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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic was first reported in Wuhan, China in December 2019, moved across the globe at an unprecedented speed, and is having a profound and yet still unfolding health and socioeconomic impacts. SARS-CoV-2, a β-coronavirus, is a highly contagious respiratory pathogen that causes a disease that has been termed the 2019 coronavirus disease (COVID-19). Clinical experience thus far indicates that COVID-19 is highly heterogeneous, ranging from being asymptomatic and mild to severe and causing death. Host factors including age, sex, and comorbid conditions are key determinants of disease severity and progression. Aging itself is a prominent risk factor for severe disease and death from COVID-19. We hypothesize that age-related decline and dysregulation of immune function, i.e., immunosenescence and inflammaging play a major role in contributing to heightened vulnerability to severe COVID-19 outcomes in older adults. Much remains to be learned about the immune responses to SARS-CoV-2 infection. We need to begin partitioning all immunological outcome data by age to better understand disease heterogeneity and aging. Such knowledge is critical not only for understanding of COVID-19 pathogenesis but also for COVID-19 vaccine development., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Alzheimer's disease is one of the most devastating neurodegenerative diseases. The exact cause of the disease is still not known although many scientists believe in the beta amyloid hypothesis which states that the accumulation of the amyloid peptide beta (Aβ) in brain is the initial cause which consequently leads to pathological neuroinflammation. However, it was recently shown that Aβ may have an important role in defending the brain against infections. Thus, the balance between positive and negative impact of Aβ may determine disease progression. Microglia in the brain are innate immune cells, and brain-initiated inflammatory responses reflected in the periphery suggests that Alzheimer's disease is to some extent also a systemic inflammatory disease. Greater permeability of the blood brain barrier facilitates the transport of peripheral immune cells to the brain and vice versa so that a vicious circle originating on the periphery may contribute to the development of overt clinical AD. Persistent inflammatory challenges by pathogens in the periphery, increasing with age, may also contribute to the central propagation of the pathological changes seen clinically. Therefore, the activation status of peripheral innate immune cells may represent an early biomarker of the upcoming impact on the brain. The modulation of these cells may thus become a useful mechanism for modifying disease progression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As is apparent from the many contributions to this Special Issue of the Journal, the impact of age on immunity is nefarious, with all manner of dysregulated responses attributed to "immunosenescence". These range from poorer responses to vaccination, lower capacity to mediate anti-cancer responses, more inflammation and tissue damage, along with autoimmunity and loss of control of persistent infections. Given the grave clinical implications of altered immune status in aged people, it is of paramount importance to understand the nature of and mechanisms responsible for "immunosenescence". As in any rapidly developing research area, certain paradigms establish themselves early on, by necessity based on earlier and fewer data, and have a disproportionate influence on how investigators think about the subject, especially investigators from other disciplines. It may therefore be appropriate to reconsider our basic knowledge at this juncture, asking exactly what do we mean by the term "immunosenescence"? This is attempted in this contribution to the Special Issue., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As age increases, immune responses and consequently protection following vaccination to seasonal influenza is commonly believed to decrease. Possible drivers of this immune dysfunction include immunosenescence, repeated exposure to the same seasonal influenza antigens, and prior infection with cytomegalovirus (CMV). Here, to determine immune parameters distinguishing vaccine humoral responders (R) from non-responders (NR) following vaccination, we surveyed broad peripheral blood "cellular immune correlates" of older adults vaccinated with Fluad® (an adjuvanted subunit influenza vaccine containing strains H1N1, H3N2 and B). Phenotyping included αβ-T-cells, γδ-T-cells, B-cells and myeloid cells. The frequencies of most of these lymphocyte phenotypes were found to be similar in R and NR, although perhaps counterintuitively, one of the few differences seen between the two groups was higher frequencies of regulatory T-cells in R. These differences were more prominent for responses to the vaccine strains H1N1 and H3N2 than to the B strain, and in CMV-seropositive than CMV-seronegative elderly. Further, frequencies of early-differentiated CD4+ T-cells tended to be higher and frequencies of memory CD4+ T-cells tended to be lower in R than NR. There were also differences in B-cells, with higher frequencies in R compared to NR. To the best of our knowledge, these results are the first to report such differences in elderly people responding or failing to respond to adjuvanted seasonal influenza vaccination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In addition to measures already used in clinical practice, molecular measures have been proposed to assess health status, but these have not yet been introduced into clinical practice. We aimed to test the association of functional capacity measures used in current practice and molecular measures with age and health status. The cohort consisted of 178 middle-aged to old participants of the Leiden Longevity Study (range 42-82years). We tested associations between functional capacity measures (physical tests: grip strength, 4-meter walk, chair stand test; cognitive tests: Stroop test, digit symbol substitution test and 15-picture learning test) with age and with cardiovascular or metabolic disease as a measure of the health status. These associations with age and health status were also tested for molecular measures (C reactive protein (CRP), numbers of senescent p16INK4a positive cells in the epidermis and dermis and putative immunosenescence (presence of CD57+ T cells)). All functional capacity measures were associated with age. CRP and epidermal p16INK4a positivity were also associated with age, but with smaller estimates. Grip strength and the Stroop test were associated with cardiovascular or metabolic disease, as was epidermal p16INK4a positivity. All associations with cardiovascular or metabolic disease attenuated when adjusting for age. In conclusion, in middle-aged to old persons, the molecular measures tested here were more weakly associated with age and health status than functional capacity measures. Whether these molecular measures associate more closely with health status in the elderly or in specific groups of patients needs to be explored further., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

