The Number of Activating KIR Genes Inversely Correlates with the Rate of CMV Infection/Reactivation in Kidney Transplant Recipients

Viral infection is a common complication after kidney transplantation. The role of natural killer cells (NK cells) in this setting remains unknown. NK cells express activating and inhibitory killer cell immunoglobulin-like receptors (KIR). We analyzed whether activating KIR genes carried by kidney transplant-recipients influence the rate of viral infection during the first year after transplantation. In patients with a KIR A/A genotype (n = 40, KIR2DS4 only activating KIR) the rate of cytomegalovirus (CMV) infection and reactivation was 36%, as compared to 20% in transplant recipients with more than one activating KIR gene (KIR B/X genotype, n = 82, p = 0.04). Adjusting for other risk factors in Cox regression, the relative risk of B versus A genotype patients was 0.34 (95% CI 0.15–0.76, p = 0.009). The degree of protection increased with the number of activating KIR genes. Symptomatic CMV disease was only observed in four individuals, all carrying a KIR A/A genotype. As for viral infections other than CMV, and for bacterial infections, no KIR-linked protective effect could be detected. Also, graft function and the rate-rejection episodes were similar in KIR A/A and KIR B/X genotype individuals. This study supports a role for activating KIR in the control of CMV infection after kidney transplantation .     

Telomeric Rather than Centromeric Activating KIR Genes Protect from Cytomegalovirus Infection after Kidney Transplantation

Cytomegalovirus (CMV) infection is a common complication after organ transplantation. Previous studies have demonstrated that activating killer‐cell immunoglobulin‐like receptors (KIR) may reduce the rate of CMV infection. KIR genes can be divided into haplotype A (containing a fixed set of inhibitory receptors) and haplotype B (carrying additional activating KIR genes). The KIR locus is divided into a centromeric and a telomeric portion, both of which may carry A or B haplotype motifs. We studied a cohort of 339 kidney transplant recipients to elucidate which KIR genes protect from CMV infection. CMV infection occurred in 139 patients (41%). Possession of telomeric (hazard ratio 0.64, 95% confidence interval 0.44–0.94, p = 0.02) but not centromeric (HR 0.86, 95% CI 0.60–1.23, p = 0.41) B motifs was associated with statistically significant protection from CMV infection. Due to linkage disequilibrium, we were not able to identify a single protective gene within the telomeric B complex (which may contain the KIR2DS1, KIR3DS1, KIR2DL5A and KIR2DS5 genes). The presence of known or putative ligands to activating KIR did not significantly modify the influence of telomeric B group genes. We confirm that B haplotypes protect from CMV infection after kidney transplantation and show that this arises from telomeric B haplotype genes.     

Natural killer cells and their receptors

Natural killer (NK) cells have been known for a long time to be a very important component of the innate immune system. However, it is only during the last 10 years that knowledge of their receptors has emerged. Described in the present review are those receptor families killer inhibitory receptor (KIR) (belonging to the immunoglobulin superfamily), and killer lectin like receptor (KLR) CD94/NKG2, that both use HLA as a ligand and have inhibiting and activating types of receptors, and natural cytotoxic receptors (NCR) which do not associate with HLA. Association of the receptor gives rise to either an inhibiting or activating signal leading to either failure or success in lysing a target cell. The KIR receptors are very polymorphic both in the number of genes expressed in an individual and the alleles present for a gene. They would appear to have had a rapid evolution compared to the CD94/NKG2 receptors. The roles that NK cells and their receptors have with various facets of transplantation, disease, pregnancy and control of virus infection in humans are described.     

