Correlation of Killer Immunoglobulin like Receptor Genes with the Rate of Cytomegalovirus Infection in Renal Transplantation Cases

C l i n M e d International Library Citation: Chauhan TS, Prakash S, Sharma RK, Agrawal S (2014)Correlation of Killer Immunoglobulin like Receptor Genes with the Rate of Cytomegalovirus Infection in Renal Transplantation Cases. J Genet Genome Res 1:003 Received: September 10, 2014: Accepted: October 01, 2014: Published: October 04, 2014 Copyright: © 2014 Chauhan TS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chauhan et al., J Genet Genome Res 2014, 1:1 ISSN: 2378-3648


Introduction
Cytomegalovirus (CMV) infection is the most common viral complication following solid organ as well as hematopoietic stem cell transplantation [1,2].While specific cytotoxic T-cell immunity represents a key factor to clear CMV [3,4], very little is known about the potential antiviral role of Natural Killer (NK) cells after transplantation.Solid organ transplantation is of special interest because current immuno suppressive protocols target the activation of T cells and therefore impair specific antiviral immunity [5].In contrast, NK cell function appears to be unaffected by such therapeutic regimens [6,7] and could therefore potentially play an important role in the anti-CMV immune response.
The clusters of KIR and HLA genes have been identified on two different chromosomes i.e., 19 and 6 respectively, leading to the possibility that an individual can lack the corresponding HLA ligand for a given KIR receptor as each can segregate independently.The balance of activating and inhibitory signals is crucial in regulating NK cell effector function, and the absence or down-regulation of MHC class I expression (missing KIR ligand) can induce susceptibility of a potential target cell to NK cytotoxicity [8,9].
HLA-C1 and HLA-Bw6 homozygote recipients and the presence of certain activating KIRs, could protect patients from CMV infection.

Material and Methods
We have investigated 286 cases (Males=245 (86%), Female=41 (14%)) and their respective donors (Male=60 (21%), Female=226 (79%)) in the present study.All patients and controls were residing in the state of Uttar Pradesh and adjoining states, since last three generations.Patients who have undergone renal transplantation were included in this study.All patients were maintained on triple immunosuppressive therapy that incorporated a calcinurin inhibitor (cyclosporine or tacrolimus), anti-metabolite (MMF or azathiorpine) and prednisolone.Some patients were on m-TOR inhibiter (everolimus) in place of calcinurin inhibitor.For each patient, information was collected for baseline factors like age, gender, creatinine, urinary protein level, blood urea nitrogen, blood pressure, complete lipid profile, sodium, potassium, calcium, inorganic phosphate, alkaline phosphate and tested for significance with the values obtained.DNA was extracted from blood of ESRD patients and controls collected in EDTA coated collection vials using Quiagen kits (Brand GMbH and Co KG, Cat # 51104).Quantitative CMV PCR was performed using DNA isolated from peripheral blood leukocytes to confirm the CMV status among cases.This work was approved by the ethics committees of SGPGIMS, Lucknow and Department of Biotechnology, Government of India, New Delhi.Informed consent of individuals was obtained as per the Declaration of Helsinki.

KIR and HLA genotyping
All DNA samples of transplant patients and controls were typed for KIR genes and HLA alleles basing on one of our previous study [13].

KIR/HLA ligand incompatibilities associated with graft outcome:
Number of matches and mismatches for KIRs with known ligands (KIR2DL1/HLA-C2, KIR2DL2/HLA-C1, KIR2DL3/HLA-C1, KIR3DL1/HLA-Bw4) and assumed HLA ligands (KIR2DS1/HLA-C2, KIR2DS2/HLA-C1, KIR2DS3/HLA-C1) have been calculated.A condition is considered to be mismatched when the recipient displays a certain KIR receptor but the donor graft does not have the corresponding HLA ligand.Similarly the case where a defined KIR receptor is expressed by the recipient and the corresponding HLA ligand is displayed by the allograft is considered to be matched.A missing condition has been assigned when the allograft has a certain HLA allele but the recipient is lacking the corresponding receptor, or when recipient and allograft both are lacking the receptor as well as the corresponding HLA ligand [14].

Statistical analysis
Gene frequency of KIR was determined by direct counting.Frequency differences between the CMV infected and non-CMV infected cases for individual inhibitory and activating KIR genes and the haplogroups were tested for significance at 95% confidence limits using two-tailed Fisher's exact test with Bonferroni correction.To test whether a certain KIR gene profile is associated with CMV infection in a well characterized renal allograft recipient context, binary logistic regression was applied.p-value ≤ 0.05 was considered significant.Magnitude of effect was estimated by odds ratios and their 95% confidence intervals.Graft survival was calculated according to the principle of Kaplan and Meier using SPSS (Statistical Package for the Social Sciences software-version 16.0) and is indicated as percent ± standard error.Statistical significance was estimated using the logrank test.

Demographic and biochemical criteria
Samples were collected from patients with complete clinical details and were on a follow-up since last twelve years in the tertiary care unit of Department of Nephrology, SGPGIMS, Lucknow.Various biochemical parameters associated with renal function have been studied between CMV and non-CMV infected cases (Supplementary Table 1).

KIR gene frequency
Upon analyzing the KIR gene frequencies for their statistical associations with CMV infected cases when compared to non-CMV infected cases, we did not find any significant association of KIR genes with CMV infected renal transplant cases (Table 1).On combining HLA-B homozygous and heterozygous groups protective association with KIR3DL1 (p=0.0201,OR=0.60, 95%CI=0.40-0.92)for CMV infected cases (Table 2) has been noted.

