The genetic predisposition of natural killer cell to BK virus associated nephropathy in renal transplant patients

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1 & 213 International Society of Nephrology see commentary on page 233 The genetic predisposition of natural killer cell to BK virus associated nephropathy in renal transplant patients Hanna Trydzenskaya 1,2, Karsten Juerchott 1, Nils Lachmann 3, Katja Kotsch 4, Kristina Kunert 4, Benjamin Weist 1, Constanze Schönemann 3, Ralf Schindler 5, Peter Nickel 5, Matthias F. Melzig 2, Christian Hugo 6, Oliver Thomusch 7, Avidan U. Neumann 1, Petra Reinke 1,5 and Nina Babel 1,5 1 Berlin-Brandenburg Center for Regenerative Therapies, Charité University Medicine Berlin, Campus Virchow Clinic, Berlin, Germany; 2 Institute of Pharmacy, Freie University of Berlin, Berlin, Germany; 3 Center for Tumor Medicine, HLA Typing Laboratory, Charité University Medicine Berlin, Campus Virchow Clinic, Berlin, Germany; 4 Department of Visceral, Transplantation and Thoracic Surgery, Medical University Innsbruck, Innsbruck, Austria; 5 Department of Nephrology, Charité University Medicine Berlin, Campus Virchow Clinic, Berlin, Germany; 6 Department of Nephrology, University Clinic Carl Gustav Carus, Dresden, Germany and 7 Department of Visceral Surgery, University Clinic Freiburg, Freiburg, Germany BK virus (BKV) infection represents a serious complication in renal transplant patients resulting in BKV-associated nephropathy and subsequent allograft loss. Natural killer cells are crucial in the antiviral immune response; however, an understanding of the role of natural killer cells in protection against BKV is limited. To elucidate whether killer-cell immunoglobulin-like receptors and their interaction between donor-/recipient-related ligands have a role in BKV infection, we performed genotyping analysis in 48 kidney transplant recipients with a history of severe BKV infection/ BKV-associated nephropathy and 11 recipients with stable renal function and no BKV reactivation. Of interest, we found that telomeric gene content motif was significantly associated with severe course of BKV infection/bkvassociated nephropathy and detected significantly higher percentage of patients with BKV-associated nephropathy carrying low numbers of activating receptors compared with the control group. Detailed analysis of each single receptor revealed significantly lower frequencies of the activating receptor KIR3DS1 in patients with BKV infection/nephropathy as compared with the controls. Thus, our study supports protective effects of activating receptors in BKV infection and suggest natural killer-cell related genetic predisposition to the development of BKV-associated nephropathy. Kidney International (213) 84, ; doi:1.138/ki ; published online 13 March 213 KEYWORDS: kidney transplantation; nephropathy; virology Correspondence: Nina Babel, Department of Nephrology, Charité University Medicine Berlin, Campus Virchow Clinic, Augustenburger Platz 1, Berlin 13353, Germany. nina.babel@charite.de Received 17 April 212; revised 7 December 212; accepted 21 December 212; published online 13 March 213 Polyomavirus BK (BKV) infection represents a serious complication in kidney transplant recipients (KTRs) resulting in BKV-associated nephropathy (BKVAN) and subsequent allograft loss. Innate immunity has an important role in antiviral defence. However, the data on involvement of innate immunity in protection against BKV are very limited. Natural killer (NK) cells are key effector cells of the innate immune system, and as such are crucial in the antiviral immune response. NK cell activity and functions are controlled by a complex repertoire of cell surface molecules, including the killer-cell immunoglobulin-like receptors (KIRs). 1 3 Previous genetic studies revealed an extensive diversity in genomic region of KIRs giving rise to more than 2 haplotypes and genotypes. 4 One haplotype can exhibit from 8 to 14 KIR genes and pseudogenes. KIRs are a family of 14 closely linked genes and 2 pseudogenes located on chromosome 19q13.4 within the leukocyte receptor cluster, which encode both inhibitory and activating receptors that are expressed by NK cells. 5 Certain KIRs transmit either inhibitory or activating signals to the NK cell, upon engagement with specific human leukocyte antigen (HLA) class I ligands, thereafter effector function is considered to result from the balance of these contributing signals that initiate or supress cell activation. 6 8 The role of activating KIRs was previously analyzed in cytomegalovirus (CMV) infection and some other infections during the first posttransplant year. 9,1 However, the role of inhibitory KIRs or impact of receptor ligand KIR/HLA interactions have not been considered in these studies. Here, we explored the role of KIRs and their interaction between donor- and recipient-related HLA antigens for the development of severe BKV infection/bkvan in kidney transplant patients. Kidney International (213) 84,