The association between killer‐cell immunoglobulin‐like receptor (KIR) and KIR ligand genotypes and the likelihood of BK virus replication after kidney transplantation

BK virus is a common opportunistic post‐transplantation viral infection. Although some risk factors have been studied in this context, the contribution of NK cells has not been assessed in detail. In a group of kidney transplant recipients, we studied the association between (i) the likelihood of BK virus replication during the two‐year period after kidney transplantation and (ii) the genotypes of the killer cell immunoglobulin‐like receptor (KIR) repertoire and their human leukocyte antigen (HLA) ligands. Other clinical factors (such as defective organ recovery and immunosuppressive treatment) were also assessed. BK virus replication was observed in 43 of the 103 recipients (41%). Patients with BK virus replication in the plasma were more likely to display defective organ recovery in the first seven days post‐transplantation. BK virus replication was not associated with Missing KIR ligands. However, BK virus replication was more frequent in patients with responsive NK cells (i.e. when a ligand for activating KIRs was not homozygous in the recipient and present in the donor). Our results suggest that defective organ recovery and the recipient's activating KIR repertoire may be related (depending on HLA ligands present in the couple recipient / donor) to the reactivation of BK virus replication after kidney transplantation.     

Natural killer cells and killer cell immunoglobulin-like receptors in solid organ transplantation: Protectors or opponents?

Among all the cells of innate immunity, natural killer (NK) cells are well-known for the fight against tumors and virally-infected cells. NK cells have been implicated in the pathogenesis of immune-mediated allograft damage, but mounting evidence suggests they can potentially promote allograft tolerance as well. In addition, NK cells express a wide variety of activating and inhibiting receptors, and the signals sent by these molecules, particularly killer cell immunoglobulin-like receptors (KIRs), determine their ultimate function. The role of KIRs and their human leukocyte antigen (HLA) class I ligands have been extensively investigated in hematopoietic stem cell transplantation (HSCT). Previous studies have suggested that, in the setting of solid organ transplantation, having certain KIR genes or KIR/HLA combinations probably affects allograft survival. Therefore, it may be helpful to analyze KIR/HLA combinations in donors and recipients to choose the optimal donor, anticipate harmful effects post-transplantation, and develop NK cell-based immunotherapies to enhance the success of solid organ transplantation. In this review, we will discuss the dual function of NK cells in solid organ transplantation, followed by a brief introduction to KIRs and the association of KIR and HLA genes with kidney, liver, and lung transplant outcomes.     

KIR/HLA ligand incompatibility in kidney transplantation

BACKGROUND: The polymorphic family of killer-cell immunoglobulin-like receptors (KIRs) consists of activating and inhibitory receptors expressed by natural killer (NK) cells and effector T cells that recognize human leukocyte antigen (HLA) class I ligands. It has been suggested that KIR/HLA incompatibility exerts beneficial effects in hematopoietic stem cell transplantation. METHODS: To elucidate whether certain receptor-ligand combinations between recipient KIR and donor HLA antigens lead to enhanced alloreactivity of NK cells associated with acute rejection (aRx) after kidney transplantation, we analyzed the entirety of matches/mismatches between KIR genes and known HLA ligands for aRx patients (n=105) compared to patients with stable renal function (n=119). RESULTS: Whereas HLA-C ligand incompatibility between donor and recipient has no influence on aRx, grafts derived from donors homozygous for HLA-C group 2 alleles seem to demonstrate a better outcome (P=0.052). Additionally, a higher number of inhibitory receptors in the recipient's genotype (P=0.042), a significant higher number of matches for the receptors KIR2DL2/DS2 (P=0.004), as well as a higher number of mismatches for KIR2DL3 (P=0.014) could be observed for patients with stable renal function. CONCLUSION: Our data illustrate that certain KIR/HLA class I ligand combinations between donor and recipient might influence graft short-term outcome after renal transplantation.     

Potential role of CC chemokine receptor 6 in prediction of late‐onset cytomegalovirus infection following solid organ transplant

CCR6 is a chemokine receptor involved in homing memory T cells, particularly Th17 cells, to sites of mucosal inflammation. Despite the critical role of memory T cells in long‐term protective immunity against cytomegalovirus (CMV), a virus that reactivates at multiple mucosal sites, the ability of CCR6 or other Th17 marker expression to predict CMV reactivation following transplantation is not clear. Using 11‐color flow cytometry, in this prospective single‐center pilot study, we measured the expression of CCR6 and other markers of T‐cell function in peripheral blood samples obtained from 21 SOT recipients at the time of discontinuation of anti‐CMV prophylaxis. CMV viremia was monitored on a monthly basis after discontinuation of prophylaxis. Eleven patients (52%) developed CMV viremia during the six‐month follow‐up period. Late‐onset CMV infection was preceded by an immune phenotype characterized by increased CCR6 expression on bulk CD4⁺ T cells and a reduced number of circulating CMV IE‐1‐specific Th1 (CD4⁺ IFN‐γ⁺) cells. Among the markers evaluated, CCR6 was the best single predictor of late‐onset CMV infection. Our results suggest that CCR6 expression at the time of discontinuation of antiviral prophylaxis might be a useful predictor of late‐onset CMV reactivation and provide the basis for future larger prospective studies.     