The vertebrate adaptive immune system is remarkable for its possession of a very broad range of antigen receptors imbuing the system with exquisite specificity, in addition to the phagocytic and inflammatory cells of the innate system shared with invertebrates. This system requires strict control both at the level of the generation the cells carrying these receptors and at the level of their activation and effector function mediation in order to avoid autoimmunity and mitigate immune pathology. Thus, quality control checkpoints are built into the system at multiple nodes in the response, relying on clonal selection and regulatory networks to maximize pathogen-directed effects and minimize collateral tissue damage. However, these checkpoints are compromised with age, resulting in poorer immune control manifesting as tissue-damaging autoimmune and inflammatory phenomena which can cause widespread systemic disease, paradoxically compounding the problems associated with increased susceptibility to infectious disease and possibly cancer in the elderly. Better understanding the reasons for slippage of immune control will pave the way for developing rational strategies for interventions to maintain appropriate immunity while reducing immunopathology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

"Immunosenescence" is a loosely descriptive designation for age-associated alterations to most measures of immunity, which can be seen in all mammals examined in any detail. Both innate and adaptive immunity are affected in a manner assumed to be deleterious, but often the clinical consequences of the assessed changes are unclear or not even investigated. The mechanisms accounting for these changes, and biomarkers of immunosenescence, are currently the subject of intensive research. Cross-sectional studies have established hallmark age-associated differences between adaptive immune factors in young and old people, particularly a lower number and percentage of naïve T cells, especially CD8+ T cells, and accumulations of late-differentiated CD8+ T cells. The latter but not the former is strongly affected by infection with the persistent ß-herpesvirus HHV5 (cytomegalovirus, CMV). Only limited longitudinal studies have so far investigated whether these differences actually reflect age-associated changes at the individual level. The Swedish OCTO/NONA-Immune studies identified a set of immune parameters including infection with CMV which predicted survival in people over 85 at baseline. Moreover, the Leiden 85+ study showed that T cell-mediated pro-inflammatory specific for CMV antigens was enriched in very old survivors, suggesting the overarching necessity of maintaining effective immunosurveillance of this virus. Here, the disparate impact of CMV on "immunosenescence" and survival in human populations under different condition is reviewed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Influenza vaccination is less effective in the elderly compared to the young. Studies that have attempted to identify immune parameters correlating with satisfactory vaccine responses have yielded inconclusive results. Here, we correlate the distribution of different circulating CD4+ and CD8+ T-cell phenotypes with the humoral response to vaccination with Intanza, an intradermal seasonal vaccine, in 54 individuals of different ages. Subjects were stratified according to age (below or over 60) and presence of a latent infection with Cytomegalovirus (CMV). CMV-seropositivity was significantly associated with a lower response rate to the vaccine in people over but not below 60 yr of age. Unlike reported data, late-differentiated (CD45RA+CCR7-CD27-CD28-) CD4+, but not CD8+ T-cells associated with a poorer vaccine response. Thus, latent CMV infection has a deleterious effect on influenza antibody responses in the elderly, which might be mediated through CD4 T-cells lacking CCR7, CD27 and CD28 and re-expressing CD45RA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Influenza remains the single most important cause of excess disability and mortality during the winter months. In spite of widespread influenza vaccination programs leading to demonstrated cost-savings in the over 65 population, hospitalization and death rates for acute respiratory illnesses continue to rise. As a person ages, increased serum levels of inflammatory cytokines are commonly recorded (TNF-α, IL-1, IL-6). Termed "inflammaging", this has been linked to persistent cytomegalovirus (CMV) infection and immune senescence, while increased anti-inflammatory cytokines (IL-10, TGF-β) are possibly associated with more healthy aging. Paradoxically, a shift with