2 H Trydzenskaya et al.: Natural killer genes in BK virus infection RESULTS Patients Forty-eight KTRs with a history of severe, prolonged BKV reactivation and biopsy-proven BKVAN were included in the study. One hundred and ten KTRs with no signs of BKV infection and stable graft function were used as controls. Studied group included patients with a homogenous genetic background (Caucasians). The clinical characteristics of the KTRs are summarized in Table 1. Univariate analysis of demographical and clinical patient data showed that patients with severe BKV infection/bkvan received triple immunosuppressive regimen (tacrolimus or cyclosporin A þ mycophenolate mofetil or azathioprine þ prednisolon) significantly more frequently than the control group (Po.1). Minimizing the possibility of control patients being misallocated for BKV reactivation (false-negative results), only patients with a longer (46 years) post transplant follow -up were included into control group. Thus, the minimal time since transplant (transplant age) in the control group (75 months) did not overlap with the latest time for BKV diagnosis post transplant in the BKVAN group (74 months) (Supplementary Figure S1 online). Caused by this study design, transplant age in the control group was significantly higher than the one of the BKVAN group (Po.1). In addition, gender was significantly different between BKVAN and control groups with the male sex as an independent risk factor for BKV infection and BKVAN development (Po.1). There was no statistically significant difference between the groups with regard to the patient age. Frequency distribution of KIR haplotypes Analysis of KIR genotypes and haplotypes can help to define the influence of specific KIR genes and their polymorphisms on the development of infections. Major haplotypes were identified based on the previous studies. 4,11 According to the previous definition, group A haplotype most commonly carries only one activating receptor KIR2DS4 plus inhibitory receptors KIR2DL1, KIR2DL3, KIR3DL1, as well as framework KIR genes common to all haplotypes (i.e., KIR3DL3, KIR2DL4, and KIR3DL2), and pseudogenes. 4 No other KIR genes can be assigned for haplotype A. In contrast, the group B haplotype is more variable and contains more than one activating KIR genes but other than KIR2DS4. To analyze whether the recipient KIR haplotype might influence outcome of BKV reactivation after kidney transplantation, we genotyped all study patients for six activating receptors: 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1, and eight inhibitory receptors: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 3DL1, 3DL2, and 3DL3. We categorized the patients as either having an A/A or B/X haplotype based on the presence or absence of multiple activating KIRs. Our analyses showed no correlation between the distribution KIR haplotypes and the occurrence of BKV infection/bkvan (Table 2). In addition, each haplotype can be further defined as centromeric (Cen) and telomeric (Tel) A/A and B/X haplotype motifs according to the position of KIR genes within the KIR locus. 4 KIR3DL1, KIR2DS4, KIR3DS1, and KIR2DS1 genes are localized telomerically of the gene KIR2DL4 and are referred as Tel haplotype fragment. KIR2DS2, KIR2DL2, and KIR2DL3 group of genes are localized centromerically of the gene KIR2DL4 and are classified as Cen haplotype motif. KIR gene assignment to certain haplotype motifs and their frequencies are presented in Table 2. Of interest, we found that Tel part of KIR haplotypes was associated with BKV infection/bkvan (odds ratio.4, 95% confidence interval.2.9, P ¼.3). There was no difference in the Cen part gene content motifs between BKV and control groups (Table 2). The absolute amount of activating genes present in KIR haplotypes was correlated with BKV infection In general, NK cell responses are controlled by a complex balance of signals derived from activating and inhibitory