 biopsies and 165 blood samples of a large cohort of renal transplant patients with precisely characterized pathologies. Immunoproteasome beta subunit 10 was found to be specifically increased in the graft and blood samples during chronic active antibody-mediated rejection and was also significantly increased in rat cardiac allografts undergoing acute rejection as well as chronic active antibody-mediated rejection. This syndrome is characterized by chronic transplant vasculopathy associated with diffuse C4d staining and circulating donor-specific antibodies. Using this animal model, we found that administration of the proteasome inhibitor, Bortezomib, delayed acute rejection and attenuated the humoral response in both the acute phase and established state of this syndrome in a dose-dependent manner. Following treatment with this reagent, donor-specific antibodies and C4d deposition were reduced. These studies highlight the role of the proteasome in chronic rejection and identify this molecule as a marker of this syndrome. Long-term graft loss remains the bane of kidney transplantation. More recently, much attention has been paid to the ‘humoral’ theory of chronic allograft rejection.1.Terasaki P.I. Humoral theory of transplantation.Am J Transplant. 2003; 3: 665-673Crossref PubMed Scopus (488) Google Scholar Evidence for the involvement of a humoral arm of the immune response to allografts has come from studies analyzing the impact of anti-human leukocyte antigen (HLA) antibodies on graft outcome,2.Terasaki P.I. Ozawa M. Predicting kidney graft failure by HLA antibodies: a prospective trial.Am J Transplant. 2004; 4: 438-443Crossref PubMed Scopus (363) Google Scholar,3.Terasaki P.I. Ozawa M. Predictive value of HLA antibodies and serum creatinine in chronic rejection: results of a 2-year prospective trial.Transplantation. 2005; 80: 1194-1197Crossref PubMed Scopus (128) Google Scholar and evidence of complement cascade activation within kidney grafts diagnosed by intragraft deposition of the complement split product C4d.4.Mauiyyedi S. Pelle P.D. Saidman S. et al.Chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by C4d deposits in peritubular capillaries.J Am Soc Nephrol. 2001; 12: 574-582PubMed Google Scholar,5.Regele H. Bohmig G.A. Habicht A. et al.Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection.J Am Soc Nephrol. 2002; 13: 2371-2380Crossref PubMed Scopus (371) Google Scholar These data were recently reinforced when the definition of chronic active antibody-mediated rejection (CAMR) was introduced into the Banff classification of kidney graft injury as an association of specific histological lesions associated with diffuse C4d deposition in peritubular capillaries and circulating donor-specific anti-HLA (DSA) antibodies.6.Solez K. Colvin R.B. Racusen L.C. et al.Banff’05 Meeting report: Differential Diagnosis of Chronic Allograft Injury and Elimination of Chronic Allograft Nephropathy (‘CAN’).Am J Transplant. 2007; 7: 518-526Crossref PubMed Scopus (865) Google Scholar Current therapies to combat the acute form of antibody-mediated rejection aim to reduce antibody titers through the use of intravenous immunoglobulin,7.Jordan S.C. Vo A.A. Toyoda M. et al.Post-transplant therapy with high-dose intravenous gammaglobulin: applications to treatment of antibody-mediated rejection.Pediatr Transplant. 2005; 9: 155-161Crossref PubMed Scopus (38) Google Scholar plasmapheresis,8.Akalin E. Dinavahi R. Friedlander R. et al.Addition of plasmapheresis decreases the incidence of acute antibody-mediated rejection in sensitized patients with strong donor-specific antibodies.Clin J Am Soc Nephrol. 2008; 3: 1160-1167Crossref PubMed Scopus (73) Google Scholar or B-cell-targeting antibodies such as Rituximab,9.Faguer S. Kamar N. Guilbeaud-Frugier C. et al.Rituximab therapy for acute humoral rejection after kidney transplantation.Transplantation. 