Previous studies have associated activating Killer cell Immunoglobulin-like Receptor (KIR) genes with protection from cytomegalovirus (CMV) replication after organ transplantation. Whether KIR-associated protection is operating in the context of primary infection, re-activation, or both, remains unknown. Here we correlated KIR genotype and CMV serostatus at the time of transplantation with rates of CMV viremia in 517 heart (n=57), kidney (n=223), liver (n=165) or lung (n=72) allograft recipients reported to the Swiss Transplant Cohort Study. Across the entire cohort we found B haplotypes—which in contrast to A haplotypes may contain multiple activating KIR genes—to be protective in the most immunosuppressed patients (receiving anti-thymocyte globulin induction and intensive maintenance immunosuppression) (hazard ratio after adjustment for covariates 0.46, 95% confidence interval 0.29–0.75, P=0.002). Notably, a significant protection was detected only in recipients who were CMV-seropositive at the time of transplantation (HR 0.45, 95% CI 0.26–0.77, P=0.004), but not in CMV seronegative recipients (HR 0.59, 95% CI 0.22–1.53, P=0.28). These data indicate a prominent role for KIR—and presumably natural killer (NK) cells—in the control of CMV replication in CMV seropositive organ transplant recipients treated with intense immunosuppression.
Cytomegalovirus (CMV) accounts for some of the most relevant infectious disease complications after solid organ transplantation (SOT).1 Patients at high risk for CMV replication and disease are seronegative individuals receiving an organ from a seropositive donor, or those receiving intensive and strongly T-cell-depleting immunosuppression, such as antithymocyte globulin (ATG).2 Natural killer (NK) cells are of special interest in SOT, since studies have shown that these innate lymphocytes may be less susceptible to immunosuppressive agents such as calcineurin inhibitors.3, 4 In agreement with a role of NK cells in antiviral immunity after SOT, polymorphisms within the Killer cell Immunoglobulin-like Receptor (KIR) gene complex have been correlated with the risk of CMV replication after solid organ or hematopoietic stem cell transplantation.5, 6, 7, 8, 9, 10, 11, 12 KIR are trans-membrane proteins expressed by NK cells and small subsets of T cells.13 Depending on their intracellular domain, KIR can have activating or inhibitory function. Inhibitory KIR mediate NK cell self-tolerance by binding to human leukocyte antigen class I molecules. In contrast, the function of activating KIR is less clear, and binding to human leukocyte antigen class I has only been documented for KIR2DS1 and KIR2DS4.14, 15 NK cells (the primary KIR-expressing effector cells) belong to the innate arm of the immune system. As a paradigm, innate responses were thought to follow an identical response pattern when triggered on consecutive occasions. However, in mouse models, primary CMV infection was shown to induce memory NK cells able to subsequently respond more efficiently to a second infectious challenge.16 Notably, also in humans infection with CMV permanently alters the NK cell-receptor repertoire,17, 18, 19, 20 and human memory NK cells have recently been shown to expand upon re-exposure to CMV.21 Here we aimed to relate KIR-associated protection and CMV primary infection versus reactivation, by assessing KIR genotype and CMV serostatus at the time of transplantation with rates of CMV viremia and disease in allo-transplant recipients prospectively followed within the Swiss Transplant Cohort Study.22
Incidence of CMV viremia and disease
One or more episodes of CMV viremia occurred in 223 of 517 patients in this study, with a cumulative incidence (CI) of 45±3% at 2 years of follow-up. The median time to first CMV viremia was 53 days. Of the 230 first episodes of documented CMV viremia, the majority was asymptomatic (n=196, 88%), whereas viral syndrome (n=14, 6%) and CMV disease (n=13, 6%) were rare. The 2-year CI of CMV viremia differed by transplanted organ, reaching 57±7% in heart, 47±3 in kidney, 41±4% in liver and 42±6% in lung transplant recipients, respectively.
CMV viremia/disease in haplotype AA versus BX patients
The frequencies of KIR genes and KIR haplotypes were comparable to those found in healthy Caucasians23 and are summarized in Supplementary Figure 1. Analysis of the incidence of CMV viremia in patients grouped by transplanted organs revealed significant protection of BX versus AA haplotype patients receiving lung transplantation (2-year cumulative incidence of CMV viremia 33±7% versus 67±12%, in BX versus AA patients, P=0.005), and a trend toward protection in recipients of heart allografts (2-year CI 48±9% versus 72±11%, P=0.07), whereas no differences were noted in kidney (2-year CI 47±4% versus 46±6%, P=0.62) or liver allografts (2-year CI 42±5% versus 38±8%, P=0.62, Figure 1a).