receptors. It determines whether or not NK cell effector functions are initiated. We therefore tested the hypothesis, whether the number either of activating or inhibitory KIRs in the recipient s genome might influence BKV infection outcome. We determined the absolute number of activating Table 1 Clinical and demographical characteristics of the study patients Patient characteristics BKVAN group (N ¼ 48) Control group (N ¼ 11) P-value Age (yr), median (range) 59 (31 76) 61 (32 84) NS Gender (male/female) 37/11 /5.8 Time since transplant/transplant age (mo), median (range) 55 (18 137) 1 (75 176) o.1 Diagnosis of BKV reactivation posttransplant (mo), median (range) 4 (1 74) NA NA Maximum BKV load a (copies/ml serum), median (range) 238,516 (2,38 33,374,) NA NA PRA pretransplant, n (%) 9 (19) 2 (18) NS HLA mismatches A/B/DR, median (range) 4 ( 6) 3 ( 6) NS HLA mismatches DR, median (range) 1 ( 2) 1 ( 2) NS IS initial, n (%) Tac or CsA þ MMF or AZA þ Pred 48 (1%) 91 (83%) o.1 Tac or CsA þ none þ Pred (%) 19 (17%) Abbreviations: AZA, azathioprine; BKVAN, BK virus associated nephropathy; CsA, cyclosporin A; HLA, human leukocyte antigen; IS, immunosupression regimen; KTRs, kidney transplantrecipients; MMF, mycophenolatemofetil; mo, months; NA, notapplicable; NS, notsignificant; PRA, panelreactiveantibody; Pred, prednisolone; Tac, tacrolimus; yr, years. a Maximum BKV serum load. 3 Kidney International (213) 84,

3 H Trydzenskaya et al.: Natural killer genes in BK virus infection Table 2 Distribution of KIR haplotypes and their centromeric and telomeric gene content motifs BKVAN group, n (%) Control group, n (%) OR 95% CI P- value Adj. P- value N 48 (1) 11 (1) KIR haplotype A/A 19 (39.6) 38 (34.5) B/X 29(.4) 72(65.4) Centromeric motif Cen-A/A 23 (47.9) 54 (49.1) Cen-B/X 25 (52.1) 56 (5.9) a Percentage of patients with activating KIRs, % BKVAN group Control group Telomeric motif Tel-A/A 38 (79.2) 66 () Tel-B/X 1 (2.8) 44 () Abbreviations: BKVAN, BK virus associated nephropathy; CI, confidence interval; KIR, killer-cell immunoglobulin-like receptor; OR, odds ratio. a Percentage of patients, % and/or inhibitory receptors for each study groups and compared these numbers between the groups (Figure 1). We did not observe differences between BKVAN group and control group for the number of inhibitory KIRs. In contrast, analysis of activating receptor genotypes showed significantly higher percentage of patients bearing low number (p3) of activating KIR genes in the BKVAN group as compared with control patients (odds ratio.5, 95% confidence interval 1, P ¼.4). Negative association between activating receptor KIR3DS1 and development of BKVAN Previous disease-association studies revealed a link between certain KIRs and autoimmune diseases or infections. Therefore, we further focused on individual/single activating and b Percentage of patients, % 1 BKVAN group Control group Number of activating receptors Number of inhibitory receptors Figure 1 Low amount of activating killer-cell immunoglobulinlike receptors (KIR) genes is associated with BK virus associated nephropathy (BKVAN). a and b show the percentage of patients with the low or high numbers of activating (a) or inhibitory (b) KIR receptors/patient in the BKVAN and control groups, respectively. Statistical analyses showed significantly higher percentage of patients bearing low number (p3) of activating KIR genes in the BKVAN group as compared with control patients (P ¼.4). Onesided Fisher s exact test was performed. Error bars represent 95% confidence intervals based on the binomial distribution. 8 2 b Percentage of patients with inhibitory KIRs, % DS1 2DS2 2DS3 2DS4 2DS5 3DS1 2DL1 2DL2 2DL3 2DL4 2DL5 3DL1 3DL2 3DL3 Figure 2 Negative association between activating receptor KIR3DS1 and development of BK virus associated nephropathy (BKVAN). Panels demonstrate the percentage of patients with activating (a) and inhibitory (b) killer-cell immunoglobulin-like receptors (KIR) genes among BKVAN and control groups. We found significantly higher percentage of patients bearing activating receptor KIR3DS1 in the control group compared with patients with BKVAN (P ¼.2). w 2 or two-sided Fisher s exact tests were performed. Error bars represent 95% confidence intervals based on the binomial distribution. inhibitory KIR genes present in our study groups. For this purpose, we analyzed the frequency distribution of each individual activating and inhibitory KIR genes, respectively (Figure 2). While the