2007; 83: 1277-1280Crossref PubMed Scopus (163) Google Scholar but these have not gained notoriety in the chronic form. Moreover, these strategies do not seem to have an effect on plasma cells, the source of antibodies. Thus, the development of more effective strategies would benefit patients suffering from this type of late graft rejection with such a poor prognosis.10.David-Neto E. Prado E. Beutel A. et al.C4d-positive chronic rejection: a frequent entity with a poor outcome.Transplantation. 2007; 84: 1391-1398Crossref PubMed Scopus (31) Google Scholar The identification of molecular markers associated with CAMR would not only facilitate its diagnosis, but could also help to understand the pathophysiology of CAMR and thus aid in the design of new therapeutic strategies. Currently, CAMR diagnosis depends on the triad of graft lesions together with intragraft C4d and DSA mentioned above. Neither one of these alone can diagnose CAMR; DSA are predictive of graft loss but have not yet been shown to tightly correlate with intragraft lesions, and C4d has recently been shown to be absent in up to 50% of CAMR cases.11.Sis B. Jhangri G.S. Bunnag S. et al.Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining.Am J Transplant. 2009; 9: 2312-2323Crossref PubMed Scopus (355) Google Scholar Here, using a gene-set comparison approach, we describe the identification of a well-known molecule, the immunoproteasome-β subunit 10 (PSMB10, also known as MECL-1) as a potential marker of CAMR in humans. Moreover, we obtained concordant results in a rat heart allograft model that we have recently described as a pertinent model of CAMR (histological lesions of chronic vascular damage, persisting antidonor antibodies and diffuse C4d deposits on the graft vasculature).12.Ballet C. Renaudin K. Degauque N. et al.Indirect CD4+ TH1 response, antidonor antibodies and diffuse C4d graft deposits in long-term recipients conditioned by donor antigens priming.Am J Transplant. 2009; 9: 697-708Crossref PubMed Scopus (19) Google Scholar Here we show that, in this model, inhibition of the proteasome significantly prolongs allograft survival by preventing acute rejection, and also attenuates established humoral immune responses (decreases DSA and C4d deposition) in both short- and long-term surviving recipients. Our data suggest the implication of the immunoproteasome in CAMR with PSMB10 as a potentially useful marker and point toward proteasome inhibition as a means of treating both acute rejection and CAMR. In order to identify potential diagnostic markers of chronic graft injury in humans, we compared several microarray data sets published in the literature in the context of human kidney transplant biopsies with chronic lesions (referred to as CAN or chronic rejection in the studies in question; Table 1). In total, less than 20 molecules were found to be common between at least two data sets (not shown). Among them, PSMB10 was found to be upregulated in chronic rejection in a study by Donauer et al.13.Donauer J. Rumberger B. Klein M. et al.Expression profiling on chronically rejected transplant kidneys.Transplantation. 2003; 76: 539-547Crossref PubMed Scopus (49) Google Scholar and in Banff grade 3 versus Banff grade 0 in a study by Flechner et al.14.Flechner S.M. Kurian S.M. Solez K. et al.De novo kidney transplantation without use of calcineurin inhibitors preserves renal structure and function at two years.Am J Transplant. 2004; 4: 1776-1785Crossref PubMed Scopus (262) Google Scholar PSMB10 was chosen from among the others because of its function as an instrumental member of the immunoproteasome and the availability of reagents to test it as a potential therapeutic target. In fact, the immunoproteasome was shown to be involved in antigen processing for presentation,15.Tanaka K. Kasahara M. The MHC class I ligand-generating system: roles of immunoproteasomes and the interferon-gamma-inducible proteasome activator PA28.Immunol Rev. 1998; 163: 161-176Crossref PubMed Scopus (253) Google Scholar and proteasome inhibition was shown to blunt antibody responses in mice.16.Cascio P. Oliva L. Cerruti F. et al.Dampening Ab responses using proteasome inhibitors following in vivo B cell activation.Eur J Immunol. 