aging toward an anti-inflammatory (IL-10) response and decline in the IFN-γ:IL-10 ratio in influenza-challenged peripheral blood mononuclear cells is associated with a decline in the cytolytic capacity of CD8+ T cells responsible for clearing influenza virus from infected lung tissue. Thus, it is seemingly counter intuitive that the immune phenotype of healthy aging predicts a poor cell-mediated immune response and more serious outcomes of influenza. Herein we postulate a mechanistic link between the accumulation of late-stage, potentially terminally differentiated T cells, many or most of which result from CMV infection, and the immunopathogenesis of influenza infection, mediated by granzyme B in older adults. Further, adjuvanted influenza vaccines that stimulate inflammatory cytokines and suppress the IL-10 response to influenza challenge, would be expected to enhance protection in the 65+ population., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Decreased immune responsiveness associated with aging is generally termed "immunosenescence". Several theories have been proposed to explain age-related declines in immune responses. Here, we will focus on and describe potential defects in T cell signal transduction from the membrane to the nucleus, leading to changes in the type, intensity and duration of the response as a major factor contributing to immunosenescence. We will first detail T cell signaling through the T cell receptor (TCR), CD28 and IL-2 receptor (IL-2R) and then discuss the observed age-related alterations to these signaling pathways. The role of membrane rafts in T cell signaling and T cell aging will be described. These factors will be considered in the context of the notion that age-related changes to T cell signaling may be attributed to changes in the functionality of the T cells due to shifts in T cell subpopulations with age. For this reason, we conclude by highlighting the application of multiparametric signaling analysis in leukocyte subsets using flow cytometry as a means to obtain a clearer picture with respect to age-related changes to immune signaling., (Copyright © 2010 Elsevier B.V. All rights reserved.)

"Immune senescence" is a descriptive term for the deleterious age-associated changes to immunity observed in all mammals studied so far. While all components of innate and adaptive immunity are changed with age, the clinical impact of these changes is not clear, and mechanisms of and markers for immunosenescence are controversial. In humans, several cross-sectional studies have demonstrated that the major accepted age-associated changes to parameters used to assess adaptive immune status are markedly influenced by infection with cytomegalovirus (CMV). In the very limited longitudinal studies thus far carried out, a cluster of immune parameters associating with 2-, 4- and 6-year survival of the very elderly has been identified and termed the "immune risk profile" (IRP). This cluster includes seropositivity for CMV and is characterised by accumulations of clonal expansions of late-differentiated CD8+ T cells, many of which are specific for CMV antigens. Here we review the impact of CMV on "immune senescence" in humans., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Cell culture and in vitro models are the basis for much biological research, especially in human immunology. Ex vivo studies of T cell physiology employ conditions attempting to mimic the in vivo situation as closely as possible. Despite improvements in controlling the cellular milieu in vitro, most of what is known about T cell behavior in vitro is derived from experiments on T cells exposed to much higher oxygen levels than are normal in vivo. In this study, we report a reduced proliferative response and increased apoptosis susceptibility after T cell activation at 2% oxygen compared to in air. To explain this observation, we tested the hypothesis of an impaired efficacy of intracellular protective mechanisms including antioxidant levels, oxidized protein repair (methionine sulfoxide reductases), and degradation (proteasome) activities. Indeed, after activation, there was a significant accumulation of intracellular oxidized proteins at more physiological oxygen levels concomitant with a reduced GSH:GSSG ratio. Proteasome and methionine sulfoxide reductase activities were also reduced. These data may explain the increased apoptotic rate observed at more physiological oxygen levels. Altogether, this study highlights the importance of controlling oxygen levels in culture when investigating oxygen-dependent phenomena such as oxidative stress., (Copyright 2009 Elsevier Inc. All rights reserved.)

