Binggeli, Simone

Descœudres, Bernard

Egli, Adrian

Hirsch, Hans H.

Mayr, Michael

Mihatsch, Michael J.

Schaub, Stefan

Steiger, Jürg

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Case ReportTransient allograft dysfunction from immune reconstitution ina patient with polyoma BK-virus-associated nephropathyStefan Schaub1, Michael Mayr1, Adrian Egli2, Simone Binggeli2, Bernard Descœudres1,Ju¨rg Steiger1, Michael J. Mihatsch3 and Hans H. Hirsch2,41Clinic for Transplantation Immunology and Nephrology, University Hospital Basel, 2Transplantation Virology andMolecular Diagnostics, Department of Clinical and Biological Sciences, Institute for Medical Microbiology, University ofBasel, 3Institute for Pathology, University Hospital Basel and 4Clinic for Infectious Diseases & Hospital Epidemiology,University Hospital Basel, Basel, SwitzerlandKeywords: immune reconstitution syndrome; polyomaBK-virus nephropathy; renal allograft rejectionIntroductionDuring the last 10 years, polyoma BK-virus associatednephropathy (PVAN) has emerged as a seriouscomplication in renal transplant recipients [1]. Due tothe establishment of an accurate non-invasive screen-ing procedure measuring polyomavirus BK-viraemia,BK-viruria and decoy cells in urine, PVAN can bediagnosed at early stages [2]. This allows for timelytherapeutic intervention, which has significantlyreduced the incidence of severe PVAN courses includ-ing graft loss [3,4].Management of PVAN is mainly based on areduction of the immunosuppressive drugs, whilethe impact of anti-viral therapy is not yet clear [5].This strategy bears the inherent risk that allograftrejection may arise, which is difficult to differentiatefrom an immune response to the BK-virus, becauseboth entities can present as morphologically andmolecularly indistinguishable, with interstitial infil-trates and tubulitis [1,5–7]. Therefore, more dataregarding the natural course of PVAN under reducedimmunosuppression might be helpful to illuminate thescope of post-intervention responses.Case reportA 37-year-old woman had end-stage renal failure dueto a nephropathy of unknown origin. She was highlysensitized as a consequence of two blood transfusionsand two pregnancies (peak CDC-PRA 78%, peakFlowPRATM class I 93%, FlowPRATM class IInegative). After being on haemodialysis for 9 years,she received a kidney from a 7-year-old deceaseddonor. There were three HLA-mismatches (recipient:HLA-A3/24, B7/55, DR4/13; donor: HLA-A2/24,B7/38, DR11/13) and the recipient had two donor-specific HLA-antibodies (DSA) detectable in threehistoric sera (A2 and B38; determined by FlowPRATMsingle-antigen flow-beads). Flow-cytometric T- andB-cell cross-matches were positive with historic sera,but negative with the current one. The patient wasconsidered to be at high risk for rejection and receivedan induction therapy consisting of polyclonal anti-T-lymphocyte globulin (ATG-Fresenius) as well asintravenous immunoglobulins (IvIg) [8]. Maintenanceimmunosuppression consisted of tacrolimus, mycophe-nolate mofetil (MMF) and steroids.Figure 1 summarizes the course of immunosuppres-sive therapy, allograft function and BK-virus activity;Figure 2 demonstrates the histology of the fourallograft biopsies obtained in this patient. The allografthad an immediate good function and serum creatininedropped to 140 mmol/l by day 10 post-transplant.On day 21, serum creatinine rose to 308 mmol/l andthe first allograft biopsy was obtained. The diagnosisof antibody-mediated rejection was made based onthe presence of thrombotic microangiopathy, diffuseC4d-staining in peritubular capillaries and reappear-ance of both remote DSA (A2 and B38) in highquantities (Figure 2; picture 1A and 1B). The patientreceived another course of IvIg, six steroid pulses andfour plasmapheresis treatments. Subsequently, serumcreatinine declined to 110 mmol/l.Eight weeks post-transplant, increasing BK-virusreplication in the urine was noted along with