The effect of a reduced oxygen tension culture system (6% O2) on TCCs has been examined. Specifically, the effects of the altered culture conditions on DNA damage levels, in vitro lifespan and proliferative capacity were assessed in six independently-derived human CD4+ TCCs. DNA damage levels over the entire lifespan were significantly lowered by reducing oxygen tension. However, lifespan (total population doublings (PDs) achieved) and proliferative capacity (PDs/week) was reduced for all clones under reduced oxygen tension (mean: lifespan 74.9 PD, proliferative capacity 2.1 PD) when compared to standard culture (20% O2 conditions; mean: lifespan 56.3 PD, proliferative capacity 1.0 PD, p<0.05). The effect of a synthetic superoxide dismutase mimetic (10µM EUK-134) on TCCs has also been examined. The effects of 10µM EUK-134 on in vitro lifespan and proliferative capacity were assessed in six independently-derived human CD4+ TCCs. Lifespan and proliferative capacity were were significantly reduced (mean: lifespan 53.3 PD, proliferative capacity 0.6 PD) when compared to standard culture (20% O2 conditions; mean: lifespan 74.2 PD, proliferative capacity 2.0 PD, p<0.05). Preliminary data suggests that DNA damage levels over the entire lifespan were significantly lowered in the presence of EUK-134, analysis will be complete by 31/12/2005. To further investigate these results, the TCC were examined to determine the fraction of the populations which were actively proliferating (using a colorimetric ELISA BrdU incorporation assay), the fraction of the populations which were actively undergoing apoptosis (using a flow cytometry-based Annexin V assay) and cell cycle distribution analysis of the populations (using classical PI flow cytometry technique). Analysis will be complete and results available by 31/12/05. Results may suggest that reduced oxygen tensions induced in the culture system had an effect on the overall redox balance of TCCs (this is partially verified by the reduced levels of oxidative DNA damage in these clones). This may have led to a detrimental effect on ROS-mediated signalling pathways in the TCCs and thus contributed to the reduced proliferation of the TCCs. Western blot analysis was utilised to investigate redox sensitive signalling pathways, purified proteins from six independently-derived human CD4+ TCCs were probed at the beginning and end of their in vitro lifespans for the relative levels of phosphorylated- and dephosphorylated-JNK 1/2 MAPK, phosphorylated- and dephosphorylated-p38 MAPK and phosphorylated- and dephosphorylated-ERK MAPK. Preliminary data suggests that there are age-related alterations in MAPK-mediated signalling in TCCs under the various culture conditions employed in WP10. Analysis will be complete and results available by 31/12/05. The gene for a hsp 70 stress protein (hsp72a1b) has been cloned and preparations made for stable expression in TCC. HTERT constructs have been generated using GFP marker but so far the infection rate of TCC has been much too low to generate any hTERT transfectants. Use of cytokine cocktails to extend lifespan has focussed on IL 15, found to be of benefit for some TCC when combined with IL 2.
Identification of immunogenetic factors (HLA and cytokine gene polymorphisms) involved in successful aging
Genetic epidemiology studies were performed on samples from randomly selected healthy elderly individuals (age 66-99 years) compared to unrelated young controls (age 21 - 35 years) and families with longevity members from the Bulgarian population. The following genetic markers were analyzed: HLA class I (A, B) and class II (DRB) loci, IL-2, IL-6, IL-10, IL-12B, IFNã, TNF-A, and TGF-B1 genes. Additionally, HLA-DQB1 and DPB1 polymorphism was assessed in samples selected based on their HLA and cytokine gene profiles. Allele and haplotype frequency analyses were performed in order to identify alleles and extended haplotypes associated with immunosenescence. Prevalence of protective for autoimmune diseases DRB1*1101 and DRB1 x 1601 associated haplotypes was observed in healthy elderly Bulgarians. IL-10 -1082, -819, -519 genotypes related to a high level of expression were also positively associated with longevity in Bulgarians. Additionally, investigation of extended profiles based on several SNPs in TNF-A gene with possible relation to the level of expression revealed decreased pro-inflammatory genotypes in elderly individuals. Extended genotypes, including several polymorphic positions of IL-12B and TGF-B1 relevant for successful aging were identified. Significance of the observed IL-12B genotypes was further confirmed in samples from the Italian population. Additionally, new single nucleotide polymorphisms were identified in the prompter and intron 1 regions of TNF-A with possible functional significance. Sequences of these new gene variants are available. A set of data concerning genetic background of aging is provided and genetic markers associated with successful aging are identified. These data will allow drawing conclusions on a correlation between gene variants and human longevity and will contribute to formulation of recommendations for biomarkers with possible clinical relevance.