2008; 38: 658-667Crossref PubMed Scopus (50) Google Scholar In healthy individuals, PSMB10 is expressed constitutively by the immune system (the spleen, peripheral blood, and lymph nodes; Supplementary Figure S1A) and by lymphocytes and monocytes, where its expression is regulated by cell activation (Supplementary Figure S1A and B). It can also be induced through exposure to interferon-γ.17.Van den Eynde B.J. Morel S. Differential processing of class-I-restricted epitopes by the standard proteasome and the immunoproteasome.Curr Opin Immunol. 2001; 13: 147-153Crossref PubMed Scopus (161) Google ScholarTable 1Studies used to identify common biomarkers of late graft injuryFirst author, journal/yearSubjectSampleMicroarray platformGene listHotchkiss, Transplantation/2006‘Chronic allograft nephropathy’ (CAN)BiopsiesAffymetrixSelected upregulated genes in CAN (gene list published)Flechner, Am J Transplant/2004Banff grade 3 vs Banff 1BiopsiesAffymetrixGenes upregulated in Banff 3 vs Banff 01 (gene list available on the authors’ website)Scherer, Transplantation/2003‘Chronic Rejection’ (CR)BiopsiesAffymetrixGenes upregulated in biopsies at 6 months which went on to develop CR at 1-year vs biopsies at 6 months with no CR at 1-year (exhaustive gene list kindly provided by the authors)Donauer, Transplantation/2003‘Chronic Rejection’ (CR)Biopsies‘Homemade’ according to Stanford protocolUpregulated in CR vs normal and polycystic kidneys (gene list published) Open table in a new tab Download .jpg (.05 MB) Help with files Supplementary Figure S1 We analyzed PSMB10 in graft biopsies with different histological diagnoses (Table 2). As shown in Figure 1a, PSMB10 mRNA was specifically upregulated in biopsies with CAMR (P<0.001). Of note, PSMB10 expression was not correlated with proteinuria (r=-0.16; P=0.63). Receiver operator characteristic (ROC) curve analysis revealed that PSMB10 mRNA had an excellent capacity to discriminate CAMR from the other histological diagnoses, with an area under the curve of 0.92 (P<0.0001, 95% confidence interval of 0.84–0.97). At a cut-off of 1.95, there was a sensitivity of 0.85 and a specificity of 0.83 (Figure 1b). Thus, PSMB10 shows potential as an intragraft marker of CAMR in humans.Table 2Patients included in analysis of biopsies (Nantes and Hanover biopsies pooled; see Materials and Methods section).GroupNIF/TACNI toxCAMRaThe DSA were directed against class II (n=6), class I (n=2), or both (n=5).ACRbThese were grade Ia (n=3), Ib (n=2), and grade IIa (n=4 with moderate intimal arteritis-vi) – all were C4d-negative and DSA-negative.n131614139Recipient age, years: median (range)40.0 (18–69)49.5 (24–66)51.5 (29–66)48 (24–71)45 (25–61)Recipient gender ratio (M/F)9:49:78:67:65:4Donor age, years: median (range)35.0 (12–69)51.0 (23–77)41.5 (16–56)34 (14–75)34 (5–72)Time post-transplant years: median (range)cBiopsies for cause only.12 (1–3)5 (1–11)11 (1–18)0.67 (0.08–5)Donor gender ratio (M/F)8:510:68:65:6 (n=2 NA)8:1HLA incompatibilities: mean±s.d.2.5±1.72.5±1.22.4±1.43.5±1.43.1±1.1% First transplant100100799278Cockroft creatinine clearance (ml/min): mean±s.d.75.9±26.449.9±17.052.4±24.125.8±16.732.6±16.2Proteinuria (g/24 h): median (range)0.10 (0.03–0.43)0.11 (0.01–6.37)0.09 (0.03–4.47)1.94 (0.80–4.00)0.46 (0.21–0.49)Banff c grade: mean±s.d.0.00±0.001.25±0.580.80±1.032.27±0.791.5±0.53Immunosuppression protocol (% of patients)MMF: 69%MMF: 63%MMF: 64%MMF:46%MMF: 78%at the time of biopsyAza: 8%Aza: 13%Aza: 14%Aza: 15%Aza: 0%FK506: 62%FK506: 63%FK506: 29%FK506:38%FK506: 67%CsA: 23%CsA: 13%CsA: 71%CsA: 54%CsA: 0%Rapa: 0%Rapa: 13%Rapa: 7%Rapa: 0%Rapa: 22%Steroids: 54%Steroids: 69%Steroids: 57%Steroids: 