The Impact of Infection on Chronic Allograft Dysfunction and Allograft Survival After Solid Organ Transplantation

Infectious diseases after solid organ transplantation (SOT) are a significant cause of morbidity and reduced allograft and patient survival; however, the influence of infection on the development of chronic allograft dysfunction has not been completely delineated. Some viral infections appear to affect allograft function by both inducing direct tissue damage and immunologically related injury, including acute rejection. In particular, this has been observed for cytomegalovirus (CMV) infection in all SOT recipients and for BK virus infection in kidney transplant recipients, for community‐acquired respiratory viruses in lung transplant recipients, and for hepatitis C virus in liver transplant recipients. The impact of bacterial and fungal infections is less clear, but bacterial urinary tract infections and respiratory tract colonization by Pseudomonas aeruginosa and Aspergillus spp appear to be correlated with higher rates of chronic allograft dysfunction in kidney and lung transplant recipients, respectively. Evidence supports the beneficial effects of the use of antiviral prophylaxis for CMV in improving allograft function and survival in SOT recipients. Nevertheless, there is still a need for prospective interventional trials assessing the potential effects of preventive and therapeutic strategies against bacterial and fungal infection for reducing or delaying the development of chronic allograft dysfunction.     

Donor and recipient HLA/KIR genotypes do not predict liver transplantation outcome

Whether or not Natural Killer (NK) cells affect the immune response to solid organ allografts is still controversial. Main determinants of NK‐cell activation are specific HLA/killer‐cell immunoglobulin‐like receptors (KIR) interactions that, in transplantation, may induce NK‐cell alloreactivity. So far, in liver transplantation (LTX) donor‐versus‐recipient alloreactivity has not been investigated; in addition, studies of predicted recipient‐versus‐donor NK‐cell alloreactivity have led to contradicting results. We typed a cohort of LTX donors and recipients for HLA‐C/Bw4 and KIRs. We estimated the effect of NK‐cell alloreactivity, as predicted by classically used models, in the donor‐versus‐recipient direction. The results indicate that HLA/KIR mismatches in the donor‐versus‐recipient direction do not predict graft rejection nor graft or patient survival, suggesting that donor‐derived NK cells do not play a major role in LTX outcome. In addition, when considering predicted NK‐cell alloreactivity in the reverse direction (recipient‐versus‐donor), we first confirmed that donor HLA‐C genotype was not associated with acute rejection, graft or patient survival and secondly we found that none of the models describing NK‐cell alloreactivity could predict LTX outcome. Overall our observations suggest that, in contrast to what is shown in haematopoietic stem cell transplantation, donor‐derived NK cells may not contribute in preventing liver graft rejection, and that recipient‐versus‐donor NK‐cell alloreactivity does not predict LTX outcome.     

CMV‐infected kidney grafts drive the expansion of blood‐borne CMV‐specific T cells restricted by shared class I HLA molecules via presentation on donor cells

We aimed to determine the role of cytomegalovirus (CMV)‐infected donor cells in the development of a CMV‐specific immune response in kidney transplant recipients. We assessed the CMV pp65‐specific immune response by using interferon‐? ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R? 31, D+R + 44, D?R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN‐?‐producing anti‐CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV‐specific CD8⁺ T cells after transplantation, we compared the number of HLA‐A2–restricted CMV‐specific CD8⁺ T cells in primo‐infected recipients who received an HLA‐A2 or non–HLA‐A2 graft. The median of anti‐CMV pp65 T cells restricted by HLA‐A2 was very low for patients who received a non–HLA‐A2 graft vs an HLA‐A2 graft (300 [0‐14638] vs. 17972 [222‐85594] anti‐CMV pp65 CD8⁺ T cells/million CD8⁺ T cells, P = .001). This adds new evidence that CMV‐infected kidney donor cells present CMV peptides and drive an inflation of memory CMV‐specific CD8⁺ T cells, likely because of frequent CMV replications within the graft.     