percentage of patients exhibiting any single inhibitory KIR gene did not differ significantly between the groups, we found significantly higher percentage of patients bearing activating receptor KIR3DS1 in the control group (39%) compared with patients with BKVAN (19%) (odds ratio.4, 95% confidence interval.1.9, P ¼.2). Moreover, the calculation of Kaplan Meier estimators revealed a significant difference in the BKV-free survival between KIR3DS1 groups (Supplementary Figure S2A online). Role of KIR/HLA interactions for the BKVAN development The overall effect of NK cells appears to be determined not only by the number and balance between activating and inhibitory receptors but also or even rather by the possibility of certain KIRs to interact with their ligand. The typical KIR ligands are HLA class I molecules known to be highly polymorphic. This generates diversity in the number of potential inhibitory interactions that the NK cells from a Kidney International (213) 84,

4 H Trydzenskaya et al.: Natural killer genes in BK virus infection single individual may have. Therefore, in genetic studies the KIR/HLA interactions should be involved into a biological model, rather than just considering KIRs in isolation. As individuals can have different number of potential ligands for inhibitory or activating receptors, we calculated the frequency distribution of donor s and recipient s HLA C1, C2, Bw4, A3, and A11 alleles between the groups (Table 3). We did not find any statistical differences for the distribution of certain alleles between the groups. To analyze the role of the certain receptor ligand combinations between recipient KIRs on NK cells and donor/or recipient HLA antigens for the development of BKVAN, we calculated the number of matches for certain KIRs with known HLA ligands: HLA-C2 and KIR2DL1 or KIR2DS1; HLA-C1 and KIR2DL2 or KIR2DL3; HLA-Bw4 and KIR3DL1; HLA-A*3,*11 and KIR3DL2 (Table 3). We did not find any associations between certain KIR/HLA matches and BKVAN. Lack of KIR3DS1 and telomeric KIR gene motif are gender-, age-, and immunosuppression-independent risk factors of BKVAN Our univariate analysis revealed six significant parameters associated with BKV reactivation and BKVAN development: gender, immunosuppression, transplant age, Tel KIR gene motif, number of activating KIR genes, and activating receptor KIR3DS1. We first analyzed the telomeric KIR gene motif, the number of activating KIR genes, and KIR3DS1 together in a conditional logistic regression model with age and gender as covariates. None of the three markers were independently associated with the risk for BKVAN in this model. The same was true for pairwise models of these three markers. This is caused by the fact that KIR3DS1 is always present in the telomeric part of the KIR haplotype and also highly correlated to other activating receptors, resulting in a very high correlation of these three markers. We therefore tested these three markers each alone in conditional logistic regression models with age and gender. In this context, the lack of the activating receptor KIR3DS1 as well as the telomeric gene motif occurred as gender- and age-independent risk factors of severe BKV infection/bkvan (P ¼.2 and P ¼.2, respectively). Next, we tested the influence of immunosuppressive therapy on the KIR3DS1 and the telomeric KIR gene motif as risk factors of BKV reactivation. Patients that received triple immunosuppressive therapy tacrolimus þ mycophenolate mofetil þ prednisolon were selected from both BKVAN and control groups to match patients by the immunosuppression regimen. Time-to-event analysis using Kaplan Meier estimator (P ¼.2 and P ¼.2, respectively) as well as conditional logistic regression models with gender and age as covariates (P ¼.4 and P ¼.4, respectively) supported the significance of the KIR3DS1 and the telomeric KIR gene motif as gender-, age-, and immunosuppression-independent risk factors for BKV infection and BKVAN development (Supplementary Figure S2 online). The telomeric KIR motif harbors three KIR genes (KIR2DS1, KIR2DS4, and KIR3DL1) in addition to KIR3DS1. We tested each of these three genes together with KIR3DS1, age, and gender in conditional logistic regression models. In contrast to KIR3DS1, none of the three other genes were significantly associated with BKVAN, neither in all studied patients nor in the immunosuppression-matched groups. Taken together, these observations suggest a stronger influence of KIR3DS1 compared with the other genes in the telomeric KIR gene motif. DISCUSSION Studies that associated KIR genotypes with diseases have identified these links mainly with viral infections and autoimmune diseases. 