3.3. T-cell subsets

The values of various T-cell subsets, including CD28 þ , CD28 2 , and CD57 þ phenotypes were examined in elderly, middle-aged and young randomly selected healthy individuals (Table 5). Significantly lower relative and absolute numbers of CD3 þ 8 þ ðp , 0; 02Þ and CD8 þ 28 þ ðp , 0: 01Þ cells were found in elderly people compared to both middle-aged and young ones. Decreased CD8 þ 57 þ cells ðp , 0: 05Þ were observed in old individuals in comparison with middle-aged subjects. The lower percentage of CD8 þ T cells was more pronounced (,12%) in individuals aged over 85 years. No significant changes in the values of CD8 þ 28 2 , CD4 þ 28 þ and CD4 þ 28 2 cell subsets in the peripheral blood

E. Naumova et al. / Experimental Gerontology 39 (2004) 637–644

Table 5

Phenotype distribution of T-cell subsets (%, cells/mm3) in elderly compared to middle-aged and young subjects

CD3 þ T-cells CD3 þ 4 þ CD3 þ 8 þ CD8 þ 57 þ CD8 þ 28 þ CD8 þ 28 2 CD4 þ 28 þ CD4 þ 28 2 Elderly 70.4 ^ 6.45 49.9 ^ 10.15 17.62 ^ 4.82**3 7.5 ^ 2.61* 10.1 ^ 3.33*** 10.33 ^ 4.33 46.4 ^ 12.4 3.4 ^ 3.3 ðn ¼ 17Þ 1229 ^ 456 850 ^ 289 12 ^ 149** 124 ^ 65* 177 ^ 96*** 172 ^ 63 728 ^ 264 53 ^ 42 Middle-aged 74.7 ^ 7.1 46.9 ^ 8.6 28.0 ^ 5.9 11.4 ^ 5.1 16.9 ^ 3.8 12.6 ^ 7.2 44.9 ^ 6.9 3.2 ^ 3.1 ðn ¼ 26Þ 1499 ^ 517 948 ^ 316 548 ^ 208 223 ^ 125 324 ^ 114 235 ^ 142 881 ^ 312 59 ^ 57 Young 74.18 ^ 7.37 47.6 ^ 6.26 27.3 ^ 4.24 9.11 ^ 4.73 19.0 ^ 4.05 9.66 ^ 3.08 44.4 ^ 3.65 2.2 ^ 1.3 ðn ¼ 21Þ 1432 ^ 329 927 ^ 248 515 ^ 109 184 ^ 91 332 ^ 127 162 ^ 49 776 ^ 165 36 ^ 17

Values are given as mean ^ SD; *p , 0:05; **p , 0:02; ***p , 0:01:

were divided into 3 subgroups: old (proband, aged .65 years), middle aged (first generation, age 37–57 years) and young (second generation, aged 18–24 years) individ- uals. The immunophenotype analysis of lymphocyte subsets showed that although most pronounced in the elderly group, lower numbers of CD8 þ T cells were also found in middle- aged and young members of the families compared to the age matched controls. A progressive CD8 þ 28 þ cell subsets decline was seen with aging, with significant difference between the old subjects on one hand and the middle-aged ðp , 0: 02Þ and young ðp , 0: 01Þ individuals on the other hand (Fig. 2).

in longevity. Our study in extended families with longevity members and matched for geographical distribution controls from the Bulgarian population showed a slightly increased frequency of HLA-B*35, DRB1*12 alleles and correspond- ing haplotypes. Positive association of DRB1*12 with longevity has been previously observed in the Japanese population (Akisaka and Suzuki, 1998). Further it has been shown that life span prolongation is associated with delay of immunodeficiency of normal aging or with possible amelioration of the autoimmunity that develops with age. In agreement with this in elderly and their family members we did not observed the common alleles and haplotypes, positively associated with autoimmunity in Bulgarians. Additionally, gene polymorphisms within known or puta- tive regulatory regions have been demonstrated to be associated with different disorders, including age related (Haukim et al., 2002). Pro-inflammatory cytokines have been shown to play a pathogenic role (Bruunsgaard, 2001). Some of cytokine gene polymorphisms have been demon- strated to affect gene transcription causing variations in cytokine production. However, recent publications showed contradicting results regarding the association of cytokine gene/haplotypes and the level of expression (Bidwell et al.,

4. Discussion

In this pilot study we investigated how immunological factors, particularly HLA, cytokine gene polymorphism and immunophenotype profiles, contribute to the successful aging in the Bulgarian population. For the first time we also performed family analyses in order to assess the combine effect of these three biological markers.

In the last years many studies have searched for the impact of HLA genes in human longevity. Due to the central role of HLA molecules in the immune response it has been suggested that this genetic complex could be associated with successful aging and that particular HLA alleles and haplotypes might have different distribution in longevity individuals (Caruso et al., 2000). However, the results were contradicting and no clear consensus has been reached. DRB1*07, *11, *13 alleles (Lagaay et al., 1991; Ivanova et al., 1998) as well as the ancestral haplotype HLA-B8- DR3 (Proust et al., 1982; Rea and Middleton, 1994), that confers resistance to viral infections, were shown to be increased in centenarians. Additionally, it has been suggested that HLA/longevity associations are population specific. The Bulgarian population is relatively homo- geneous and based on HLA class I and class II profile it is characterized by features of Southern European anthro- pological type with some influence of additional ethnic groups (Ivanova et al., 2001, 2002). Therefore Bulgarians

%201510CD3+8+CD8+28+ —Cpgs28-     CD8+57+YAMAOAGOA BMA OYA

Fig. 2. Phenotype distribution of CD8 þ cells in families with longevity members.

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consistent with a healthy immune status of elderly people in our study group.