theappearance of numerous decoy cells, followed byplasma BK-viral loads persisting above 10 000Correspondence and offprint requests to: Stefan Schaub, MD, MSc,Transplantation Immunology and Nephrology, University HospitalBasel, Petersgraben 4, 4031 Basel, Switzerland.Email: schaubs@uhbs.chNephrol Dial Transplant (2007) 22: 2386–2390doi:10.1093/ndt/gfm283Advance Access publication 2 June 2007 The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please email: journals.permissions@oxfordjournals.orgcopies/ml. The diagnosis of ‘presumptive’ PVAN wasmade [1] and immunosuppression reduced (tacrolimustrough levels from 10–15 ng/ml to 6–8 ng/ml, MMFdose from 2 to 1.5 g/day, further tapering of steroids).Three weeks later, a second allograft biopsy wasperformed. Dense focal lymphohistiocytic infiltrateswere seen, affecting 20% of the cortical and 35% of themedullar area with mild tubulitis. There were numerousSV40 antigen-positive tubular epithelial cells in themedulla and in the cortex (Figure 2; picture 2A and 2B).At this time-point, BK-viral loads were 3.9 1011copies/ml in urine and 5.1 106 copies/ml in plasma,respectively. The diagnosis of PVAN pattern Bwas made and immunosuppression further reduced(tacrolimus trough levels to 4–6 ng/ml, MMF dose to0.5 g/day, steroids were tapered out over 8 weeks).BK-viraemia remained constantly above 106 copies/ml for 8 weeks, but then started to decline by twolog10 units to a plateau of around 4 104 copies/ml.Within this time period, serum creatinine concentra-tions slowly rose from 140 to 180 mmol/l. A thirdallograft biopsy was performed, which showed massivediffuse lymphohistiocytic infiltrates affecting 100% ofthe cortical and medullar area with severe tubulitis.There were only few SV40 antigen positive tubularepithelial cells in the medulla and the cortex (Figure 2;picture 3A and 3B). A presumed diagnosis ofBK-virus specific immune reconstitution was made.10–15 6–8 4–6Tacrolimus[ng/mL]2000 1500 500 1500MMF[mg/day]PrednisoneATG 5 days IvIg125gSM 0.5gPP642024681012BK viral load[log co/mL]100200300400500600-- -- -Decoy cells[count per 10 HPF]Urine viral loadDecoy cells(- indicates no detection     of decoycells)050100150200250300Serumcreatinine[mmol/L]** **2 3410123Jul 05 Okt 05 Jan 06 Apr 06 Jul 06BK VLP IgGBK VLP IgMELISA[OD]Plasma viral loadFig. 1. Course of immunosuppressive therapy, allograft function, BK-virus activity and BK-virus serology within the first year aftertransplantation. Stars with appended numbers in the serum creatinine graphic indicate allograft biopsies. The corresponding allografthistologies are shown in Figure 2. PP, plasmapheresis; SM, solumedrol i.v.; IvIg, intravenous immunoglobulins; BK-virus-specific IgG andIgM antibodies were measured in human sera using virus-like particles (VLP) as antigens coated to microtitre plates. BK-virus-like particleswere purified by density gradient centrifugation of lysates from Sf9 insect cells infected with recombinant BK-virus-VP1 Bac-to-Bacexpression vectors (Invitrogen). Antigen coating and serum dilutions provided optimal OD results at 50 ng and 1: 400, respectively(S. Bodaghi and H.H. Hirsch, unpublished data).Immune reconstitution in PVAN 2387Immunosuppression consisting of tacrolimus withtrough levels of 4–6 ng/ml and MMF 0.5 g/day wasmaintained and no additional therapy introduced.Retrospective analysis of serum samples at thistime-point revealed newly produced BK-virus specificIgM together with increasing IgG antibodies (Figure 1,bottom panel) consistent with an emerging BK-virusspecific immune response.Fig. 2. Allograft histology graded according to the Banff classification [16] and a recently proposed semi-quantitative assessment for PVAN[1]. All allograft biopsies consisted of two cores obtained with a 16-gauge needle. First biopsy, 21 days post-transplant (cy0, i0t0, ci0ct0): thetubulointerstitial compartment was completely normal, but there were fibrin thrombi in peripheral