1,2 Some previous studies identified a relationship between KIR genotypes and outcomes of several infections, such as human immunodeficiency virus, 5,12 CMV, 13 hepatitis B virus, 14 and hepatitis C virus. 15,16 Our data demonstrate for the first time the role of KIRs in the pathogenesis of BKV infection. NK cell function is regulated by a complex balance between activating and inhibitory receptors such that the net signal derived from these receptors is integrated to determine whether or not NK cell effector functions are initiated. 6 8 Therefore, we first analyzed the overall frequencies of activating and inhibitory receptors. Our study supports the role of activating KIRs in the control of BKV infection and development of BKVAN after kidney transplantation. So, we found a significantly higher percentage of patients with BKVAN carrying low numbers of activating KIRs compared with control group. Similar to other recent studies, we identified two major KIR haplotypes A and B according to the presence or absence of KIR2DS4 gene. The assessed frequencies of A and B haplotypes in both studied groups were similar to the results obtained from the Caucasian population. 4,11,17 In addition, we classified haplotypes into Centromeric (Cen) and Telomeric (Tel) A/A and B/X haplotype motifs according to the position of KIR genes within the KIR locus. In line with previous study on patients with CMV reactivation after kidney transplantation, 18 we were able to identify a protective role of Tel part of B KIR gene content motifs for BKV reactivation and BKVAN. Detailed analysis of each single KIR allele revealed significantly lower frequencies of the activating receptor KIR3DS1 in patients with BKVAN. In genetic studies, a role for KIR3DS1 have been suggested in the outcome of multiple human diseases associated with viral infection. Protective role of KIR3DS1 genotype was found also for such viral infections as human papilloma virus and hepatocellular carcinoma with chronic hepatitis C virus infection. 16,19 In addition, the presence of KIR3DS1 showed a slowdown of human immunodeficiency virus disease progression in human immunodeficiency virus-1- infected population as compared with patients without KIR3DS1 allele. 2,21 Our data are in line with the data on 362 Kidney International (213) 84,

5 H Trydzenskaya et al.: Natural killer genes in BK virus infection Table 3 No association between KIR/HLA interactions and the development of BKVAN A HLA ligand BKVAN group, n (%) Control group, n (%) OR 95% Cl P-value Adj. P-value N 48 (1%) 11 (1%) Donor HLA þ C1C1 1 (32.3) (36.4) C2C2 6 (19.4) 22 (2.) C1C2 15 (48.4) 48 (43.6) Bw4 27 (77.1) 71 (64.5) A3, A11 16 (38.1) 37 (33.6) Recipient HLA þ C1C1 17 (45.9) (36.4) C2C2 7 (18.9) 22 (2.) C1C2 13 (35.1) 48 (43.6) Bw4 21 (.) 69 (62.7) A3, A11 15 (36.6) 38 (34.5) B Genetic factor BKVAN group, n (%) Control group, n (%) OR 95% Cl P-value Adj. P-value N Recipient KIR þ /donor HLA þ 48 (1%) 11 (1%) 2DL1 46 (95.8) 11 (1.) N.43 N DL1 þ C1C2 14 (45.2) 48 (43.6) DL1 þ C2C2 6 (19.4) 22 (2.) DL2/2DS2 25 (52.1) 56 (5.9) DL2/2DS2 þ C1C2 9 (29.) 26 (23.6) DL2/2DS2 þ C1C1 7 (22.6) 21 (19.1) DL3 (83.3) 11 (91.8) DL3 þ C1C2 12 (38.7) 45 (.9) DL3 þ C1C1 8 (25.8) 36 (32.7) DS1 1 (2.8) 39 (35.5) DS1 þ C1C2 4 (12.9) 16 (14.5) DS1 þ C2C2 () 9(8.2) N.57 N DS3 12 (25.) 36 (32.7) DS3 þ C1C2 3 (9.7) 18 (16.4) DS3 þ C1C1 3 (9.7) 13 (11.8) DL1 47 (97.9) 18 (98.2) DL1 þ Bw4 26 (74.3) 71 (64.5) DL2 48 (1) 14 (94.5) DL2 þ A3, A11 16 (38.1) 34 (3.9) Recipient KIR þ /recipient HLA þ 2DL1 46 (95.8) 11 (1.) N.43 N DL1 þ C1C2 13 (35.1) 48 (43.6) DL1 þ C2C2 6 (16.2) 22 (2.) DL2/2DS2 25 (52.1) 56 (5.9) DL2/2DS2 þ C1C2 7 (18.9) 25 (22.7) DL2/2DS2 þ C1C1 1 (27) 2 (18.2) DL3 (83.3) 11 (91.8) DL3 þ C1C2 11 (29.7) 46 (41.8) DL3 þ C1C1 15 (.5) 36 (32.7) DS1 1 (2.8) 39 (35.5) DS1 þ C1C2 1 (2.7) 16 (14.5) DS1 þ C2C2 3 (8.1) 8 (7.3) DS3 12 (25) 36 (32.7) DS3 þ C1C2 3 (8.1) 17 (15.5) DS3 þ C1C1 4 (1.8) 12 (1.9) DL1 47 (97.9) 18 (98.2) DL1 þ Bw4 2 (57.1) 68 (61.8) DL2 48 (1) 14 (94.5) DL2 þ A3, A11 15 (36.6) 35 (31.8) Abbreviations: BKVAN, BK virus associated nephropathy; CI, confidence interval; HLA, human leukocyte antigen; KIR, killer-cell immunoglobulin-like receptor; OR, odds ratio. posttransplant CMV course showing that extent of protection against CMV infection corresponds to the number of activating KIR genes. 9,1 A new aspect of the study was the evaluation of KIR allele frequencies in context of donor and recipients HLA alleles, respectively. In the present study, we did not Kidney International (213) 84,