Expansion of CD28null effector memory and effector T cells expressing higher levels of NK associated receptors in healthy elderly
We have previously studied that the T cells show a decrease in the expression of CD28 in the elderly, associated with an increase of the expression of some NK-R in the CD28 negative subsets. The aim of this work is to analyse the telomere length in the different lymphocyte subsets from PBMCs from healthy elderly donors compared with young. Our results show that the CD28 negative subsets are significantly increased in the elderly. This increase is associated with a shortening in the telomere length when compared with the CD28 negative subset from young donors. We have also found no significant differences in the NK phenotype between young and elderly. This result is completed by the finding that there are no differences between telomere lengths from the healthy elderly NK subsets when compared with the young NK subset. When the CD28 positive subset was analysed, we found that the telomere length was significantly longer both in young and elderly donors comparing with the CD28 counterpart. These results support the hypothesis that these cells are cytotoxic T cells that has undergone a process of replicative senescence. We have also analysed the different memory subsets based on simultaneous staining for CD45RA and CCR7 , lymphocytes described as subsets that could be segregated into so called naive (CD45RA+CCR7+), central-memory (CD45RA-CCR7+), effector-memory (CD45RA-CCR7-) and effector (CD45RA+CCR7-) T lymphocytes. The aim of this work is to analyse the expression of CD56, CD94, CD244, CD28 on CD8 T cells associated with the memory phenotype (based on CD45RA and CCR7 phenotype). The results show us that in healthy donors the CD8+CD28+ subset is mainly represented by the naive subset, while the CD8+CD28- subset, are mainly represented by the effector subset. We have also found that the CD244 cytotoxicity surface marker is significantly expressed on the memory effector subset, while the expression of CD56 and CD94 markers is higher in the effector subset and lower in the naive subset. When the CD8+CD56+ subset was analysed, we found that this cytotoxic subset is mainly represented by the effector phenotype.
We have sought immunological biomarkers of ageing informative in elderly free-living humans. Sets of biomarkers must have predictive value for clinical endpoints: we have selected death as the endpoint in longitudinal studies of people over 80 years old. The panel of biomarkers showing 2, 4, and 6 year predictive value is termed the "Immune Risk Profile", or IRP. It is being constantly refined and tested also on chronologically younger people, as well as patients with cancer and other diseases involving an element of chronic antigenic stress. Modelling T cell clonal expansion and contraction under chronic antigenic stimulation enables us to model these in vivo events, we believe, quite accurately. In vitro interventions using supplements, cytokines, antibodies, as well as genetic engineering approaches, are providing us with tools to eventually intervene in vivo as well, in order to modulate or even ablate immunosenescence, which materially contributes to morbidity and mortality in the elderly. Panels of biomarkers established in longitudinal studies in vivo and cultures in vitro will enable subjects to be accurately monitored for the impact of said interventions.
Genomic studies were carried out on T cell samples in collaboration with Unilever group in Colworth. The experimental design consisted in collecting total RNA from donors of different classes of age and compared them using microarray technology in order to evaluate differences in gene expression related to age. To avoid that gene expression data will be complicated by intrinsic intra-individual variation in samples we formerly conducted a pilot study of intra and inter-individual gene variability in young healthy donor-derived CD3+ T-cells. In fact the concept of individual variability was identified as a relevant problem when performing microarrays analysis and is crucial for the best evaluation of mRNA transcript levels when these determinations are performed employing fresh blood cells. PCA analysis, performed on one subject in a six-month period, revealed that a very small portion of gene expression profiles varies over-time. In fact the vast majority (>94%) of gene transcripts are stable over time of blood draw. Moreover, an in-depth investigation of gene expression profiles allowed us to quantify the individual variability using variance ratio or F statistic by dividing the variance of the expression levels among individuals by the variance within individuals and further confirmed using ANOVA. Finally we extracted a list of the most interesting genes, which identified an "individual molecular signature". Additionally, to take into account the different percentage of T cells present in the blood of each donor at each time and for better understanding the contribution of the various T cell subsets to RNA expression profile, phenotypic analyses was assessed on part of the T cells used for microarray analysis. In particular, we assessed T cell subsets relevant to immunosenescence as the percentage of CD4+ or CD8+ and percentage of peripheral and central memory on CD4+ or CD8+ T cell subpopulations. Statistical results showed significant differences in lymphocytes composition between the various donors (P<0,01), and no significant differences for the same donor samples at different times (P>>0,01). In conclusion, each subject shows its own pattern of T cell subpopulations that it seem not to change over-time. These results allowed us to progress with our experimental design aimed at characterizing T cells gene expression related to age.
