abnormal cells that either lack the expression, or express inadequate amounts, of human leukocyte antigen (HLA) class I molecules (Ljunggren and Karre, 1990). The ability of NK cells to discriminate between normal and HLA class I-deficient cells is due to the expression of clonally distributed HLA class I-specific inhibitory receptors (Radaev and Sun, 2003). Subsequent NK cell-mediated lysis of HLA class I deficient cells arises from the expression of HLA class I-specific activating receptors by NK cell clonal populations (Biassoni et al., 2000).

The KIR loci map to chromosome 19q13.4 within a region termed the leukocyte receptor complex (LRC) (Wagtmann et al., 1997). At present, approximately 15 expressed KIR genes have been identified and these are classified into four major groups (KIR2DS, 3DS, 2DL, 3DL) on the basis of the structural organisation and function of the receptors they encode (Fig. 1) (Marsh et al., 2002). In addition, two KIR pseudogenes have been identified (Martin et al., 2000; Wilson et al., 2000). These are named KIR2DP1 and KIR3DP1, reflecting the high sequence homology they share with two-domain and three-domain KIR genes, respectively.

NK cells express two structurally distinct classes of receptor that control effector function through HLA class I interactions; the C-type lectin-like heterodimer receptors (Lazetic et al., 1996; Carretero et al., 1997) and receptors belonging to the immunoglobulin superfamily (Ig-SF) (Arm et al., 1997; Meyaard et al., 1997; Cantoni et al., 1999; Fanger et al., 1999). Whilst members of the CD94/NKG2 lectin-like receptors are encoded by a relatively conserved set of genes, many of the NK cell receptors belonging to the Ig-SF display considerable genetic variation, in particular, the killer cell immunoglobulin-like receptors (KIRs).

In the present study, a PCR–SSOP typing system was implemented to determine the KIR genotype of a cohort of healthy aged Irish subjects in comparison to a younger control group to ascertain whether a particular repertoire of receptors is associated with successful ageing.

2. Materials and methods

The KIR gene family represents a highly diverse class of receptor-encoding genes. Within the human population, remarkable genetic diversity at the KIR region has been observed (Crum et al., 2000; Hsu et al., 2002a; Norman et al., 2002; Uhrberg et al., 2002). This phenomenon arises from variation in KIR gene type and number, which stems from KIR polygenism, coupled with allelic diversity at individual loci (Shilling et al., 2002). It is postulated that the occurrence of random point mutations and recombination between homologous KIR genes represent two mechanisms underlying KIR polymorphism (Witt et al., 1999).

2.1. Subjects

One hundred unrelated control samples (59% female, 41% male with an age-range of 19–45 years old) were chosen at random from the DNA bank of anonymous healthy Caucasian individuals from the Northern Ireland population, stored at the Northern Ireland Histocompat- ibility and Immunogenetics Laboratory (NIHIL), Belfast City Hospital. Ninety-three unrelated aged samples (70% female, 30% male with an age-range of 80–97 years old)

L.D. Maxwell et al. / Experimental Gerontology 39 (2004) 1223–1232

Table 1

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

Target Primer 50 Sequence 30 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 TTCCAGGTC(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

The X denotes the presence of the universal base inosine nucleotide and closed brackets indicate either nucleotide, conferring their generic/broad range nature. Nucleotides highlighted in bold correspond to intron sequences. The PCR cycling conditions for the above primer combinations are as follows: a96 8C/5 min; 60 8C/30 s; 72 8C/90 s (35 cycles); b,c,d96 8C/5 min; 60 8C/30 s; 72 8C/1 min (35 cycles).

was confirmed using gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide (0.5 mg/ml).

STudy (BELFAST) were also included in the study (Rea et al., 2000). A second subset of 89 unrelated healthy control samples (69% female, 31% male with an age-range of 19–45 years) from the NIHIL DNA bank and 89 unrelated aged samples (70% female, 30% male with an age-range of 80–97 years) from the BELFAST study were analysed in an attempt to confirm any significant findings, following analysis of the first cohort of samples. All aged DNA samples were from healthy elderly Caucasian individuals from the BELFAST study of the Northern Ireland population displaying no signs of age-related disorders, still living independently and approximating SENIEUR status (Ligthart et al., 1984, 1990). Ethical approval was obtained from the Queen’s University of Belfast ethics committee.

membrane as previously described (Middleton et al., 1995). Hybridisations, stringent washes and chemiluminescence detection of the hybridised digoxigenin-labelled probes were carried out as previously reported (Williams et al., 1999). The DNA sequences of the probes and their empirically determined stringent wash temperatures and concentrations are given in Table 2.

2.3. Statistical analysis