6 H Trydzenskaya et al.: Natural killer genes in BK virus infection find any correlations between KIR/recipient HLA or KIR/donor HLA compatibilities and BKV infection, respectively. While the interactions between KIRs and their corresponding HLA ligands were analyzed in some previous studies in non-transplant population, the effect of their interactions was not considered in most studies on viral infections in transplant populations. To our knowledge, this is the first study analyzing the impact of KIR bindings to their corresponding HLA ligands on donor as well as on recipient cells. Taken together, our data demonstrate genetic predisposition to BKV infection and BKVAN on NK cell level and suggest protective effect of activating KIRs and in particular KIR3DS1. The mechanism behind the protective effect of activating KIRs is uncertain. The specific recognition of HLA class I ligands by the activating KIRs is not well defined and it is at a substantially lower affinity as compared with inhibitory KIRs. 22 It is also possible that activating KIRs have ligand-independent functions. Alternatively, some studies suggest that the activating KIRs may also directly recognize viral proteins ligands If, however, as a result of viral infection, the target cell loses the expression of major histocompatibility complex class I molecules, then owing to missing-self recognition the major histocompatibility complex-binding inhibitory receptors are not engaged anymore and the activating receptors have a very important role in the protection against infection. Our study has also some limitations including relatively small BKVAN group size as well as study design-caused differences in the immunosuppressive regimen between the groups. To alleviate the limitations, multiple statistical tests were performed on the whole study population as well as on the matched subgroups confirming KIR3DS1, telomeric gene motif, and low number of activating receptors as risk factors. In our study, different immunosuppression regiments did not influence the association between KIR gene repertoire and BKV infection/reactivation: telomeric gene motif and KIR3DS1 revealed as an independent risk factors of BKV reactivation and BKVAN development. Current immunosuppression therapy in kidney transplantation targets mainly T-cell activation and therefore impairs patient s virus-specific immunity. 26 In contrast, NK cell function appears to be unaffected by such therapeutic regimens and could be therefore very essential in the antiviral immune response after transplantation. However, BKV has developed some mechanisms to evade from NK cell s recognition. Recently, Bauman et al. has shown that viral mirna of BKV specifically downregulates the stressinduced ligand ULBP3, which results in the reduced NKG2Dmediated killing of viral-infected cells by the innate immune system. 3,31 Further analysis of kidney allograft-infiltrating NK cells with respect to their KIR repertoire is required to elucidate the role of its interactions with ligand-expressing cells for the control of BKV replication and BKVAN development. MATERIALS AND METHODS Patients This study was approved by the local ethical review committee in compliance with the Declaration of Helsinki Principles, and informed consent was obtained from all patients. The study included 158 patients. Group 1 involved 48 KTRs with a recovery from severe, prolonged BKV reactivation and biopsy-proven BKVAN. All 48 KTRs are alive with a functioning graft. Group 2 involved 11 KTRs with stable graft function with no BKV infection/reactivation. In order to exclude false-negative BKV results in the control group caused by a later manifestation of BKV reactivation in follow-up, only long-term transplant patients (more than 6 years) were included in the control group. Detailed group characteristics are presented in Table 1. Caused by the study design with a longer follow-up/higher transplant age in the control group, there were differences in the immunosuppressive therapy between the groups. In order to exclude that these differences influence study results, all statistical analyses were run additionally for both groups matched with regard to the immunosuppression regimen. For this purpose, patients that received triple immunosuppressive therapy (tacrolimus þ mycophenolate mofetil þ prednisolon) were selected from both BKV (42 out of 48) and control ( out of 11) groups. Determination of BK viral load BKV-DNA analysis was performed by TaqMan Real Time polymerase chain reaction (PCR) as described previously. 