In the present study we have verified and extended our previous on the immune risk phenotype in the elderly. To this end, we have conducted a thorough longitudinal study of nonagenarian, and showed that the number of expanded CD8 T cell clones is higher in non-IRP than IRP nonagenarians or middle-aged controls. Nonetheless, MHC/peptide multimer staining indicated that the number of CMV specific T cells was greater in nonagenarians than in middle-aged controls, although the ratio of functionally-intact cells was significantly lower. The lowest ratio of functional CMV specific T cells was found in an IRP individual. Data from a thorough longitudinal analysis of the CMV specific T cells in nonagenarians, showed a very stable pattern both with respect to frequency, phenotype and clonal composition. We hypothesize that the number of CD8 T cell clones expands as the individual ages, possibly as a compensatory mechanism to control latent infections, e.g. CMV, but eventually reach a point upon which clonal exhaustion leads to shrinkage of the CD8 clonal repertoire, associated with decreased survival. These data are in press in The Journal of Immunology, and thus will disseminate to the scientific community. Although there is no immidiate commercial implementation use, a future use of these data in terms of generating therapeutic interventions to protect the elderly from infection seems likely. Moreover, the methedological combination of pheno- and clonotype analyses may set the stage for future monitoring of immune status using additional parameters. Together, our data may have an impact on the scientific community, and the health status and quality of life, particularly in the elderly.
T-cell senescence occurs during ageing of the immune system and is an important contributor to dysregulation and dysfunction of cellular processes observed in ageing. These include alterations in cellular growth, replication, metabolism, cell-cell communication, signalling, cytokine secretion, DNA repair and apoptosis. However, little is known about the mechanisms and molecular alterations underlying these functional changes. In the current study, we have applied surface-enhanced laser desorption/ionisation (SELDI)-ToF-MS ProteinChip Array technology for protein profiling of early and senescent T-cells in search of protein or peptide bio-markers of immunosenescence. We used the Ciphergen ProteinChip® PCS4000 SELDI system, a combination of affinity chromatography and mass spectrometry, to study protein profile changes in T-cell lysates that occur during in vitro ageing and immunosenescence with an aim to find protein or peptide biomarkers of immunosenescence. H50 chips (reversed-phase chromatography) and Q10 chips (anion-exchange chromatography) were used to target hydrophobic and negatively charged proteins respectively in T-cell lysates. Biomarker analysis using the CiphergenExpressTM software identified differential expression of a variety of peaks associated with in vitro T-cell ageing. A consistent pattern of differential protein expression was observed between both early and late passage T-cell clones grown in vitro, and from T-cell clones derived from young and old donors. The corresponding proteins were subsequently identified by a combination of SELDI-TOF-MS, peptide mass fingerprinting MALDI-TOF-MS and Nanospray-IonTrap-MS/MS. Through a combination of SELDI/MALDI-TOF-MS and nano-ESI-MS/MS we have identified several candidate proteins that are differentially expressed in ageing and senescent T-lymphocytes. These factors belong to a variety of functional processes including lipid metabolism, cytoskeleton remodelling, histone organisation, calcium signalling and protein degradation and represent potential bio-markers of T-cell ageing and immunosenescence. Of interest, several actin polymerising/ depolymerising factors were differentially expressed in ageing T-cells, indicating that alterations in cytoskeleton remodelling may be an important step in progression toward cellular senescence.