Natural killer-cell activity after human renal transplantation in relation to killer immunoglobulin-like receptors and human leukocyte antigen mismatch

BACKGROUND: Natural killer (NK) cells use killer immunoglobulin-like receptors (KIR) that bind to self-class I major histocompatibility complex (MHC) molecules to prevent killing of autologous cells. Mismatched allografts, which do not express recipient MHC class I molecules, can therefore be potential targets for NK-cell killing. In our living related-unrelated renal transplantation program, donor-recipient pairs vary in the amount of both HLA and KIR genes they share. This provides us with a unique opportunity to dissect the influence of KIR on NK-cell function after transplantation. METHODS: Recipient NK cells were used in a cytotoxicity assay against donor peripheral blood mononuclear cells 2 days before, on the day of, and 3 days after transplantation. Results were correlated to HLA-KIR compatibility between donor and recipient. RESULTS: NK killing, in a direct ex vivo setting, was demonstrated to be HLA mismatch dependent. Recipient NK antidonor cytotoxicity was unaltered despite having received 2 days' treatment with cyclosporine A before transplantation. However, cytotoxicity increased 3 days after transplantation in 71% of recipients. Recipients exhibiting increased NK cytotoxicity against their donors after transplantation were found to possess more activating KIR genes specific for donor class I MHC molecules than those in whom killing activity did not increase (P<0.04). CONCLUSIONS: NK cells are activated after transplantation despite quadruple immunosuppression, suggesting that recipient NK-cell cytotoxicity against the donor may be a previously unrecognized area of the rejection process, especially in poorly matched donor-recipient pairs where the recipient may not express the correct repertoire of inhibitory receptors to prevent killing of donor cells.     

No Impact of KIR‐Ligand Mismatch on Allograft Outcome in HLA‐Compatible Kidney Transplantation

Natural killer (NK) cell function can be modulated by the killer cell immunoglobulin‐like receptors (KIR) which interact with human leukocyte antigen (HLA) class I molecules on target cells. KIR‐ligand mismatching has recently been shown by van Bergen et al. (American Journal of Transplantation 2011; 11(9): 1959–1964) to be a significant risk factor for long‐term graft loss in HLA‐A, ‐B and ‐DR compatible kidney transplants. To verify this potentially important finding, we performed genotyping of 608 deceased‐donor kidney graft recipients and their HLA‐A, ‐B and ‐DR compatible donors for KIR and HLA, using samples and clinical data provided by the Collaborative Transplant Study. Graft survival of KIR‐ligand‐matched and ‐mismatched transplants was compared. We found no impact of KIR‐ligand mismatching on 10‐year graft survival in HLA‐A, ‐B, ‐DR compatible kidney transplants. Further analysis did not reveal a significant effect of recipient activating/inhibitory KIR or KIR genotypes on graft survival. Our data do not support the concept that KIR‐HLA matching might serve as a tool to improve long‐term renal allograft survival.     

KIR‐Ligand Mismatches Are Associated With Reduced Long‐Term Graft Survival in HLA‐Compatible Kidney Transplantation

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system with the ability to detect HLA class I disparities via killer‐cell immunoglobulin‐like receptors (KIR). To test whether such KIR‐ligand mismatches contribute to the rejection of human solid allografts, we did a retrospective cohort study of 397 HLA‐DR‐compatible kidney transplantations and determined the KIR and HLA genotypes of recipients and the HLA genotypes of donors. In transplantations compatible for HLA‐A, HLA‐B and HLA‐DR (n = 137), in which a role for T cells and HLA antibodies in rejection was minimized, KIR‐ligand mismatches were associated with an approximately 25% reduction in 10‐year death‐censored graft survival (p = 0.043). This effect was comparable to the effect of classical HLA‐A and HLA‐B incompatibility, and in HLA‐A,‐B‐incompatible transplantations (n = 260) no significant additional effect of KIR‐ligand mismatches was observed. Multivariate Cox regression analysis confirmed the effect of KIR‐ligand mismatching as an independent risk factor in HLA‐A,‐B,‐DR‐compatible transplantations (hazard ratio 2.29, range 1.03–5.10, p = 0.043). This finding constitutes the first indication that alloreactive NK cells may thwart the success of HLA‐compatible kidney transplantations, and suggests that suppression of NK‐cell activity can improve the survival of such kidney grafts.     