glomerular capillary loops (arrow)consistent with thrombotic microangiopathy (1A). Peritubular capillaries (PTC) were diffuse positive for C4d by immunofluorescence (IF)(1B). Staining for SV40 antigen by immunohistochemistry was negative. Second biopsy, 3 months post-transplant (cy3, i2t1, ci0ct0): densefocal lymphohistiocytic infiltrates in the cortical and medullary interstitial space with mild tubulitis. Note enlarged nuclei (arrow) withoutclear-cut inclusion bodies (2A). Glomeruli and arteries were completely normal. Staining for SV40 antigen revealed numerous infected tubularepithelial cells in the cortex and medulla (2B). Staining for C4d in PTC and HLA-DR in tubular epithelial cells were negative. Third biopsy,6 months post-transplant (cy1, i3t3, ci0ct1): massive diffuse lymphohistiocytic infiltrates affecting the whole cortical and medullary space withsevere tubulitis (3A). Immune phenotyping of the infiltrate revealed a very dominant fraction of T-cells (CD3þ) with equal amounts of CD4þand CD8þ cells, few macrophages (CD68) and few B-cells (CD20). Glomeruli and arteries were completely normal. Only few tubularepithelial cells were positive for SV40 antigen (3B). Staining for C4d in PTC and HLA-DR in tubular epithelial cells were negative. Fourthbiopsy, 10 months post-transplant (cy0, i0t0, ci0ct0): both biopsy cores showed perfectly normal renal tissue (4A and 4B). No tubularepithelial cells were positive for SV40 antigen. Staining for C4d in PTC and HLA-DR in tubular epithelial cells were negative.2388 S. Schaub et al.Ten weeks later, BK-viraemia became negative,followed by clearance of the virus in the urine. Serumcreatinine declined and stabilized at 110–120mmol/l.A follow-up biopsy showed normal allograft tissuewithout any tubulointerstitial infiltrates, no tubularatrophy and interstitial fibrosis and no SV40 antigenpositive tubular epithelial cells (Figure 2; picture 4Aand 4B). Testing for BK-virus-specific T-cells byinterferon-g ELISpot at this time-point revealeda response against BK-virus large T and VP1 proteins.Intracellular staining for interferon-g showed a promi-nent large T- directed CD8þ T-cell response consistentwith an established cellular immune responseas reported previously (Figure 3) [9].DiscussionThe majority of patients with PVAN demonstratetubulointerstitial inflammation [6,10], which caneither indicate concurrent allograft rejection or aninflammatory response to the BK-virus. The relativecontribution of these two entities is unknown andspecific single markers to separate them are currentlylacking. The clinico-pathological course of our patientis intriguing and may serve as an index case ofBK-virus-specific immune reconstitution syndrome.At the time of the third allograft biopsy showingdiffuse massive interstitial infiltrates and severetubulitis, circumstantial evidence suggested that acuterejection was less likely as the cause. First, extensiveinfiltrates were coincident with a significant decline ofBK-virus replication, indicated by a decreasing plasmaBK-viral load of >2 log10 units and a substantialclearance from the allograft based on a significantreduction of SV40 antigen-positive tubular epithelialcells. Second, the increase of serum creatinine was onlymoderate, despite severe tubulointerstital pathology.Third, staining of tubular epithelial cells for HLA-DRin frozen sections was available, which served as anadjunctive tool to differentiate between tubulitis dueto rejection and PVAN. HLA-DR positivity is a typicalfinding in Banff Ia/Ib rejection whereas it is mostlynegative in PVAN, as in this case and describedearlier [11]. However, the strongest argument againstan alloimmune-related inflammation was the clinico-pathological course. Indeed, massive tubulointerstitialinfiltrates developed after immunosuppression wassignificantly lowered, but they subsequently resolvedcompletely while maintaining the same low immuno-suppression without having