Immunosenescence contributes materially to the decreased ability of the elderly to control infectious disease, which is also reflected in their generally poor response to vaccination. This state is influenced by many genetic and environmental factors, but it has become increasingly clear over the past year that the pathogen load to which individuals are exposed throughout life impacts greatly on immune performance in late life. The link to accelerated immunosenescence in this respect may be via the enhanced pro-inflammatory status observed in the elderly and in chronic disease at any age. Here, we summarise some of the developments in this area and in other important areas of immunosenescence research over the past year.

Herpes viruses (particularly CMV and to some extent EBV) might play a role in accelerating the deterioration of immune functions with age. Indeed, it has been demonstrated that chronic infection with CMV causes an expansion of specific CD8 T lymphocytes and that this is related to a shrinkage of the T cell repertoire in very elderly people, predicting mortality. We have analysed CD8 T cells in young and old healthy Sicilians who were both CMV- and EBV-seropositive. Our data confirm expansions of T cells specific for the HLA-A2-restricted pp65 (495-503) CMV epitope up to nearly 14% of total peripheral CD8 cells in certain elderly individuals (range 0-14%). However, the mean percentage of CMV-specific cells in the elderly was not greater than the young (range 0.2-3%). The CMV-specific CD8 cells in the elderly were predominantly CD45RA+, but in the young they were mostly CD45RO+. Our findings are somewhat different from published reports from Northern European populations, both in terms of mean numbers and surface phenotypes. These findings may reflect disparate hygienic and nutritional conditions 70-90 yr ago, which were very different in Northern and Southern Europe at that time, as well as a different genetic background.

During the immune response T cell function is influenced by extrinsic factors, some of which lead to increased protein and DNA damage and are thought to play a role in age-related immune dysfunction. Damage is in part due to reactive oxygen species produced as a result of aerobic metabolism during a vigorous immune response, but in the in vitro models commonly used to study human immunity may also be due to culturing cells under hyperoxic conditions, i.e., in air. Reactive oxygen species (ROS) are ubiquitously generated but an imbalance between ROS production and protection against ROS may severely affect T cell activation. Controlling and modulating oxidative stress in the extracellular milieu may influence T cell signalling and activation. Here, we discuss the relevance of oxidative stress modulation to prevent T cell dysfunction. We draw attention to some technical, but critical, aspects of T cell culture under hyperoxic conditions.

In the present NONA immune longitudinal study, we investigate the previously identified Immune Risk Profile (IRP), defined by an inverted CD4/CD8 ratio and associated with persistent cytomegalovirus infection and increased numbers of CD8+CD28- cells, relative 6-year survival and age in NONA individuals. These subjects have now reached age 92, 96, and for the first time in this study, 100 years at follow-up. A 55 year old middle-aged group was used for comparison. Immunological monitoring included the analysis of numbers of lymphocytes and neutrophils, the T-cell subsets CD3+CD4+, CD3+CD8+, CD8+CD28+, CD8+CD28-, and the CD4/CD8 ratio. Longitudinal data were analysed by multivariate analyses of variance (MANOVA) from four measurement occasions at 2-year inter-intervals. One-way ANOVA was used for cross-sectional comparisons at baseline and the 6-year follow-up. The results confirmed the importance of the IRP as a major predictor of mortality in this population of very old. Moreover, the results suggested that survival to the age of 100 years is associated with selection of individuals with an "inverted" IRP that was stable across time, i.e., maintenance of a high CD4/CD8 ratio and low numbers of CD8+CD28- cells. The results underlines the importance of a longitudinal study design in dissecting immune parameters predictive of survival and show for the first time that centenarian status is associated with avoidance of the IRP over at least the previous 6 years and probably throughout life.

Immunosenescence resulting in decreased ability to control infectious disease contributes to morbidity and mortality not only in the very elderly, but in all likelihood already from middle age. Studying immunity in humans is therefore essential for developing treatments to restore dysregulated immune responses and assure healthy longevity. The past year has seen many significant advances in our knowledge of age-associated alterations to immunity in elderly people, only some of which can be briefly reviewed here.

