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To date, there are limited data on the relationship between killer‑cell immunoglobulin‑like receptor (KIR) genes and BK polyomavirus–associated nephropathy (PyVAN). In this study, we focus on the impact of KIRs and their interaction with human leukocyte antigen ligands on the risk of PyVAN, and evaluate other possible risk factors for the occurrence of PyVAN in a population of kidney transplant recipients.Thus far, the risk stratification for posttransplant PyVAN has been primarily based on the assessment of characteristic histology on a renal biopsy and / or detection of BK polyomavirus (BKPyV) DNA in the urine and plasma. In our opinion, this strategy could be improved by implementing other biomarkers predictive of PyVAN after kidney transplantation. It would allow for more personalized management. The assessment of KIR2DL3 prior to transplantation could potentially be used in combination with checking for BKPyV DNA.

Introduction

In at least 80% of the adult population, BK polyomavirus (BKPyV) infection occurs early in childhood and remains latent within renal tubular cells and the uroepithelium, with minimal clinical manifestations.¹ In kidney transplant recipients, the virus reveals its pathogenicity most commonly by causing a form of tubulointerstitial nephropathy, referred to as BK polyomavirus–associated nephropathy (PyVAN).² Apart from kidney transplantation itself, one of the strongest determinants of PyVAN evolution is the recipient’s level of immunosuppression. The risk of PyVAN currently ranges from 1% to 10%, and the risk of graft loss is 10% to 50% within 2 years post‑diagnosis, reflecting the importance of this infectious complication.³ There is currently no specific antiviral therapy against BKPyV, and no therapeutic approach has been found superior. The most important component of anti‑PyVAN treatment is the reduction of immunosuppressive treatment strength. However, complete withdrawal of immunosuppression is impossible, and proper identification of other PyVAN risk factors is necessary to discover new modalities helpful in minimizing the negative impact of this complication in the kidney transplant population.⁴ Within the last decade, several factors have been shown to affect the risk of BKPyV replication and the evolution of PyVAN, including some donor characteristics (eg, older age, the presence of BKPyV viruria, cytomegalovirus [CMV] serostatus, and the absence of HLA‑C7), and recipient characteristics (eg, prior sensitization, obesity, sex, and BKPyV serostatus).^5-7 Additionally, some factors that are related to the transplantation procedure itself were found to play a role, including the time of cold ischemia, the presence of a ureteral stent, and graft function.^8,9 Among immunological parameters, the type of immunosuppressive treatment and occurrence of acute rejection episodes were also shown to impact the risk of PyVAN occurrence.⁹ In recent years, the list of relevant risk factors has grown through the identification of several genetic markers. In recipients, the presence of KIR3DS1, HLA‑C7, HLA‑A2, HLA‑B44, HLA‑B51, and HLA‑DR15, as well the g‑interferon gene polymorphism, were identified to play a role. In donors, the presence of HLA‑C7 and MICA A5.1 were also shown to be relevant.¹⁰

The immune response to BKPyV infection is complex, and the role of both adaptive and innate responses has not been well established in the transplant recipients. Natural killer (NK) cells have been shown to affect the pathophysiology of BKPyV infection and nephropathy. Killer immunoglobulin‑like receptors (KIRs) are surface receptors that are expressed by NK cells and play a central role in immunity. Together with their HLA ligands, KIRs regulate the function of NK cells, thereby determining whether they are to be activated or inhibited from killing the cell being interrogated.¹¹ Inhibition and activation of the NK cell response is determined by the balance between activating and inhibitory signals from KIRs‑HLA interactions.

KIRs that have a short intracytoplasmic tail activate lymphocytes. Inhibitory KIRs have a long intracellular region and may possess 2 or 3 immunoglobulin‑like extracellular domains.^12-14 There are 2 groups of KIR haplotypes: group A and group B. Group A includes mostly inhibitory KIRs, of which only KIR2DS4 and KIR2DL4 activate NK cells.¹⁵

The genetic variability of KIRs and / or HLA ligands in solid organ transplants might help to determine the clinical outcomes and protection against viral infections. To date, there are limited data on the relationship between the KIR genes and PyVAN. In this study, we focused on the impact of KIRs and their interaction with HLA ligands on the risk of PyVAN, and evaluated other possible risk factors for the occurrence of PyVAN in a population of kidney transplant recipients.