applied any rejectiontreatment. A drug-induced interstitial nephritis isalso very unlikely, because no drugs were added ordiscontinued within this time frame, with the exceptionof tapering out prednisone and reducing tacrolimusand MMF. Therefore, BK-virus-specific immunereconstitution is the most probable explanationfor the observed clinico-pathological course, and tothe best of our knowledge there is no consistentalternative diagnosis.Support for this interpretation comes from retro-spectively measured BK-virus-specific antibody titresas a surrogate marker of the emerging immuneresponse to the BK-virus, as has been reportedpreviously [12]. A marked increase of BK-virus specificIgM and IgG was noted following the decline ofplasma BK-virus load at the time of severe tubulo-interstitial inflammation. Longitudinal analysis ofBK-virus specific cellular immune responses or char-acterization of cellular infiltrates in the allograft wouldhave been of particular interest in this case [13],but were not available. However, we analysed thefrequency of BK-virus specific interferon-g producing0.45%CD3+CD4+ CD3+CD8+IFNg%IFNg%BK LT peptidesCD69RPMI mediumCD69B0.92%0.07%0.04%                     A 100,00010,0001,000100101SEB BK LT BK VP1SFU per million PBMCFig. 3. BK-virus-specific interferon-g production in PBMC atresolution of PVAN (i.e. at the time point of the fourth allograftbiopsy). The 15-mer peptide pools of 11 amino acid overlapsspanning BK-virus large T-antigen (LT) and VP1 capsid protein(VP1) were used for PBMC stimulation and interferon-g detection byELISpot assay (A) or by intracellular cytokine staining/flowcytometry (B) as described previously [9]. Staphylococcus enterotoxinB (SEB, SIGMA, Buchs, Switzerland, 1 mg/ml) and cell culturemedium served as positive and negative control, respectively.The number of spot forming unit (SFU) per well was calculatedfrom triplicates after subtractions of negative control. By flowcytometry, at least 30 000 CD3þ cells were acquired and analysed ona FACS-Canto (Becton Dickinson). The frequency of BK-virus-specific cellular immune responses was determined for each antigenand expressed as percent of interferon-g positive cells amongCD3þCD4þCD69þ or CD3þCD8þCD69þ gated lymphocytes,respectively.Immune reconstitution in PVAN 2389T-cells by ELISpot and flow cytometry after resolutionof PVAN, which were consistent with an establishedcellular immune response against the BK-virus and inline with our previous observation [9]. The reportedcase suggests that integration of longitudinal dynamicsof BK-virus replication together with allograft func-tion, histology and possibly markers of virus-specificcellular and humoral immunity may currently be themost valuable parameter for differentiation of tubu-lointerstitial allograft rejection vs a BK-virus-specificimmune response.An interesting finding in this case was the detectionof completely normal renal tissue in the last biopsydespite severe tubulointerstitial inflammation andmany BK-virus infected tubular epithelial cells inprevious biopsies. This finding is surprising and mayindicate that the end-stage of PVAN characterized byprogressive tubular atrophy and interstitial fibrosis isthe result of ongoing tubular cell necrosis unbalancedby regeneration [10]. Therefore, the good outcome inthe reported case could be related to an early andefficient clearance of the BK-virus, or to a highcapacity of the allograft from a 7-year-old donor tosustain injury and to regenerate. Although this singlecase suggests that even severe inflammation due toimmune reconstitution may not be harmful, the valueof immunomodulatory drugs including steroids andleflunomide as a treatment option to limit the extent ofinflammation is not clear yet. Notably, the immunereconstitution syndrome is well-known in HIV/AIDSpatients and steroids are often considered when theinflammation becomes damaging to an organ [14,15].In conclusion, we report a