Discussion
Cytomegalovirus (CMV) is a significant cause of morbidity among patients receiving chronic maintenance immunosuppression and is often considered to show viral infection. 15There exist welldefined risk factors for CMV disease, such as CMV seropositivestatus of the donor and recipient, the immunosuppressive regimen, and the type of transplanted organ [16].Cytomegalovirus (CMV) seropositivity is known to have a major impact on the repertoire of antigen specific T cells as an estimated 10% of circulating T cells is CMV-antigen specific in seropositive individuals [17].In addition, the changes generally observed with an aged immune system, like a decreased CD4/CD8 ratio and expansion of CD28 negative CD8 positive T cells seem to be related to CMV sero-positivity [18,19].In our study, all of the donors and recipients undergo live related transplantation were CMV IgG sero-positive.This was due to the higher incidence of CMV infection among Indian population [20].We were not able to differentiate between CMV Donor-ve/Recipientve, Donor-ve/Recipient+ve, Donor+ve/Recipient-ve categories due to lack of seroreactive positive cases.
Several infectious diseases have been confirmed to be associated with certain KIR receptor/HLA ligand combinations [21].Human NK cells that have matured in the presence of HLA ligands for KIR receptors developed more potent cytotoxic capacity than those maturing in the absence of such ligands through a mechanism called 'licensing' [22].Therefore, in vitro at least, the lysis of CMV-infected cells by NK cells could be more complex than a simple missing KIR ligand mechanism [23].It has been suggested that inflammatory signals induced by infection could break NK-cell tolerance and even in the absence of the HLA ligand, unlicensed NK cells could be activated to kill the virus-infected cells.The contribution of non-KIR family receptors could also become predominant in the immune response of NK cells to viral infections.When the allograft has a certain HLA allele but the recipient is lacking the corresponding receptor, or when recipient and allograft both are lacking the receptor as well as the corresponding HLA ligand.
Cumulative incidence of cytomegalovirus infection in kidney transplant recipients stratified by KIR AA and Bx haplotype.In the present study inhibitory receptor KIR2DL1 showed protective effect for CMV infected renal transplant cases.The combinatorial analysis revealed significant protective associations against KIR2DL2-HLAC1, KIR2DL3-HLA-C1 and KIR3DL1-HLA-Bw4 combinations for CMV infected cases.KIR, the best characterized group of NK receptors are allotype and isotype specific.KIR and HLA are present on different chromosomes and therefore are differently inherited.NK-cell activity is probably a result of the balance between activating and inhibitory receptors [24].
CMV causes a latent infection in majority of infected individuals.In immune-compromised individuals it can reactivate, and cause a life threatening illness.Earlier reports have emphasized upon the importance of NK cells and their KIR receptor in controlling CMV reactivation after hematopoietic cell transplant (HCT).More specifically, donors with more than one activating KIR gene were associated with 65% reduction in CMV reactivation [25] and the same effect has been seen in kidney transplants [26].In the HCT setting, it was determined that the number of activating KIR genes in the donors but not in the recipients, was associated with protection from CMV reactivation and that the protective effect was highest when the donor genotype contained >5 activating KIR genes or at least a combination of KIR2DS2 and KIR2DS4 [27].Although the frequency of KIR2DS2 and 2DS4 is prominent in the present study among CMV infected individuals but it did not reveal any significance.
Studies on murine NK cells [28,29] have shown that if mature NK cells from wild-type are transferred into a host with a different MHC class I environment, the cells can be reprogrammed to their new environment.This may explain the increased expression of activating KIR after transplant reported in several earlier studies.Mature NK cells can be re-educated as their new environment dictates, the new environment being allo-transplantation and /or CMV reactivation.In the matched relative donor transplant typically do encode for the same HLA type in donor and recipient but, it is possible that minor histocompatibility antigens or other HLA (e.g.non classical HLA-G-E) could serve as ligand that could influence this enhancement of expression.
In lieu of the hypothesis that presence of activating KIR genes may downplay the incidence of CMV infection we have found prolonged graft survival among individuals possessing KIR Bx haplotype which possesses number of activating KIR genes.However, the question is what specific CMV-associated factors might influence KIR gene expression?KIR genes are known to be regulated by epigenetic factors [30,31], and it is possible that either viral or host factors have some effect.A cohort of 286 kidney allograft recipients in the present study have demonstrated evidence of protective effect of activating KIR on the graft survival rate among CMV infected cases.While our data is in concordance with recent evidence from hematopoietic stem cell transplant recipients, the major limitation of our results is that all the cases under this study were seropositive for CMV.Further the data for CMV recurrence is very less so Donor/Recipient combinations were not highly informative.

Table 1 :
KIR gene frequency distribution among cytomegalovirus and noncytomegalovirus infected renal transplantation cases .

Table 2 :
KIR receptor-HLA ligand association among cytomegalovirus and noncytomegalovirus infected renal transplantation cases.

Table 4 :
Effect of KIR-HLA ligand matches/mismatches on graft survival among CMV infected renal transplant cases.

Table 3 :
KIR-HLA ligand matches/mismatches among CMV and non-CMV cases using binary logistic regression.KIR receptor is expressed by the recipient and the corresponding HLA ligand is displayed by the allograft.Mismatch: recipient displays a certain KIR receptor but the donor graft does not have the corresponding HLA ligand.KIR/HLA ± (Missing):