23%Steroids: 33%Abbreviations: ACR, acute cellular rejection; Aza, azathioprine; CAMR, chronic antibody-mediated rejection; CNI tox, calcineurin inhibitor toxicity; CsA, cyclosporin A; FK 506, tacrolimus; DSA, donor-specific anti-HLA; HLA, human leukocyte antigen; IF/TA, interstitial fibrosis and tubular atrophy; MMF, mycophenolate mofetil; N, normal histology; TG, transplant glomerulopathy.a The DSA were directed against class II (n=6), class I (n=2), or both (n=5).b These were grade Ia (n=3), Ib (n=2), and grade IIa (n=4 with moderate intimal arteritis-vi) – all were C4d-negative and DSA-negative.c Biopsies for cause only. Open table in a new tab Abbreviations: ACR, acute cellular rejection; Aza, azathioprine; CAMR, chronic antibody-mediated rejection; CNI tox, calcineurin inhibitor toxicity; CsA, cyclosporin A; FK 506, tacrolimus; DSA, donor-specific anti-HLA; HLA, human leukocyte antigen; IF/TA, interstitial fibrosis and tubular atrophy; MMF, mycophenolate mofetil; N, normal histology; TG, transplant glomerulopathy. Given that ACR is becoming a rare phenomenon, we next focused on CAMR, to determine whether the specific regulation of PSMB10 was reflected in the peripheral blood. PSMB10 was analyzed in 150 kidney transplant recipients with stable graft function and 15 patients with CAMR (Table 3). As shown in Figure 2, patients with CAMR had significantly higher levels of PSMB10 than those with stable graft function (P<0.01). Again, PSMB10 expression was not correlated with proteinuria (r=-0.01; P=0.98).Table 3Patients included in analysis of PBMCsGroupSTACAMRaThe DSAs were directed against class II (n=11), class I (n=1), or both (n=3).n15015Recipient age in years: median (range)53 (20–85)51 (29–76)Recipient gender (% males)6247Donor age in years: median (range)36 (9–69)41 (14–74)Donor gender (% males)7560Time post-transplant in years: median (range)8 (5–21)6 (1–25)HLA incompatibilities: mean±s.d.3.4±1.33.5±1.7% First transplantations10080Cockroft creatinine clearance (ml/mn): mean±s.d.65.6±14.234.3±19.7Proteinuria (g/24 h): median (range)0.16 (0.04–0.16)2.61 (1.95–11.52)IS protocol at blood samplingMMF: 51%MMF: 60%Aza: 26%Aza: 7%FK506: 26%FK506: 67%CsA: 72%CsA: 20%Steroids: 15%Steroids: 27%Abbreviations: Aza, azathioprine; CAMR, chronic antibody-mediated rejection; CNI, calcineurin inhibitor; CsA, cyclosporin A; DSA, donor-specific antibody; FK 506, Prograf; HLA, human leukocyte antigen; MMF, mycophenolate mofetil; NA, not applicable; PBMC, peripheral blood mononuclear cell.a The DSAs were directed against class II (n=11), class I (n=1), or both (n=3). Open table in a new tab Abbreviations: Aza, azathioprine; CAMR, chronic antibody-mediated rejection; CNI, calcineurin inhibitor; CsA, cyclosporin A; DSA, donor-specific antibody; FK 506, Prograf; HLA, human leukocyte antigen; MMF, mycophenolate mofetil; NA, not applicable; PBMC, peripheral blood mononuclear cell. To assess whether PSMB10 could potentially serve as a minimally invasive clinical decision-making tool, ROC curve analysis was performed. The results (Figure 2b) showed that this molecule analyzed in recipient blood could still discriminate CAMR from the other groups of patients well, albeit less than in the biopsies, with an area under the curve of 0.72 (P<0.01; 95% confidence interval of 0.57–0.84). At a cut-off of 0.96, there was a sensitivity of 0.67 and a specificity of 0.64. We next performed a multivariate analysis on the 150 stable patients to evaluate the potential impact of clinical and demographic factors on the expression of PSMB10 in the peripheral blood (Table 3). Of all the parameters tested (legend to Figure 2), the statistically significant parameters identified as being associated with PSMB10 mRNA expression were recipient gender (P<0.05) and time post-transplant (P<0.01). Thus, creatinine clearance, proteinuria, donor and recipient age, number of HLA incompatibilities (A+B+DR), and maintenance