Monitoring of CMV‐specific cell‐mediated immunity with a commercial ELISA‐based interferon‐γ release assay in kidney transplant recipients treated with antithymocyte globulin

Monitoring for cytomegalovirus (CMV)‐specific cell‐mediated immunity (CMV‐CMI) may be useful for individualizing valganciclovir (VGCV) prophylaxis after kidney transplantation (KT). We performed a commercial ELISA‐based interferon (IFN)‐γ release assay (QTF‐CMV) from posttransplant months 2‐5 (362 points) in 120 CMV‐seropositive KT recipients that received antithymocyte globulin as induction therapy and VGCV prophylaxis (median of 92 days). Forty‐seven patients (39.3%) had CMV infection after discontinuation of prophylaxis. The QTF‐CMV assay was reactive, nonreactive, and indeterminate in 264 (72.9%), 90 (24.9%), and 8 points (2.2%). The QTF‐CMV assay at prophylaxis discontinuation exhibited suboptimal accuracy for predicting protective CMV‐CMI (sensitivity: 77.4%; specificity: 34.3%; positive predictive value [PPV]: 64.1%; negative predictive value [NPV]: 50.0%), with no differences in 1‐year CMV infection rates between patients with negative (nonreactive or indeterminate) or reactive results (45.8% vs 36.1%; P = .244). Specificity and PPV to predict protective CMV‐CMI improved by elevating the IFN‐γ cutoff value to 1.13 IU/mL (65.7% and 71.4%) and 7.0 IU/mL (85.7% and 76.2%), although NPVs decreased. The QTF‐CMV assay as per manufacturer's interpretative criteria performed poorly to predict protection from CMV infection following discontinuation of VGCV prophylaxis among ATG‐treated CMV‐seropositive KT recipients. This performance is slightly improved by modifying the IFN‐γ positivity threshold.     

The Time Course of Development and Impact From Viral Resistance Against Ganciclovir in Cytomegalovirus Infection

(Val)ganciclovir is used to treat cytomegalovirus (CMV) infection following solid organ (SOT) or hematopoietic stem cell (HSCT) transplantation. Treatment failures occur, but the contribution from 39 known ganciclovir‐related mutations (GRMs) in the CMV‐UL97 gene remains controversial. We propose a categorization of these GRMs potentially useful when interpreting sequence analyses in clinical settings. The UL97 gene was sequenced from first/recurrent CMV infections among consecutive SOT or HSCT recipients during 2004–2009. GRMs were categorized as: Signature GRM (sGRM) if in vitro ganciclovir IC₅₀ ratio for mutated versus wild‐type virus >2 (n = 24); polymorphic GRM (pGRM) if ratio <2 (n = 15). (Val)ganciclovir treatment failure was defined as persistent viremia for 30 days or switch to foscarnet within this period. Of 99 (49 HSCT and 50 SOT) recipients with one CMV infection episode, 15 (13 HSCT and 2 SOT) experienced a total of 19 recurrent infection episodes. The prevalence of sGRM was 0% at start of first episode, whereas at start of recurrent episodes, prevalence was 37%. Only one sGRM was present at a time in individual patients. Patients with CMV containing an sGRM (vs. wild type)—but not with a pGRM—were at excess risk of treatment failure (odds ratio = 70.6 [95% CI:8.2–609.6]; p < 0.001). sGRMs emerged only following longer termed use of antiherpetic drugs and usually in recurrent CMV infection episodes. Risk of ganciclovir treatment failure was raised if an sGRM was detected.     