T cells undergo rapid clonal expansion upon antigenic stimulation to produce an effective immune response. Any defect in the DNA mismatch repair (MMR) system may have a detrimental effect on T cell proliferation. This study employed an in vitro model of human CD4+T cell ageing to investigate MMR capacity at various stages of T cell lifespan. A novel modification of the alkaline comet assay, which utilised T4 endonuclease VII to detect single base DNA mismatches, was used to assess DNA mismatch frequency. No clear pattern in DNA mismatch frequency with increasing culture age was observed. However, the ability to repair induced DNA mismatches (following treatment with acridine mutagen ICR-191) revealed an age-related decline in the efficiency of the MMR system in clones derived from a 26 and a 45-year-old donor, but not from an 80-year-old very healthy SENIEUR donor. This study suggests that unchallenged, dividing human T cell clones have variable levels of DNA mismatches throughout their lifespan, not affecting proliferation. However, when challenged with supra-physiological levels of DNA mismatches, deficiencies were found in ageing T cell clones in MMR capacity, with the exception of T cell clones from a SENIEUR donor previously shown to maintain effective DNA excision repair.

Many immune functions decline with age and may jeopardize the elderly, as illustrated, for example by the significantly higher mortality rate from influenza in old age. Although innate and humoral immunity are affected by aging, it is the T cell compartment, which manifests most alterations. The mechanisms behind these alterations are still unclear, and several explanations have been offered including thymic involution and Telomere attrition leading to cell senescence. Age related accumulation of mutations has been documented and could serve as an additional mechanism of T cell dysfunction. One effective repair mechanism capable of rectifying errors in DNA replications is the mismatch repair (MMR) system. We previously reported a comparative examination of individual DNA samples from blood cells obtained at 10 year intervals from young and old subjects. We showed significantly higher rates of microsatellite instability (MSI), an indicator of MMR dysfunction in older subjects, compared to young. In the present study we confirm this result, using direct automated sequencing and in addition, we demonstrate that as CD8 lymphocytes from aged individuals, undergo repeated population doublings (PDs) in culture, they develop MSI. CD4 clones that also undergo repeated PDs in culture develop significant MSI as well. Elucidation of this previously unexplored facet of lymphocyte dynamics in relation to aging may help identify novel mechanisms of immunosenescence and pathways that could serve as targets for interventions to restore immune function.

Large clonal expansions of peripheral CD8(+) T cells carrying receptors for single epitopes of CMV are common in the elderly and may be associated with an immune risk phenotype predicting mortality. To study the effect of ageing on the ability of CMV-specific CD8(+) T cells to produce type 1- and type 2-cytokines, interferon-gamma-and IL-10-producing, CD8(+) T cell responses in the presence of CMV peptide antigen were measured in CMV-seropositive old and young donors. We found that large expansions of A2/NLV-specific CD8(+) T lymphocytes in the elderly are accompanied by a partial loss of antigen responsiveness as reflected in a greatly decreased frequency of antigen-specific IFN-gamma-and IL-10-producing cells. Thus, despite carrying specific antigen receptors, the majority of the clonally expanded CMV-specific CD8(+) cells in the elderly was dysfunctional according to these criteria. Our data indicated a bias towards a more anti-inflammatory response in the elderly. The accumulation of dysfunctional CMV-specific cells might fill the 'immunological space' and decrease the available repertoire of T cells for novel antigens. This might account for the increased incidence of many infectious diseases in the elderly.

Oxidative DNA damage has been suggested to contribute to the decline in T cell clone (TCC) function with increased age in vitro. To test this hypothesis the effect of a reduced oxygen tension culture system (6% O(2)) on TCCs was examined. Specifically, the effects of the altered culture conditions on DNA damage levels, in vitro lifespan and proliferative capacity were assessed in five independently derived human CD4+ TCCs. DNA damage levels over the entire lifespan were significantly lowered by reducing oxygen tension. Lifespan (total population doublings (PDs) achieved) and proliferative capacity (PDs/week) were reduced for all clones under reduced oxygen tension when compared to standard culture conditions. This observed tendency warrants further investigation using a greater number of clones from donors of all age groups before definitive conclusions regarding the effect of low oxygen tension on the lifespan and proliferative capacity of TCC can be made. However, these results may suggest that the reduced oxygen tension culture system has interfered with some aspect of T cell biology not yet examined within the remit of this study.