immunosuppression were not confounders, neither were presence or absence of anti-HLA. Thus, PSMB10 is not simply a reflection of presence of anti-HLA antibodies. These data are modeled in Figure 2c, where predicted values of PSMB10 are expressed according to time post-transplant and recipient gender. PSMB10 was thus significantly higher in the peripheral blood mononuclear cells (PBMCs) of male recipients compared with female recipients (P<0.05) and displayed a distinctive inverse bell-shaped relationship with time post-transplant. However, these potentially confounding factors could not explain the difference in PSMB10 between stable (STA) and CAMR, because there was no difference in time post-transplant or recipient gender between the STA and CAMR groups (P=not significant). Thus, overall these data show that PSMB10 may also be a peripheral blood biomarker of CAMR in humans, but some potential confounding factors may exist and need to be taken into consideration. To determine whether we could reproduce the above data in a rodent model, PSMB10 mRNA was analyzed in the grafts and PBMCs of rat recipients of cardiac allografts undergoing acute rejection or CAMR. We found that PSMB10 was significantly increased in the cardiac allografts during both acute rejection (at day (D)7 post-transplant) and CAMR (analyzed at D100 post-transplant) compared with syngeneic controls (Figure 3a; P<0.01 and P<0.0001, respectively). A different expression profile was observed in the PBMCs, where PSMB10 displayed no change during acute rejection, suggesting an effect of DST priming on PBMCs. Moreover, similar to in humans, a significant increase was observed during CAMR at D100 post-transplant (Figure 3b; P<0.05). Thus, PSMB10 shows potential as an intragraft and peripheral blood marker of CAMR in rats and humans. Given this upregulation of PSMB10 in acute rejection and CAMR in this rodent model, we set out to determine whether proteasome inhibition could influence graft outcome. In the context of acute rejection, recipients of major histocompatibility complex (MHC)-mismatched cardiac allografts were treated with Bortezomib every other day from D0–D20. The data presented in Figure 4a show that Bortezomib dose-dependently prolonged cardiac allograft survival with an optimal effect at 0.1 mg/kg, giving a mean survival of 31.7 days (n=6) (versus 6.3 days in untreated controls (n=4); P<0.01). Moreover, at D7, there were significantly lower circulating anti-donor MHC class I and II antibodies for total immunoglobulin G (IgG) (P<0.01) as well as IgG1 (P<0.01), and IgG2c (P<0.05), with no reduction for IgG2a or IgG2b (Figure 4b). In the context of CAMR, Bortezomib was initially administered every other day from D80 to D100 at 0.1 mg/kg in rat recipients of cardiac allografts that had received DST before transplantation and were surviving long-term. The DST+Bortezomib recipients had significantly reduced levels of circulating anti-donor MHC class I and II antibodies for total IgG as well as IgG1, IgG2a, IgG2b, and IgG2c (Figure 5a). Bortezomib-treated recipients tended to display less intra-graft complement deposition (Table 4). In fact, although the grafts of DST-treated animals displayed diffuse linear expression of C4d (Figure 5b, left-hand panel and magnified insert), two Bortezomib-treated animals showed only vague background staining, whereas the others displayed minimal staining (Figure 5b, right-hand panel and magnified insert). There were no differences between the two groups in terms of infiltrate, fibrosis, vascular obstruction, vascular lesions, and number of affected arteries; however, myocyte necrosis was absent in four of the five animals in the Bortezomib-treated group (Table 4 and examples in Figure 5c, far left and middle panels). We thus performed further experiments in which treatment was initiated earlier at D60 and continued until D100. Again, a significant decrease in circulating