2.2. KIR genotyping by PCR-SSOP

PCR amplification was performed using the appropriate primer combinations for each target region (Table 1) in a 100 ml reaction volume containing 67 mM Tris-HCl pH 8.8; 16 mM (NH4)2SO4; 2.0 mM MgCl2; 0.01%(w/v) Tween-20; 200 mM of each dNTP; 0.2 mM of each primer; 3 units Taq polymerase (Bioline, London, UK); 0.1-0.5 mg DNA. After an initial denaturation step of 96°C for 5 min, the cycling conditions specified in the legend of Table 1 were employed for each target region. After the final cycle, the samples were held at 72°C for 5 min and then at 15°C until removed from the PCR machine. Successful amplification was confirmed using gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide (0.5 mg/ml).

Table 1: PCR primer combinations used for specific amplification of KIR genes from genomic DNA

Target Primer 5' Sequence 3' PCR product KIR genes amplified region name size(bp) Trans/cyt 
TMF1 GCTGTGATTAGGTACTCAGTGGCC 930 3DL1-3, 3DS1, 2DL1-5, 2DL1*004, 2DS1-5, Domaina 
TMF2 GTTCTGATTGGGACCTCAGTGGTC 
TMF3 ATTCTGATTGGGACCTCAGTGGCT 
DmcytR TTGAGACAGGGCT(GA)TTGTCTCC 
D2 Ig D2F TTCCAGGTCA(TC)ATXTG(AG)GAAACCTTC 302 3DL1-3, 3DS1, 2DL1-5, 2DS1-5, 2DP1, 3DP1 Domainb 
D2R ACCTGTGAC(AG)GAAACAAGCAGTGG 
D2R2 ACCTGTGACAGAAAC(GA)(GA)GCAGTGG 
D1 Ig D1F AGAGAXXGTCATCCTGCA(AG)TGTTGGTC 241 3DL1-3, 3DS1, 2DL1-3, 2DS1-5, 2DP1, 3DP1 Domainc 
D1R GATCACXATGTCCAG(AG)GG(GA)TCACTGG 
D0 Ig D0F-1 GTGGTCAGGACAAXCCCTTXCT(GC)TCTGCC(CT)GGC 265 2DL4-5, 3DL1-3, 3DS1, 2DP1, 3DP1 Domaind 
D0F-2 GTGGTCAGGACAAGCCCTTCTGCTCTGCCTGGC 
D0RR1 GGGTTGCTGGGTGC(TC)GACCACC 
D0RR2 GGGTTGCTGGGTGC(TC)GACCACTC 

Thein the elderly population may suggest a role in successful ageing. However, further studies with larger sample sizes are needed to confirm these findings. It is also important to note that in this study, activating KIR genes were found at a lower frequency compared to inhibitory KIR genes in all sample groups, which is consistent with previous studies. Overall, the results suggest that KIR genes may play a role in successful ageing by optimizing NK cell activity in the elderly population.In healthy aged individuals, the activating receptor may play a role in providing improved innate immune protection against virus infections and cancer. The exact mechanism through which this occurs is somewhat speculative. It is possible that certain activating KIR genes may be up-regulated in response to oncogenesis and infection, leading to improved NK cell-mediated killing of abnormal cells and more efficient elimination of viral infections and tumour cells. 

NK cells play a crucial role in the innate immune response by eliminating abnormal cells that display compromised HLA class I expression. This cytolytic activity is regulated by a range of activating and inhibitory receptors with HLA class I ligand specificity, such as KIRs. This study investigated the potential role of KIR diversity in immunosenescence and longevity in the Irish population. Inhibitory KIRs also modulate NK cell activity, which can provide beneficial effects for healthy ageing. Certain inhibitory KIR genes encode receptors that prevent NK cell-mediated lysis of healthy autologous cells with normal levels of HLA class I expression, which is crucial in preventing self-reactivity and autoimmune disorders. 

However, dysfunctional mechanisms controlling the expression levels of inhibitory KIR genes could lead to reduced cell surface expression of HLA class I restricted inhibitory KIRs, allowing NK cell-mediated lysis of healthy autologous cells expressing normal levels of HLA class I. This scenario may arise via the interaction of HLA class I independent activating NK receptors with their ligands, such as natural cytotoxicity receptors that possess ligands other than HLA class I expressed by normal autologous cells. Thus, certain inhibitory KIR genes may be more susceptible to aberrant expression in ageing, providing an increased disposition to autoimmune disorders.

A PCRSSOP typing system was used to determine the presence or absence of known KIR genes in a cohort of healthy aged individuals and young control subjects, but no individual KIR genotype was found to be associated with successful ageing, suggesting that the polygenic nature of the KIR loci may not be a major influence in longevity. The study encountered a high level of genotypic diversity in the sample groups, which was not entirely unexpected due to the polygenic nature of KIRs. This makes the identification of a specific KIR gene combination challenging.

Recent studies have suggested that gender is a variable concerning the genetics of ageing, and certain combinations of KIR genes may exert subtle gender-dependent effects during the ageing process. However, more research is required to elucidate the precise role that NK cells play in longevity, including larger pooled sample groups aimed at investigating whether the polymorphic nature of genes encoded within the LRC is important in ageing. 

Acknowledgements go to the R&D Office of Health and Personal Social Services (Northern Ireland) and the Department of Employment and Learning (Northern Ireland) for their support, as well as all participants in the study. Both cytokines and HLA class I molecules mediate important immunomodulatory functions for NK cell activity. Certain cytokines including IL-2, IL-15 and IL-18 induce NK cell activity while HLA class I molecules are ligands for certain activating and inhibitory KIRs. Epistatic interactions between particular cytokine, HLA, and KIR haplotypes may contribute to successful ageing or reduced lifespan.