Repair of mismatches in mammalian cell DNA is mediated by a complex of proteins that constitute the so-called mismatch repair system (MMR), the main post-replicative pathway for the correction of replication errors. Loss of MMR (as exemplified by germline mutations in some MMR genes, leading to hereditary non-polyposis colorectal cancer) results in increased mutation rates at both coding sequences and in non-coding regions such as microsatellites. In order to evaluate possible functional alterations of this repair system during ageing that could affect immune system efficiency, we studied microsatellite instability at five different loci interspersed in the genome (CD4, VWA31, Tpox, Fes/FPS and p53) in total DNA from T lymphocyte clones derived from hematopoietic stem cells, or peripheral T cells of young or elderly subjects. In addition, these clones had been maintained for different periods in vitro to represent a culture model of ageing. We observed increasing instability accumulating with increasing passages in culture, particularly in CD34+cell-derived clones, but no clear donor age relationship.

Physical stress induced in healthy volunteers by the Loughborough intermittent shuttle test (LIST) was used to validate a newly developed whole-blood cell culture system (Instant leukocyte culture system (ILCS). Exercise induced immune modulation was investigated through measurement of cytokine levels after activating leukocytes in peripheral blood ex vivo using the physiologic stimulant lipopolysaccharide (LPS). LPS induced production of three different cytokines, interferon gamma (IFNgamma), interleukin-6 (IL-6), and interleukin-10 (IL-10). IFNgamma levels were significantly decreased (P = 0.02 and P = 0.001 ) and IL-10 levels significantly increased (P= 0.04 and 0.03) after exercise. LPS induced IL-6 production was only marginally further increased by exercise. In conclusion, the ILCS system provided a reliable ex vivo method, showing common as well as subject specific features in the time course of the immune modulation caused by the LIST protocol. This system will be useful for studies of the elderly, where cytokine standardisation is notoriously difficult.

T-cell clones (TCC) derived from human peripheral blood lymphocytes of a young control, a healthy elderly (SENIEUR) donor, or from CD34(+) hematopoietic progenitor cells were utilised in this study to examine how in vivo and in vitro ageing affects T-cell apoptotic capability. The role of CD25, CD28 and the intracellular proteins, FLICE-inhibitory protein (FLIP), receptor-interacting protein (RIP) and caspase 3 were investigated. We observed an age-related decline in the expression of the IL-2 receptor alpha chain CD25, and absence of the co-stimulatory receptor CD28 on three of the four TCC studied. In young donor- and CD34 cell-derived TCC, but not in SENIEUR donor-derived TCC, we observed an age-related increase in susceptibility of the cells to mFas-L-induced apoptosis, which correlated with the age-related decrease of CD25 expression. Expression levels of full-length RIP and FLIP did not show any correlation to apoptotic susceptibility. However, expression levels of the cleaved form of RIP were greatly reduced in the SENIEUR donor-derived TCC, which together with a trend towards increased caspase 3 activity, could indicate an age-related alteration in utilisation of different apoptotic signalling pathways.

Large clonal expansions of peripheral CD8+ T cells carrying receptors for single epitopes of CMV and EBV are common in the elderly and may be associated with an immune risk phenotype predicting mortality. Here we show that the frequency of CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1), a marker of cells unable to undergo further clonal expansion, was markedly elevated in CD8+ T cells from old donors. Moreover, tetramer staining revealed that the elevated frequency of CMV-specific CD8+ T cells in the elderly was due to an accumulation of cells bearing this dominant negative receptor. The fraction of CMV-specific T cells able to secrete interferon-gamma after specific antigenic stimulation was significantly lower in the elderly than in the young, although the total number of functional cells was comparable. Therefore, the majority of the clonally expanded virus-specific CD8+ cells in the elderly was dysfunctional. Thus, T cell responses are altered in the aged by an accumulation of replicatively senescent dysfunctional T cells carrying receptors for persistent herpes viruses. The presence of clonal expansions of such virus-specific cells may shrink the available repertoire for other antigens and contribute to the increased incidence of infectious disease in the elderly.