anti-donor MHC class I and II antibodies for total IgG as well as for three of the four IgG subtypes tested was noted at D100 post-transplant in the Bortezomib-treated recipients (Figure 5d). This decrease was not simply a time-dependent effect. because recipients had significantly less DSA after the 40-day treatment versus before treatment, whereas DSA levels were unchanged in untreated animals over the same time period (Figure 5e). C4d staining at D100 in this group was heterogeneous (Table 4). A histological analysis at D100 showed that there was a tendency toward reduced vascular lesions and a significant reduction in fibrosis compared with the untreated DST group (P<0.05; Table 4 and an example Figure 5c, far right panel). Thus, in this model and at the dose and schedules studied, Bortezomib is able to significantly attenuate the humoral immune response in graft recipients undergoing CAMR with a reduction in fibrosis and a tendency to reduce the histological lesions of transplant vasculopathy.Table 4Histological analysis of DST-treated animals (D7 and D14 before transplantation) subsequently untreated or treated every other day from D80 to D100 or from D60 to D100 (CAMR lesions already established) with Velcade at 0.1 mg/kgRatGroupGraft infiltrateFibrosisNecrosisVascular lesionsVascular obstructionNo. of affected arteries (art/section)C4d1DST23*+223 (28)+++2DST23+000+++3DST34*+2413 (16)+++4DST2–32+211 (21)+++5DST2–33+113 (19)+++1DST+Bortezomib D80–D1002–33–4*+1–3413 (33)+2DST+Bortezomib D80–D10023-248 (48)-3DST+Bortezomib D80–D1001–23–4-000-4DST+Bortezomib D80–D1001–23-247 (38)++5DST+Bortezomib D80–D1001–23*-112 (16)++1DST+Bortezomib D60–D10021*+000+++2DST+Bortezomib D60–D1002–32*+000++3DST+Bortezomib D60–D1001–23Focal337 (31)+++4DST+Bortezomib D60–D10021Focal113 (8)+++5DST+Bortezomib D60–D1001–22-000-6DST+Bortezomib D60–D10022Focal000.-P=NSP<0.05P=NSP=NSP=NSAbbreviations: CAMR, chronic antibody-mediated rejection; DST, donor-specific antibody; NS, not significant.Infiltrate was graded from 0 to 4 (0, absence; 1, minimum; 2, discrete; 3, moderate; 4, abundant). Fibrosis was graded from 0 to 4 (0, absence; 1, focal; 2, diffuse-minimal; 3, diffuse-moderate; 4, diffuse-abundant; *, edema). Vascular obstruction was graded from 0 to 4 (0, 0; 1, 80%). Vascular lesions were graded from 0 to 3 (0, normal; 1, leuco-intimal adhesion; 2, inflammatory endarteritis; 3, fibrous endarteritis). C4d was graded from – to +++ (-, 75%). Necrosis was graded as presence (+) or absence (-) of myocyte damage. Statistical significance was measured using a non-parametric Kruskal–Wallis test with Dunn's Multiple Comparison test. Open table in a new tab Abbreviations: CAMR, chronic antibody-mediated rejection; DST, donor-specific antibody; NS, not significant. Infiltrate was graded from 0 to 4 (0, absence; 1, minimum; 2, discrete; 3, moderate; 4, abundant). Fibrosis was graded from 0 to 4 (0, absence; 1, focal; 2, diffuse-minimal; 3, diffuse-moderate; 4, diffuse-abundant; *, edema). Vascular obstruction was graded from 0 to 4 (0, 0; 1, 80%). Vascular lesions were graded from 0 to 3 (0, normal; 1, leuco-intimal adhesion; 2, inflammatory endarteritis; 3, fibrous endarteritis). C4d was graded from – to +++ (-, 75%). Necrosis was graded as presence (+) or absence (-) of myocyte damage. Statistical significance was measured using a non-parametric Kruskal–Wallis test with Dunn's Multiple Comparison test. Finally, as PSMB10 expression did not correlate with proteinuria in the blood or graft of humans, we wished to confirm this in a rat model. For this purpose, we analyzed PSMB10 in Buffalo/Mna rats, a well-known rat model of proteinuria due to spontaneous idiopathic nephritic syndrome of unknown origin (see Materials and Methods section for details). As shown in the Figure 6, PSMB10 was not significantly differentially expressed between rats with or without proteinuria, despite radical differences in