We hypothesized that the type of transplanted organ would be unlikely to have a major influence in modifying the KIR effect on CMV viremia itself, but might rather point to immunosuppressive regimens of different intensity. Indeed intensity of induction and maintenance immunosuppression differed substantially between the subpopulations receiving different organs (Table 1). ATG induction was used in 77% of heart transplants, and in 18% of kidney transplants, but not in liver or lung transplantation. Basiliximab induction was given to 16%, 68%, 53%, and 89% of heart, kidney, liver, and lung transplant recipients, respectively. We therefore analyzed the effect of KIR haplotype on CMV viremia in patients grouped according to the type of induction. This analysis showed that a trend towards a protective effect of B haplotypes was restricted to patients receiving induction treatment with ATG (2-year CI of CMV viremia in BX versus AA patients 43±7% versus 65±9%, P=0.06), whereas CMV viremia rates were not significantly different in patients receiving induction with the anti-CD25 antibody basiliximab (2-year CI 42±3% versus 45±5%, P=0.82, Figure 1b). Intensity of maintenance immunosuppression also differed between transplanted organs, with 81%, 94%, 64% and 94% of heart, kidney, liver and lung transplant recipients receiving three or more immunosuppressive drugs (typically a corticosteroid, a calcineurin inhibitor and an antimetabolite, P<0.001). A trend towards increased CMV replication in AA haplotype recipients was only found in patients receiving three or more immunosuppressive agents (56±3% versus 48±5%), but not in patients receiving two immunosuppressive agents only (14±7% versus 22±6%).
The notion that intensity of immunosuppression rather than the organ transplanted was associated with a differential effect of KIR genotype on CMV viremia probability was further corroborated by subgroup analysis of kidney transplant patients (where in the overall population no significant effect of KIR genotype was detected, as described above): in 40 kidney transplant recipients receiving ATG induction, 1-year cumulative incidence of CMV viremia was 38% versus 48% in BX and AA patients, respectively, which was comparable in magnitude to the effect seen in recipients of other organs receiving ATG induction. Of note, a total of 27 episodes of CMV disease occurred, of which 7 were classified as proven tissue invasive disease, 6 as probable disease (CMV viremia and typical organ dysfunction, but no biopsy) and 14 as viral syndrome. Distribution of KIR genotype in patients with symptomatic and asymptomatic CMV viremia was not different (KIR BX in 65% versus 70% for patients with symptomatic and asymptomatic CMV viremia, P=0.53). Sixty-seven patients developed recurrent CMV viremia during follow-up. The KIR haplotype distribution in these patients also was similar to that of patients with only one CMV event (KIR BX in 69% versus 70%, P=0.82).
Effect of CMV serostatus
We next assessed whether the protective effect of KIR BX depended on the CMV serostatus of recipients at the time of transplantation in the relevant high-risk population (patients undergoing either lung or heart transplantation or receiving ATG induction treatment, as defined before (total n=169, lung n=72, heart n=57, kidney n=40).
Across this entire high-risk population our data confirmed that the risk for CMV viremia was approximately half as high in BX haplotype compared to AA haplotype patients after adjustment for covariates (hazard ratio 0.46, 95% CI 0.29–0.75, P=0.002). Importantly, however, a significant protective effect was only seen in CMV seropositive recipients (n=121, HR=0.45, 95% CI 0.26–0.77, P=0.004). By contrast, in the 48 CMV-seronegative organ recipients the hazard ratio for CMV viremia conferred by a BX genotype was 0.59 (95% CI 0.22–1.53, P=0.28, univariate cumulative incidences; Figure 2).
This analysis of the effect of KIR genotype and CMV serostatus at the time of transplantation on CMV viremia after transplantation was prompted by several single-center studies demonstrating a reduced rate of ‘CMV events’ in kidney transplant recipients carrying KIR B haplotype genes,7, 9 as well as in recipients of hematopoietic stem cell grafts if the donor carried B haplotype genes.5, 6 We made use of the recently established Swiss transplant cohort study, which prospectively collects data on all patients receiving solid organ grafts in Switzerland.22 By including unselected consecutive patients who had received single-organ allografts, we investigated the previously described association of BX KIR haplotype with protection from CMV viremia/disease, specifically aiming to define the role of KIR-associated protection in CMV primary infection versus reactivation.
A significantly reduced rate of CMV viremia in kidney transplant recipients carrying KIR B haplotypes was detected in the subpopulation of kidney transplant recipients receiving ATG induction, as well as in heart transplant recipients (which predominantly received ATG induction) and lung transplant recipients (highest rate of triple- or quadruple-agent maintenance immunosuppression). Together, these data indicate that the KIR effect on CMV viremia was more prominent in patients receiving intense immunosuppression, which was formally proven by interaction analysis in this cohort.