CD4+ human T cell clones were derived from activated peripheral blood lymphocytes of healthy young adults to establish cloning efficiencies (CE) and clonal longevities. These results were compared with those obtained using cells from the very elderly, also in excellent health. CE and both maximal and average longevities under appropriate culture conditions were very similar in the two groups. Moreover, CE of CD34+ hematopoietic progenitor cells and longevities of clones derived from them were also similar. Finally, CE and longevities of clones derived from a patient with chronic myelogenous leukaemia were found to be comparable as well. Hence, T cells with absolutely no antigenic exposure in vivo prior to cloning (i.e. CD34-derived) and those potentially exposed to chronic antigenic stimulation (CML-derived) and those from young or old donors all had similar cloning and propagation properties in vitro. These results imply that the longevity of T cells in culture is more likely to be dictated by cloning conditions than any intrinsic differences between the cells studied.

Immunosenescence is an age-associated decline of immunity involving multiorgan changes. As the mean age of the population increases, an increase of diverse pathologies associated with immunosenescence has been observed in the developed countries. Age-related changes in the immune system contribute to the increased incidence and severity of infectious diseases and possibly cancer in the elderly. Moreover, in young individuals chronic activation of the immune system (as occurs in autoimmune diseases, cancer, HIV infection and other chronic infections) induces changes in the immune response that parallel those observed in elderly individuals. An interdisciplinary approach to immunosenescence including investigation of the molecular and cellular mechanisms and clinical aspects is being utilised by the EU program "Immunology and Ageing in Europe" (ImAginE, QLK6-CT-1999-02031), the second major international conference of which took place in Córdoba, Spain, 22-26th March, 2001). We briefly summarise some of the highlights of the meeting here, and bring together in this special issue a collection of peer-reviewed papers reflecting the broad approach to immunosenescence currently being applied.

An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA(4977) and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA(4977) and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA(4977) was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identified using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence.

Immunosenescence is an age-associated dysregulation of immune function which may contribute to the increased susceptibility of the elderly to infectious disease. Although age-associated changes are measurable in the innate immune system, it is the adaptive arm of the immune system which is particularly susceptible to the deleterious effects of ageing, especially the T cell compartment. In this review, the characteristics of longitudinal ageing in cultured monoclonal human T cell populations will be summarized. It will be argued that parallels between this in vitro model and T cell senescence in vivo suggest the use of such models to screen for interventions ameliorating immunosenescence in vivo.

Aging is associated with alterations of the immune system, thought to be related to an increased susceptibility to infectious diseases, and possibly to cancer and autoimmunity in the elderly. In the present paper we report data obtained on freshly collected blood from 148 healthy subjects of different ages (from cord blood to 102 years old). The subjects were divided into seven age classes (cord blood, 3-11 years, 15-39 years, 41-60 years, 61-74 years, 75-84 years, 85-102 years) and their lymphocyte subsets and the expression of the apoptosis-related molecule CD95 were evaluated. In respect of lymphocyte subsets, the major differences were found in the cord-blood samples compared with the oldest old groups. In the cord-blood group, the absolute number of all the lymphocyte subsets was enhanced, but in the oldest group, an increase of CD16+ lymphocytes was observed, whereas CD19+ lymphocytes, which progressively decrease with age, continue to decrease further in the very old. The data show that the expression of CD95 increases until age 74 years, whereas in the oldest old it tends to decrease again. The trend of CD95 expression seems to be related to the change of expression of CD95 on CD4+ lymphocytes, because the CD8+/CD95+ population rose steadily throughout the entire age range. The evaluation of CD95+/CD45R0+ lymphocytes shows similar results to those observed analyzing CD95 on total lymphocytes. Furthermore, a constant increase of CD95+/CD28+ and a related decline of CD28+ lymphocytes was observed in all age groups. These data suggest that the expression of CD95 on the different subsets of lymphocytes can be considered a good marker for studies of immunosenescence, because it may be predictive of successful aging, and can partially explain the change in lymphocytes subsets in elderly.

The term "immunosenescence" refers to an age-associated dysregulation of immune function which contributes to the increased susceptibility of the elderly to infectious disease. Although there are age-associated changes measurable in the innate immune system (Pawelec et al., 1998c), it is the adaptive, particularly T cell, system which is most susceptible to the deleterious effects of aging. In this minireview, characteristics of aging in long-term human T cell cultures will be summarized, and the parallels between the in vitro model and in vivo immunosenescence will be documented. The use of culture models to screen for ways of manipulating immunosenescence in vitro may provide a basis for intervention to ameliorate immunosenescence in vivo.