We believe that the in vitro modelling of the process of clonal expansion and contraction, or lack of it, under chronic stimulation can contribute to further definition and/or confirmation of biomarkers predictive of T cell dysfunction and provide some ideas on possible remediation in vivo. These approaches still focus on the use of cytokines and anti-oxidants which might not present too much of an ethical and logistical hurdle in the elderly. The data currently suggest that, at least for CD4 cells, certain types of anti-oxidant supplementation and manipulating the cytokine environment can extend the lifespan of T cells in vitro, and that zinc supplementation can enhance heat shock protein upregulation, thereby possibly providing more protection from stress. There are intriguing hints that stress resistance and integrity of cell signalling and DNA repair capacity change somewhat with age in culture but that the most marked differences are seen between donors from whom TCC are derived: the older the donor, the better the cell integrity, even at very high PD. This applied to hsp expression, to microsatellite instability and DNA repair and other important parameters such as membrane fluidity and CD28 signalling. Thus, monoclonal T cells derived from progenitor cells were in general less stable than those from adults, and nonagenarians and centenarians were the most stable. This apparently counter-intuitive finding is presumably an expression of the strong selective pressure for survival to 90-100 years of the donors. Genomic and proteomic screening of TCC derived from different donors cultured for shorter or longer periods provided data that were consistent with this idea. These results all point towards intrinsic genetic programs in successfully aged donors, which maintain superior immune responsiveness under conditions of chronic antigenic stress. The search for these genetic factors, includes cytokine and cytokine receptor polymporphisms as well as immunogenetic studies on HLA in populations and families. These data need to be integrated with current and future projects specifically addressed to the genetics of healthy ageing.
The effect of age in culture on the DNA excision repair capacity of five independently derived TCC lines has been analysed to date. Two of the TCC were from a 26 year old donor, two from a 45 year old donor and one from an 80 year old donor. The TCC were cultured from the PD at which they were obtained from P1 until they underwent apoptosis in vitro. Samples were taken for DNA repair analysis at the early in vitro lifespan, middle of lifespan and end of lifespan of the TCC. TCC were treated with physiologically relevant DNA damaging agents; hydrogen peroxide, N-methyl-N -nitro-N-nitrosoguanidine or 254nm ultraviolet irradiation. Following treatment, the amount of DNA damage in the clones was determined over a time course using modified comet assays. The results obtained revealed a decline related to in vitro age in the DNA repair capacity of clones derived from a 26 and a 45 year old donor. This decline may represent at least a partial explanation for the age related increase in DNA damage in these clones when cultured in vitro. In contrast, there was no evidence for a decline related to in vitro age in repair capacity in the clones derived from an 80 year old SENIEUR donor. An alternative mechanism must underlie the age related increased in DNA damage in these clones when cultured in vitro. Analysis of the activity of poly (ADP ribose) polymerase (PARP) within samples of TCCs at different stages of their in vitro lifespan and in PBMCs supplied by P6 and P1 has revealed a decline in PARP capacity in TCC derived from normal healthy donors with in vitro ageing. In contrast, SENIEUR donor-derived TCC did not show any evidence of a decline with increasing age in vitro. PBMCs from NONA (see P6) subjects were found to have higher levels of poly(ADP-ribose) than controls, although the difference was not statistically significant. These results provide some evidence in support of the view that poly(ADP-ribosyl)ation capacity appears to be an important contributor towards longevity. Modified comet assays used to assess levels of DNA mismatches and the ability of TCCs and ex vivo T cell samples to perform mismatch DNA repair did not reveal any clear pattern of change in DNA mismatch frequency with increasing in vitro age. The ability of the TCC to repair supra-physiological levels of induced DNA mismatches (following treatment with acridine mutagen ICR-191) was also investigated. The results revealed a modest age-related decline in the efficiency of the MMR system in TCC derived from a 26 and a 45 year old donor. In contrast, results obtained from TCC derived from an 80 year old SENIEUR donor showed a more efficient MMR capacity with increasing age in vitro. A novel fluorescent in situ hybridisation (FISH) technique in combination with the comet assay (Comet-FISH) was established and used to assess telomeric DNA integrity and repair in six independently-derived TCCs. Results revealed an age-related decline in telomeric integrity with increasing in vitro age of TCCs derived from a newborn and a 31 year old donor, and no age-related decline in telomeric integrity in TCC derived from a NONA donor. Further analysis of TCC and ex vivo samples will validate this novel technique, which could be a very effective means to analyse telomeric DNA integrity and repair in a host of cell types. Publication is in preparation.