proteinuria. Here we used a literature gene-set comparison approach and identified PSMB10 as a potential biomarker of chronic graft injury. Further profiling by quantitative PCR in biopsies and PBMCs of renal transplant recipients revealed this molecule to be specifically increased in the graft and blood in CAMR. PSMB10 was also significantly increased in rat cardiac allograft models with acute or chronic rejection. In the acute model, administration of the proteasome inhibitor Bortezomib not only dose-dependently delayed acute rejection but also attenuated the humoral response. In the chronic model with established CAMR, Bortezomib treatment decreased DSA and C4d deposition and improved some aspects of the chronic tissue injury. These data thus suggest PSMB10 as a potential marker and the proteasome as a therapeutic target for CAMR. Proteasomes are large protease complexes located in cytoplasm and nuclei that degrade cellular proteins in a ubiquitin-dependent and adenosine triphosphate-dependent manner present in immune cells.18.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 (438) Google Scholar In response to interferon-γ, the three catalytic subunits are replaced by their homologous subunits, PSMB 8, 9, and 10, to form the so-called immunoproteasome, which is essential for processing antigenic peptides for presentation through the class I MHC complex.15.Tanaka K. Kasahara M. The MHC class I ligand-generating system: roles of immunoproteasomes and the interferon-gamma-inducible proteasome activator PA28.Immunol Rev. 1998; 163: 161-176Crossref PubMed Scopus (253) Google Scholar Immunoproteasomes have additional effects that are independent to class I processing, for example, they inhibit T-cell proliferation, as T cells lacking immunoproteasome subunits hyperproliferate in vitro and in vivo, and KO mice have higher numbers of central memory CD8+ cells.19.Caudill C.M. Jayarapu K. Elenich L. et al.T cells lacking immunoproteasome subunits MECL-1 and LMP7 hyperproliferate in response to polyclonal mitogens.J Immunol. 2006; 176: 4075-4082Crossref PubMed Scopus (61) Google Scholar Experiments in KO mice have revealed that PSMB10 is involved in controlling homeostatic equilibrium between T-cell subsets,20.Zaiss D.M. de Graaf N. Sijts A.J. The proteasome immunosubunit multicatalytic endopeptidase complex-like 1 is a T-cell-intrinsic factor influencing homeostatic expansion.Infect Immun. 2008; 76: 1207-1213Crossref PubMed Scopus (39) Google Scholar thereby controlling the T-cell repertoire.21.Basler M. Moebius J. Elenich L. et al.An altered T cell repertoire in MECL-1-deficient mice.J Immunol. 2006; 176: 6665-6672Crossref PubMed Scopus (83) Google Scholar Given the expression of PSMB10 by lymphocytes and monocytes, it is possible that the increase in PSMB10 in the graft in CAMR may be the result of specific infiltration of these cells. B cells in particular may be involved in this because patients with chronic rejection undergo lymphoid neogenesis with the development of intragraft B-cell germinal centers.22.Thaunat O. Field A.C. Dai J. et al.Lymphoid neogenesis in chronic rejection: evidence for a local humoral alloimmune response.Proc Natl Acad Sci USA. 2005; 102: 14723-14728Crossref PubMed Scopus (194) Google Scholar Likewise, increased PSMB10 expression may come from locally present activated monocytes or mature dendritic cells, as the immunoproteasome is known to be increased in these cells.23.Whiteside T.L. Stanson J. Shurin M.R. et al.Antigen-processing machinery in human dendritic cells: up-regulation by maturation and down-regulation by tumor cells.J Immunol. 2004; 173: 1526-1534Crossref PubMed Scopus (74) Google Scholar Given that the proteasome decreases during late phase plasma cell differentiation,24.Cenci S. Mezghrani A. Cascio P. et al.Progressively impaired proteasomal capacity during terminal plasma cell
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