and KIR Frequencies in Sicilian Centenarians

Florinda List`ı,1 Calogero Caruso,1 Giuseppina Colonna-Romano,1 Domenico Lio,1 Domenico Nuzzo,2 and Giuseppina Candore1

Abstract

Several studies suggest that human longevity appears to be linked inextricably with optimal functioning of the immune system, suggesting that specific genetic determinants may reside in loci that regulate the immune re- sponse, as human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genes. It has been suggested that longevity is associated with positive selection of alleles (i.e., HLA-DR11) or haplotypes (i.e., HLA- B8,DR3) that confer resistance to infectious disease(s). On the other hand, the cytolytic activity of natural killer (NK) cells is controlled by activating and inhibitory cell-surface receptors, including KIR. The genetic diversity of the KIR loci with respect to successful aging has been analyzed only in one study performed in the Irish popu- lation. Although two KIR genes (2DS3, 2DL5) displayed an initial increased frequency in the aged group, the significance of this association was lost when repeated in a second cohort. We have evaluated by polymerase chain reaction–sequence-specific primers (PCR-SSP) HLA-DRB1 and KIR receptors=HLA ligands frequencies in cente- narians and controls from Sicily. Our results demonstrate an increase of the HLA DRB1*18 allele in male cente- narians ( p ¼ 0.0266, after Bonferroni correction). Concerning KIR, no significant difference was observed after Bonferroni correction. However, our findings suggest that HLA=KIR=longevity associations are population spe- cific, being heavily affected by the population-specific genetic and environmental history. This kind of study is important to better understand aging and longevity, hence enhancing the planning of antiaging strategies.

Introduction

with longevity in the Sardinian population. On the other hand, association studies are subjected to a number of pos- sible confounding factors (as part of the homogeneity of the population in terms of geographic origin).5,6

inextricably linked with optimal functioning of the immune system, sug- gesting that specific genetic determinants may reside in loci that regulate the immune response, as human leukocyte antigen (HLA), cytokine networks, and killer cell immuno- globulin-like receptor (KIR).1 The studies performed on the association between longevity and HLA (the human major histocompatibility complex [MHC]) are generally difficult to interpret, owing to major methodological problems. How- ever, some of these studies, which are well designed and performed and support suggestions derived from the mouse studies on MHC effects on longevity.2,3 These studies sug- gest that longevity is associated with positive selection of alleles (i.e., HLA-DR11) or haplotypes (i.e., HLA-B8,DR3) that confer resistance to infectious diseases, respectively, via peptide presentation or via antigen nonspecific control of immune response. It is noteworthy that these associations are mostly gender related.3,4

Another crucial role is played by the KIRs in the regula- tion of innate immune response. They are glycoproteins ex- pressed on the cell surface of natural killer (NK) cells and subsets of T cells. These polymorphic receptors interact with specific motifs on HLA class I molecules, modulate NK cy- tolytic activity, and are encoded by genes located on chro- mosome 19q13.4.7–9

The KIR gene family represents a highly polymorphic class of receptor-encoding genes, which have displayed considerable allelic diversity, and it is postulated that KIR polymorphism arises from both the occurrence of random point mutations and recombination between homologous KIR genes.10,11 At present, approximately 16 expressed KIR genes have been identified and these are classified into four major groups (KIR2DS, 3DS, 2DL, 3DL) on the basis of the structural organization and function of the receptors they encode.12 In addition, two KIR pseudogenes have been identified.9,13 These are named KIR2DP1 and KIR3DP1,

In our previous case–control studies, we have demon- strated that both HLA-DR and -DQ alleles are not associated

1Immunosenescence Unit, Department of Pathobiology and Biomedical Methodologies, University of Palermo, Palermo, Italy. 2Institute of Biomedicine and Molecular Immunology, National Research Council, Palermo, Italy.

reflecting the high sequence homology they share with two- domain and three-domain KIR genes, respectively.

typed for class II HLA alleles and KIR receptors =HLA li- gands by polymerase chain reaction–sequence-specific pri- mer (PCR-SSP; One Lambda, Inc. Canoga Park, CA) and KIR-TYPE=Epitop-TYPE (BAG Health Care GmbH, Lich, Germany). The KIR-TYPE kit was designed to identify 14 KIR genes (2DL1, 2DL2, 3DL1, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1), 2 pseudogenes (2DP1 and 3DP1) and the common variants of KIR2DL5 (KIR2DL5A, KIR2DL5B), the KIR2DS4 allele (*001=002 and *003), and KIR3DP1 allele (*001=002 and *003), whereas the Epitop- TYPE kit was used to detect the alleles of the HLA specifi- cities HLA-Cw Asn80, HLA-Cw Lys80, HLA-B Bw4Threo, HLA-B Bw4 Iso, and HLA-A Bw4. The PCR reaction was amplified using a PCR thermal sequencer (Mycicler, Biorad, Milan, Italy). After the PCR process, the amplified DNA fragments were separated by agarose gel electrophoresis (2–2.5%), 0.5–1% Tris=borate=EDTA buffer, and ethidium bromide (0.5mg=mL). All the subjects positive for the HLA- DR3 subtype, HLA-DRB1*17, were typed for HLA-B8 by micro-SSP (One Lambda, Inc.) to identify carriers of HLA- B8,DR3(DR17) haplotype, which is part of the 8.1 ancestral haplotype (AH 8.1) HLA-A1, Cw7, B8, TNFAB*a2b3, TNFN*S, C2*C, Bf*s, C4A*Q0, C4B*1, DRB1*0301(DR17), DRB3*0101, DQA1*0501, DQB1*0201.4