By focusing on the most intensively immunosuppressed population (those receiving heart or lung allografts, and kidney recipients receiving ATG) we were able to assess how KIR B haplotypes affected the rate of CMV events. In this population, carriers of B haplotypes showed an incidence of CMV viremia that was roughly half of that seen in AA homozygous patients. Interestingly, protection from CMV viremia associated with KIR B haplotype genes was prominent in CMV IgG seropositive recipients at the time of transplant, whereas no significant protective effect was found in individuals CMV seronegative at the time of transplant. These data are compatible with the notion that carrying B haplotype genes is not sufficiently protective to prevent primary infection in the setting of immunosuppression and organ transplantation, but may suppress CMV reactivation in patients with latent infection. The selective protection conferred by activating KIR genes in these patients may involve different factors: NK cells (as the primary KIR-expressing effector cells) might need to cooperate with virus-specific memory B- and T-lymphocytes, which is possible during reactivation, but not in the case of primary infection. However, the fact that the KIR effect was most prominent in patients receiving intensive immunosuppression somewhat argues against this notion. Alternatively, NK cells may be primed by a first CMV infection episode (and possibly by latent persistence of the virus) and may therefore be more effective in preventing CMV reactivation rather than primary infection. While it is beyond the scope of the genotype/outcome association performed here to reveal such biological intricacies, the data presented here nevertheless are compatible with models of NK cell priming or NK cell memory.
Limitations of this study include the fact that both the schedule for screening of CMV viremia and methods to detect viremia were not consistent among centers, and that the study was not powered to detect an effect on the incidence of CMV disease, which may be the more relevant clinical end point. However, even asymptomatic CMV replication continues to negatively affect graft-failure free survival, as shown in the recent Swiss Transplant Cohort Study,24 suggesting that the study of factors associated with or treatment strategies leading to reduced CMV viremia remains important.
In conclusion, the current study for the first time provides evidence that KIR B haplotypes modify the risk of CMV viremia solely in patients who had already been exposed to CMV before SOT. Postulating ‘primed’ or ‘memory-like’ NK cells as the cellular correlate for this protective effect, efforts should now focus on exploring the molecular basis and regulation of this important NK cell feature.
Patients and Methods
Five hundred and seventeen patients undergoing single SOT at six transplant centers in Switzerland between May 2008 and December 2010 were combined in this analysis (Inselspital Bern, n=75; Centre Hospitalier Universitaire Vaudois, n=82; Hôpiteaux Universitaires de Genève, n=80; Kantonsspital St. Gallen, n=10; Universitätsspital Basel, n=79; Universitätsspital Zürich, n=191). Induction and maintenance immunosuppressive regimens are summarized in Table 1 along with demographic characteristics. Transplants in which both donor and recipient were CMV seronegative were excluded from the analysis.
Screening for CMV viremia by polymerase chain reaction was performed at each transplant center as per institutional guidelines (in general screening for CMV DNAemia by PCR every 1–2 weeks during the first month post transplant and then every 2 weeks until 3–6 months post transplant using assays with detection limits between 20 and 300 copies per ml). Data on transplant characteristics and transplant outcome including infectious complications were prospectively collected and retrieved using an electronic database. Written informed consent was obtained from all study participants and the study was approved by the institutional review board at all centers.
KIR genotype was analyzed in the recipient only, using a reverse sequence-specific oligonucleotide method (OneLambda, Canoga Park, CA, USA) according to the provider’s instructions.9 KIR genotypes were grouped into AA if they contained only the canonical group A haplotype genes (KIR3DL3, KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1, KIR2DS4, and KIR3DL2). Any genotype containing additional KIR genes is referred to as a BX, as it contains at least one group B haplotype.25 Genotypes were further dichotomized into telomeric and centromeric A and B haplotype motifs according to published algorithms.26
Classification of CMV disease severity
In the Swiss Transplant Cohort Study database, four different types of CMV infection are classified: asymptomatic viremia, viral syndrome, probable disease and (biopsy) proven disease. In the current analysis, all four categories were collectively classified as viremia, the latter two as CMV disease.
Patient characteristics were compared by analysis of variance or Pearson’s chi square test, as appropriate. The cumulative incidence of CMV viremia events was estimated using death and graft loss as competing risks. Patient data were censored at the time of last contact. Cox regression was used for multivariable analyses. Unless stated otherwise, only the first CMV viremia episode in each patient was considered. In the main analysis, patients were grouped into those carrying two KIR A haplotypes (AA) versus those carrying one or two KIR B haplotypes (BX). Further analyses compared the effect of telomeric or centromeric B motifs and the impact of the presence of the single KIR genes. Multivariate analyses adjusted for donor and recipient CMV sero-constellation, type of induction and type of maintenance immunosuppression. Prophylactic valgancyclovir administration was included in the Cox models as a time-dependent covariate. All multivariable analyses included the transplant center as a stratification variable. Double-sided P-values <0.05 were considered significant. No adjustment for multiple comparisons was made.
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The Swiss Transplant Cohort Study is funded by a grant from the Swiss National Science Foundation (3347CO-108795). This work was supported by the Swiss National Science Foundation (PP00P3_128461/1 to MS, and 31003A_135677/1 to CH). This work was supported by the Freiwillige Akademische Gesellschaft, Basel.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on Genes and Immunity website
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Gonzalez, A., Schmitter, K., Hirsch, H. et al. KIR-associated protection from CMV replication requires pre-existing immunity: a prospective study in solid organ transplant recipients. Genes Immun 15, 495–499 (2014). https://doi.org/10.1038/gene.2014.39
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