32 Briefly, DNA was isolated from serum using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer s instructions. Primers and probes were designed to amplify the VP1 region of BKV. A plasmid standard containing the VP1-coding region was used to determine the copy number per milliliter. Samples exceeding detection level (41 copies/ml) were considered positive. For BKV reactivation screening reasons, all patients were monitored monthly during the first 3 months post transplantation, then every 3 months during the next 9 months, and every year thereafter. In addition, BKV load analysis was performed in each case of unexplained acute creatinine rise. In case of BK viremia, defined as positive detection of BKV-DNA in serum, patients were monitored one- to bi-monthly (depending on patient compliance). In all patients with BKV reactivation, sustained BK viremia was confirmed by control analysis of BKV load 4 weeks after the first measurement. Genomic DNA preparation Genomic DNA was prepared from donor spleen cells or from fresh anticoagulated blood from kidney recipients using the QIAamp Maxi DNA Blood Kit (Qiagen) according to the manufacturer s instructions. KIR and HLA genotyping Donors and recipients were genotyped pretransplant for HLA-A, HLA-B, and HLA-C using a combination of serological typing trays Lymphotype HLA (Bio-Rad Medical Diagnostics, Dreieich, Germany) and molecular typing using sequence-specific oligonucleotide Dynal RELI (Life Technologies, Carslbad, CA). For KIR genotyping, a reverse sequence-specific oligonucleotide method was applied according to the manufacturer s instructions (One Lambda, Canoga Park, CA). Briefly, three separate PCR reactions amplifying exon 3, 5, and 7 9 of the KIR locus were conducted for each sample 364 Kidney International (213) 84,

7 H Trydzenskaya et al.: Natural killer genes in BK virus infection using biotinylated primer sets. Each PCR product was denaturated, hybridized to complementary DNA probes coupled to fluorescencecoded microbeads, and stained with R-Phycoerythrin-conjugated streptavidin. Binding of PCR product to the microbeads was then assessed using a LABScan 1 flow analyzer (Luminex, Austin, TX). The assignment of genotypes was done using HLA Fusion software (One Lambda) based on the reaction pattern under consideration of published KIR gene sequences. Six activating receptors including 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1 were analyzed in the present study. By totaling the number of activating KIR genes, the activating receptor KIR2DS4 was included in a full-length as well as in an exon 5 (22 base pairs) deleted forms. The following all eight inhibitory receptors were included in the total number analysis of inhibitory KIR genes: KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, and KIR3DL3. Statistical methods Calculations were performed using SPSS software version 18 (SPSS, Chicago, IL) and R version The contribution of various variables as risk factors of BKVAN was evaluated by w 2 or Fisher s exact tests. Time-to-event analysis using Kaplan Meier estimator and a conditional logistic regression model, including age and gender, were used for determining the independence of the KIR3DS1 marker of BKVAN. Two-sided P-values of o.5 were considered significant. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS The study was supported by a grant from EFRE (IBB-ProFit) to NB and PR. Contributions were made possible by DFG funding through the Berlin Brandenburg School for Regenerative Therapies GSC 23. We thank Antje Lassahn for her technical assistance. SUPPLEMENTARY MATERIAL Figure S1. Minimal post transplant follow up in the control group was longer than the latest post transplant BKV reactivation in BKVAN group. Figure S2. KIR3DS1 and the telomeric KIR gene motif are risk factors for BKV reactivation. Supplementary material is linked to the online version of the paper at REFERENCES 1. Khakoo SI, Carrington M. KIR and disease: a model system or system of models? Immunol Rev 26; 214: Williams AP, Bateman AR, Khakoo SI. Hanging in the balance. KIR and their role in disease. MolInterv 25; 5: French