Several studies have documented finite lifespans of at least the vast majority of cultured human T cell lines and clones. However, there is a great deal of variation among the different preparations, ranging from < 25 PD up to > 100 PD. The cultured T cells in all these studies originated from mature T cells isolated from peripheral blood of adult donors. It was, therefore, impossible to assess the contribution of differences in in vivo age to the subsequent differences between clones in in vitro aging. In an attempt to circumvent this difficulty, we have developed a culture system that supports the differentiation of highly purified human CD34+ cells into CD3+ T cells in vitro. This features the use of a serum-free medium supplemented with the cytokines flt-3 ligand, IL 3, stem cell factor (c-kit ligand) and IL 2, together with IL 7 or oncostatin M (OM). In this way it is possible to perform "longitudinal" studies on T cells derived de novo in vitro. We show here that T cell clones derived under these circumstances also manifest variable finite life expectancies, for which the only uncontrolled (nonstochastic) effects of aging must already have occurred at the stem cell level.

The growth characteristics in vitro of interleukin 2 (IL 2)-dependent human CD4+ alpha beta-T cell receptor-positive helper T cell clones (TCC) were studied in relation to alterations in surface phenotype, cytokine responsiveness, and susceptibility to activation-induced cell death (AICD). TCC derived from peripheral blood T cells had finite lifespans averaging 33 population doublings (PD) with a recorded maximum lifespan of 80 PD (n = 208). First analyses of the TCC were undertaken at ca. 25 PD, at which time all cells of all TCC expressed high intensity CD45RO and low intensity CD45RA, as well as high intensity CD95 (fas) and MHC class II antigens. The expression of these molecules remained elevated throughout the proliferative lifespan of the clones, but for those TCC which were initially CD28+ (the majority), the density of expression of the latter was diminished in most late-passage clones. Concomitant with this, late-passage cells showed reduced responsiveness to CD28-mediated costimulation by CHO transfectants expressing human CD80 compared to early-passage cells. Additionally, the level of expression of IL 2R gamma c and IL 7R chains was commonly reduced, as was the response to IL 2 and IL 7. Despite unchanged levels of fas expression on TCC with time, late-passage cells were more susceptible to AICD than early, passage cells. These observations further document functional and phenotypic alterations in long-term cultured human T helper cells, which may be considered as biomarkers of immunosenescence. This may contribute to an improved understanding of the mechanisms underlying depressed T cell function in old age.

CD4+ TCR alpha beta+ T-cell clones were prepared from three CML-patients 4-6 weeks after allogeneic bone marrow transplantation and the effects of theophyllamine and verapamil on clonal growth then assessed. Both drugs inhibited PHA-stimulated autocrine proliferation of clones as well as proliferation of clones dependent on exogenous growth factors. Inhibition was seen when using different accessory cells (PBM or BCL) during T-cell activation, and both for IL2- and IL4-dependent proliferation of previously activated T-cells. The isomer R-verapamil inhibited PHA-stimulated proliferation as well as IL2- and IL4-dependent T-cell proliferation. The drugs also inhibited proliferation of CD8+ T-cells. Although the T-cell clones were functionally heterogeneous and were derived from different patients and priming cultures, both drugs inhibited all clones investigated. However, the degree of inhibition varied between different clones.

Dipyridamole inhibited the proliferation of functionally heterogeneous CD4+ TCR alpha beta+ T-cell clones prepared from CML-patients 4-6 weeks after allogeneic bone marrow transplantation. The effect was seen when testing concentrations corresponding to the therapeutic serum level. Dipyridamole caused a dose-dependent inhibition of PHA-stimulated proliferation both for clones dependent on exogenous IL2 and clones undergoing autocrine proliferation. The inhibition was seen when using different accessory cells (PBM or BCL), and also when dipyridamole was present during IL2- or IL4-dependent proliferation of activated T-cells. The effect of dipyridamole was also investigated for 76 T-cell clones (76 CD4+ and 7 CD8+ clones) prepared by different cloning procedures from three patients. Although these clones were heterogeneous with regard to cytotoxic function, lymphokine production or lymphokine responsiveness, dipyridamole inhibited IL2-dependent proliferation of all clones. In addition dipyridamole inhibited proliferation of CML cells.