Patients and methods

The patients (n = 134) who received a kidney transplant between March 2010 and November 2014 in the Department of General and Transplant Surgery, were enrolled in the study.

Results

Characteristics of KIR genotypes

The framework genes KIR2DL4, KIR3DP1, KIR3DL2, and KIR3DL3 were present in all of the recipients. The frequencies of the other KIR genes are presented in Figure 1.

Based on the presence or absence of multiple activating KIRs, the samples in which KIR2DS4 was the only detected KIR activating gene were assigned the genotype A/A. We categorized genotypes as B/X if they carried the KIR activating genes other than KIR2DS4, which is concordant with the approach of other investigators.^15,20 We then analyzed KIR A/A and B/X subgroup frequencies in the PyVAN(+) and PyVAN(–) study subgroups. The difference between both KIR genotypes did not reach statistical significance (P = 0.88) (Table 2).

Table 2. Association between killer cell immunoglobulin‑like receptors and killer cell immunoglobulin‑like receptor genotypes and polyomavirus‑associated nephropathy

Genetic factor		PyVAN(+), n (%)	PyVAN(–), n (%)	P value
Abbreviations: PyVAN, polyomavirus‑associated nephropathy; others, see Table 1
KIR genotype	A/A	6 (14.3)	36 (85.7)	0.88
	B/X	14 (15.2)	78 (84.8)
Number of activating KIRs	1	6 (14.3)	36 (85.7)	0.63
	2	5 (16.7)	25 (83.3)
	3	4 (23.5)	13 (76.5)
	4	4 (18.2)	18 (81.8)
	5	1 (6.3)	15 (93.7)
	6	0	7 (100)
Number of inhibitory KIRs	5	0	5 (100)	0.78
	6	9 (16.7)	45 (83.3)
	7	8 (14)	49 (86)
	8	3 (16.7)	15 (83.3)
KIR2DL1+HLA‑C2		13 (16.9)	64 (83.1)	0.45
KIR2DL1-HLA‑C2		7 (12.3)	50 (87.7)
KIR2DL2+HLA‑C1		6 (9.8)	55 (90.2)	0.13
KIR2DL2-HLA‑C1		14 (19.8)	59 (80.8)
KIR2DL3+HLA‑C1		16 (19.5)	66 (80.5)	0.06
KIR2DL3-HLA‑C1		4 (7.7)	48 (92.3)
KIR3DL1+HLA‑Bw4		11 (12.2)	79 (87.8)	0.2
KIR3DL1-HLA‑Bw4		9 (20.45)	35 (79.55)
KIR3DL2+HLA‑A3/A11		10 (17.9)	46 (82.1)	0.41
KIR3DL2-HLA‑3/A11		10 (12.8)	67 (87.2)

Discussion

We analyzed the relationship between KIR genotypes and posttransplant PyVAN in a cohort of 134 recipients of kidney transplants. The key finding of our study was the association between the presence of the inhibitory KIR2DL3 and posttransplant PyVAN. The presence of KIR2DL3 (odds ratio [OR], = 4.61; P = 0.05) was associated with the risk of PyVAN among kidney transplant recipients.

Previous studies showed that different levels of susceptibility to BKPyV may be attributable to variations of the KIR receptors and consequently the repertoire of NK cells.

Trydzenskaya et al²¹ were the first to define the role of KIRs in the pathogenesis of PyVAN. Their analysis of individual receptors revealed significantly lower frequencies of the activating receptor KIR3DS1 in the the PyVAN(+) patients than in the controls, suggesting a protective effect of activating KIRs and the genetic predisposition to PyVAN on NK cell level.²¹ A lower number of activating KIRs among the PyVAN(+) patients was later confirmed in another study,²² but no difference in any particular activating KIR gene frequency was observed. The authors also confirmed that the KIR A/A haplotype was more frequent in PyVAN.²² In another study of 103 kidney transplant recipients,²³ an analysis of the recipients’ KIR and donors’ HLA genotypes in a missing‑self context did not suggest that the absence of HLA ligands for KIR inhibitory receptors or a high number of activating KIRs were associated with a significant reduction in the incidence of BKPyV replication.²³ With regard to infections other than PyVAN, a growing body of evidence suggests that the severity of their course correlates with certain KIR genotypes.