case consistent withBK-virus-specific immune reconstitution after reduc-tion of immunosuppression for treating PVAN, lead-ing to transient severe tubulointerstitial inflammationand moderate allograft dysfunction. Clearly, no gen-eral recommendations regarding diagnosis and treat-ment of BK-virus specific immune reconstitutionsyndrome can be made based on this single case.Prospective studies with standardized management arerequired to validate our findings and to furtherelucidate aetiology and treatment of tubulointerstitialinfiltrates in patients with PVAN.Acknowledgement. S.S. is supported by Swiss National FoundationGrant 3200B0-109302. H.H.H. is supported by Swiss NationalFoundation Grant 3200B0-110040/1. and by Stiftung ForschungInfektionskrankheiten, Basel (SFI#6-2005).Conflict of interest statement. None declared.References1. Hirsch HH, Brennan DC, Drachenberg CB et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplin-ary analyses and recommendations. Transplantation 2005; 79:1277–12862. Hirsch HH, Knowles W, Dickenmann M et al. Prospective studyof polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med 2002; 347: 488–4963. Wadei HM, Rule AD, Lewin M et al. Kidney transplantfunction and histological clearance of virus following diagnosisof polyomavirus-associated nephropathy (PVAN). Am JTransplant 2006; 6: 1025–10324. Brennan DC, Agha I, Bohl DL et al. Incidence of BK withtacrolimus vs cyclosporine and impact of pre-emptive immuno-suppression reduction. Am J Transplant 2005; 5: 582–5945. Randhawa P, Brennan DC. BK virus infection in transplantrecipients: an overview and update. Am J Transplant 2006; 6:2000–20056. Celik B, Shapiro R, Vats A, Randhawa PS. Polyomavirusallograft nephropathy: sequential assessment of histologic viralload, tubulitis and graft function following changes in immuno-suppression. Am J Transplant 2003; 3: 1378–13827. Mannon RB, Hoffmann SC, Kampen RL et al. Molecularevaluation of BK polyomavirus nephropathy. Am J Transplant2005; 5: 2883–28938. Bielmann D, Honger G, Lutz D, Mihatsch MJ, Steiger J,Schaub S. Pre-transplant risk assessment in renal allograftrecipients using virtual crossmatching. Am J Transplant 2007; 7:626–6329. Binggeli S, Egli A, Dickenmann M, Binet I, Steiger J,Hirsch HH. BKV replication and cellular immune responses inrenal transplant recipients. Am J Transplant 2006; 6: 2218–221910. Drachenberg CB, Papadimitriou JC, Hirsch HH et al.Histological patterns of polyomavirus nephropathy: correlationwith graft outcome and viral load. Am J Transplant 2004; 4:2082–209211. Nickeleit V, Hirsch HH, Zeiler M et al. BK-virus nephropathy inrenal transplants-tubular necrosis, MHC-class II expression andrejection in a puzzling game. Nephrol Dial Transplant 2000; 15:324–33212. Hariharan S, Cohen EP, Vasudev B et al. BK virus-specificantibodies and BKV DNA in renal transplant recipients withBKV nephritis. Am J Transplant 2005; 5: 2719–272413. Hammer MH, Brestrich G, Andree H et al. HLA type-independent method to monitor polyoma BK virus-specificCD4 and CD8 T-cell immunity. Am J Transplant 2006; 6:625–63114. Hirsch HH, Kaufmann G, Sendi P, Battegay M. Immunereconstitution in HIV-infected patients. Clin Infect Dis 2004; 38:1159–116615. Jehle AW, Khanna N, Sigle JP et al. Acute renalfailure on immune reconstitution in an HIV-positivepatient with miliary tuberculosis. Clin Infect Dis 2004; 38:e32–e3516. Racusen LC, Colvin RB, Solez K et al. Antibody-mediated rejection criteria—an addition to the Banff 97classification of renal allograft rejection. Am J Transplant2003; 3: 708–714Received for publication: 11.4.07Accepted in revised form: 16.4.072390 S. Schaub et al.

Transient allograft dysfunction from immune reconstitution in a patient with polyoma BK-virus-associated nephropathy

Transient allograft dysfunction from immune reconstitution in a patient with polyoma BK-virus-associated nephropathy

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