Kidney transplant recipients after nonrenal solid organ transplantation show low alloreactivity but an increased risk of infection

The number of kidney transplant recipients (KTRs) after nonrenal solid organ transplantation (SOT) has increased to almost 5%. Knowledge on patient and allograft outcomes, infections, and alloreactivity, however, remains scarce. We studied 40 KTRs after nonrenal SOT. Seven hundred and twenty primary KTRs and 119 repeat KTRs were used for comparison. Samples were collected pretransplantation, at +1, +2, and +3 months post‐transplantation. Alloreactive and CMV‐specific T cells were measured by interferon‐γ ELISPOT assay. Patient survival in KTRs after SOT, primary and repeat KTRs was comparable. While death‐censored allograft survival was comparable between KTRs after SOT and primary KTRs, KTRs after SOT showed superior 5‐year death‐censored allograft survival of 92.5% compared to 81.2% in repeat KTRs. Interestingly, KTRs after SOT show less preformed panel‐reactive antibodies, frequencies of alloreactive T cells, and acute rejections compared to repeat KTRs. KTRs after SOT, however, show higher incidences of EBV viremia and PTLD, sepsis, and death from sepsis. Impaired CMV‐specific cellular immunity was associated with more CMV replication compared to repeat KTRs. Our results suggest comparable patient and allograft outcomes in KTRs after SOT and primary KTRs. The observed low alloreactivity may contribute to excellent allograft outcomes. Caution should be taken in KTRs after SOT regarding infectious complications due to overimmunosuppression.     

Potential Role of NKG2D and Its Ligands in Organ Transplantation: New Target for Immunointervention

NKG2D is one of the best characterized activating receptors on Natural Killer (NK) and CD8+ T cells. This receptor recognizes several different ligands (MICA/MICB and ULBPs) induced by cellular stress and infection. In addition to the role described in cancer surveillance, recent data highlight the importance of NKG2D and its ligands in organ transplantation. Allografts show evidence of MICA and MICB expression in both acute and chronic rejection. The presence of anti-MICA antibodies has been correlated with incidence of graft rejection. Furthermore, NKG2D-ligand engagement activates NK cells, which provides T-cell costimulation, and enhances antigen specific CTL-mediated cytotoxicity. Activated NK cells may function as a bridge between innate and adaptive immunity associated with transplantation. Activated NK cells in response to IL-15 can also trigger organ rejection through NKG2D and affect the maturation of both donor and recipient antigen presenting cells (APCs) and ultimately the T-cell allogeneic response. Regulatory T cells, which modulate T-cell responses in organ transplantation and infections, were reduced in numbers by NK cells exposed to intracellular pathogens, possibly via interaction with one NK2GD receptor. Blockage of NKG2D-NKG2D-L interactions provides a novel pathway for development of inhibitors. These studies have important clinical and therapeutic implications in solid organ transplantation.     

A prospective multicenter observational study of cell‐mediated immunity as a predictor for cytomegalovirus infection in kidney transplant recipients

T cell immunity is essential for the control of cytomegalovirus (CMV) infection after transplantation. We evaluated a CMV‐specific peptide‐based enzyme‐linked immunosorbent spot (ELISPOT) assay to determine whether assay results could predict subsequent CMV events. Adult kidney transplant recipients at 43 centers underwent ELISPOT testing to enumerate interferon gamma (IFN‐γ) binding spot‐forming units (sfu) after stimulation of cells with an overlapping peptide pool of CMV phosphoprotein 65 (pp65) and immediate early‐1 (IE‐1) protein at the end of antiviral prophylaxis (EOP) and various time points thereafter. The primary outcome was a CMV event in the first posttransplant year. In 583 kidney transplant recipients (260 seropositive donor [D+]/seronegative recipient [R?] and 277 R+), CMV events occurred in 44 of 368 eligible patients (11.8%) at a median of 227 days (range 92‐360) posttransplant. A cutoff value of >40 sfu/2.5 × 10⁵ cells for either IE‐1 or pp65 was derived as a threshold for positivity, with a negative predictive value of >97% for CMV events. CMV events were significantly lower in assay positive vs assay negative patients (3.0% vs 19.5%, P < .0001 for pp65). Time to CMV event post‐EOP was significantly greater in those with sfu >40 at EOP (P < .0001). In this large, multicenter trial of kidney transplant recipients, we show that an assessment of CMV‐specific immunity using a novel ELISPOT assay is able to predict protection from CMV infection.