The present results are consistent with a previous analysis²⁴ of KIR and HLA genotypes, suggesting that KIR2DL3 (OR, 0.610; P = 0.03), with or without the HLA‑C1 ligand and Cen‑A haplotype, is associated with chronic hepatitis C virus (HCV) infection. The authors suggested that KIR2DL3 in combination with HLA‑C1 or C1C1 contributes to a failure of spontaneous clearance of HCV when the effect of IL28B is considered. This observation was confirmed by other researchers.^25,26 Several studies also linked KIR2DL3 with the spontaneous resolution of HCV infection.²⁷ Nevertheless, this protective effect has been shown only among individuals who were homozygous for KIR2DL3 and not among KIR2DL2/KIR2DL3 heterozygotes. The discrepant findings of the earlier reports were explained by the route of HCV infection and viral load of HCV. Another possible explanation of this discrepancy might be the presence of highly deleterious single‑nucleotide polymorphisms of the KIR2DL3 gene that suggest notable changes in the protein secondary structure, instability, and conservation. These factors could affect KIR function and susceptibility to viral infections.²⁸ Similarly to our findings, the absence of the KIR2DL3 gene prevented HIV‑infected patients from contracting tuberculosis.²⁹ The authors suggested that the presence of activating KIRs, together with the lack of inhibitory KIRs (KIR2DL3 + HLA‑C1/C2 and KIR2DL1 + HLA‑C1/C2), might modulate the NK cell response. Likewise, a recent study by our group showed that the lack of KIR2DS2, the presence of KIR2DL3, and the presence of KIR2DL2–HLA‑C1 were risk factors for posttransplant CMV infection.³⁰ Few studies have been designed to assess the issue of CMV prevention in the context of risk factors for PyVAN. A post hoc analysis of data from 2 prior studies on CMV prevention sought to address potential interactions between antiviral prophylaxis and BKPyV events. The authors showed that the risk of BKPyV viremia increased in patients who received VGCV prophylaxis.³¹ Extending this finding, we investigated whether anti‑CMV therapy is associated with a higher risk of PyVAN in our cohort. Interestingly, in the present study, a lower incidence of posttransplant PyVAN was observed in the patients with no anti‑CMV antiviral prophylaxis, based on univariable and multivariable analyses. Importantly, tacrolimus and cyclosporine trough levels and steroid dose did not differ between the patients with and without PyVAN. Moreover, large prospective trials that evaluated VGCV prophylaxis vs a preemptive therapy did not observe an increased risk of BKPyV infection in the prophylaxis groups.^32,33

The possible explanation of our finding is that the lower incidence of PyVAN in the patients with no anti‑CMV prophylaxis is based on the fact that VGCV may have an immunosuppressive effect by suppressing T cell proliferation. In the absence of VGCV, there is no additional bone marrow suppression, and therefore the risk of PyVAN is lower.

Finally, in the multivariable analysis, we confirmed the previously reported and widely recognized association between male sex, age, acute rejection episodes, and the onset of PyVAN.^34-37 The present study has some limitations, mainly concerning the limited sample size, the lack of information about the expression of KIRs, the lack of analyses on NK cell subsets expressing the KIRs most relevant for PyVAN development, and the lack of matched‑pair randomized groups of recipients.

In summary, our results suggest that genotyping recipients for the KIR2DL3 gene may help predict the occurrence of posttransplant PyVAN. Other factors, such as male sex, the absence of antiviral prophylaxis, and acute rejection episodes, were independent predictive factors for the occurrence of PyVAN. The results of our study may be important for clinical practice. Further interventional studies are needed to translate these potentially relevant markers into pre- and posttransplant screening and treatment modalities.