AR, Yokoyama WM. Natural killer cells and viral infections. Curr Opin Immunol 23; 15: Hsu KC, Chida S, Geraghty DE et al. The killer cell immunoglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and allelic polymorphism. Immunol Rev 22; 19: Lopez-Vazquez A, Mina-Blanco A, Martinez-Borra J et al. Interaction between KIR3DL1 and HLA-B*57 supertype alleles influences the progression of HIV-1 infection in a Zambian population. Hum Immunol 25; 66: Moretta A, Tambussi G, Bottino C et al. A novel surface antigen expressed by a subset of human CD3- CD16 þ natural killer cells. Role in cell activation and regulation of cytolytic function. J Exp Med 199; 171: Ljunggren HG, Karre K. In search of the missing self : MHC molecules and NK cell recognition. Immunol Today 199; 11: Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol 25; 5: Stern M, Elsasser H, Honger G et al. The number of activating KIR genes inversely correlates with the rate of CMV infection/reactivation in kidney transplant recipients. Am J Transplant 28; 8: Hadaya K, de Rham C, Bandelier C et al. Natural killer cell receptor repertoire and their ligands, and the risk of CMV infection after kidney transplantation. Am J Transplant 28; 8: Uhrberg M, Valiante NM, Shum BP et al. Human diversity in killer cell inhibitory receptor genes. Immunity 1997; 7: Martin MP, Gao X, Lee JH et al. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 22; 31: Cook M, Briggs D, Craddock C et al. Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation. Blood 26; 17: Lu Z, Zhang B, Chen S et al. Association of KIR genotypes and haplotypes with susceptibility to chronic hepatitis B virus infection in Chinese Han population. Cell Mol Immunol 28; 5: Khakoo SI, Thio CL, Martin MP et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 24; 35: Lopez-Vazquez A, Rodrigo L, Martinez-Borra J et al. Protective effect of the HLA-Bw4I8 epitope and the killer cell immunoglobulin-like receptor 3DS1 gene against the development of hepatocellular carcinoma in patients with hepatitis C virus infection. J Infect Dis 25; 192: Hsu KC, Liu XR, Selvakumar A et al. Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J Immunol 22; 169: Stern M, Hadaya K, Hoenger G et al. Telomeric rather than centromeric activating KIR genes protect from cytomegalovirus infection after kidney transplantation. Am J Transplant 211; 11: Bonagura VR, Du Z, Ashouri E et al. Activating killer cell immunoglobulinlike receptors 3DS1 and 2DS1 protect against developing the severe form of recurrent respiratory papillomatosis. Hum Immunol 71: Alter G, Malenfant JM, Delabre RN et al. Increased natural killer cell activity in viremic HIV-1 infection. J Immunol 24; 173: Pascal V, Yamada E, Martin MP et al. Detection of KIR3DS1 on the cell surface of peripheral blood NK cells facilitates identification of a novel null allele and assessment of KIR3DS1 expression during HIV-1 Infection. J Immunol 27; 179: Rajagopalan S, Long EO. Understanding how combinations of HLA and KIR genes influence disease. J Exp Med 25; 21: Arase H, Mocarski ES, Campbell AE et al. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 22; 296: Berg L, Riise GC, Cosman D et al. LIR-1 expression on lymphocytes, and cytomegalovirus disease in lung-transplant recipients. Lancet 23; 361: Guma M, Angulo A, Vilches C et al. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood 24; 14: Villard J. Immunity after organ transplantation. Swiss Med Wkly 26; 136: Chiossone L, Vitale C, Cottalasso F et al. Molecular analysis of the methylprednisolone-mediated inhibition of NK-cell function: evidence for different susceptibility of IL-2- versus IL-15-activated NK cells. Blood 27; 19: Wai LE, Fujiki M, Takeda S et al. Rapamycin, but not cyclosporine or FK56, alters natural killer cell function. Transplantation 28; 85: Wang H, Grzywacz B, Sukovich D et al. The unexpected effect of cyclosporin A on CD56 þ CD16- and CD56 þ CD16 þ natural killer cell subpopulations. Blood 27; 11: Bauman Y, Nachmani D, Vitenshtein A et al. An identical mirna of the human JC and BK polyoma viruses targets the stress-induced ligand ULBP3 to escape immune elimination. Cell Host Microbe 211; 9: Raulet DH, Vance RE. Self-tolerance of natural killer cells. Nat Rev Immunol 26; 6: Babel N, Fendt J, Karaivanov S et al. Sustained BK viruria as an early marker for the development of BKV-associated nephropathy: analysis of 4128 urine and serum samples. Transplantation 29; 88: Kidney International (213) 84,