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Since associations between lifespan and certain simple immune parameters such as T cell proliferative responses to mitogens were reported in the 1970´s, efforts to dissect out immune parameters correlating with morbidity and mortality have sought to define factors predicting individual longevity. Such "immune signatures" associating with defined clinical outcomes would represent biomarkers of "immunosenescence" that might also provide mechanistic insights into the ageing process. Because appropriate immune function is necessary for a healthy old age, a better understanding of immunosenescence contributing to frailty and death might allow interventions to improve personal and public health. Here, we discuss data from our studies in several different European countries and document significant differences between overtly similar populations. These findings draw attention to the marked variation even between presumably quite homogeneous populations, which may be due to the different birth cohorts studied in addition to numerous other variables. Thus, immunological parameters, and presumably many other factors, are sensitive to context-dependent variation, making it currently difficult to extrapolate biomarkers of longevity from any one human population to another.

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.

Metastatic melanoma is the most dangerous form of skin cancer, with an ever-increasing incidence worldwide. Despite encouraging results with immunotherapeutic approaches, long-term survival is still poor. This is likely partly due to tumour-induced immune suppression mediated by myeloid-derived suppressor cells (MDSCs), which were shown to be associated with response to therapy and survival. Thus, identifying pathways responsible for MDSC differentiation may provide new therapeutic targets and improve efficacy of existing immunotherapies. Therefore, we've analysed mechanisms by which tumour cells contribute to the induction of MDSCs. Established melanoma cell lines were pre-treated with inhibitors of different pathways and tested for their capacity to alleviate T cell suppression via MDSC differentiation in vitro. Targeting HSP70/90 in melanoma cells resulted in reduced induction of immune suppressive cells on a phenotypic and functional basis, for which a more potent effect was observed when HSP90 was inhibited under hypoxic conditions. This initial study suggests a novel mechanism in tumour cells responsible for the induction of MDSC in melanoma.

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.

Since the first immunomodulatory antibody was licensed by the FDA in 2011 for treating melanoma it has remained the case that only a certain proportion of cancer patients respond favourably to a particular therapy. Recent results from combining two or more different antibodies each targeting a different immune checkpoint indicate that the proportion of responding patients can be increased, but thus far there are no such therapies routinely yielding clinical benefit in 100% of patients in any cancer type. Therefore, predicting which patients will respond to a particular therapy remains of the utmost importance in order to maximise treatment efficacy and minimise side-effects and costs. Moreover, determining biomarkers predicting responses may provide insight into the mechanisms responsible for success or failure of that therapy. This article reviews seminal papers mostly from the past two years of progress in this area of intense investigation, and mostly in melanoma, the tumour type for which the largest body of data exists thus far.

Cancer treatment remains unsatisfactory with high rates of recurrence and metastasis. Immunomodulatory agents capable of promoting cellular antitumor immunity while inhibiting the local immunosuppressive tumor microenvironment could greatly improve cancer treatment. We have developed a multi-targeted mannosylated cationic liposome delivery system containing muramyl dipeptide (DS) and low doses of the chemotherapeutic agent cytarabine (Ara-C). Immunomodulation of primary immune cells and immortalized cancer cell lines by Ara-C/DS was assessed by measuring cytokine levels and surface marker expression. As a proof of concept, the generation of targeted cellular immunity was investigated in the context of responses to viral antigens. This report is the first demonstrating that Ara-C combined with DS can modulate immune responses and revert immunosuppression as evidenced by increased IFN-γ and IL-12p40 without changes in IL-10 in peripheral blood mononuclear cells, and increased CD80 and decreased CD163 on immunosuppressive macrophages. Furthermore, Ara-C/DS increased MHC class I expression on cancer cells while increasing the production of antigen-specific IFN-γ+ CD8+ T cells in viral peptide-challenged lymphocytes from both humans and vaccinated mice. Taken together, these results are the first to document immunomodulatory properties of Ara-C linked with recognition of antigens and potentially the generation of antitumor immune memory.

It is widely accepted that the brain and the immune system continuously interact during normal as well as pathological functioning. Human aging is commonly accompanied by low-grade inflammation in both the immune and central nervous systems, thought to contribute to many age-related diseases. This review of the current literature focuses first on the normal neuroimmune interactions occurring in the brain, which promote learning, memory and neuroplasticity. Further, we discuss the protective and dynamic role of barriers to neuroimmune interactions, which have become clearer with the recent discovery of the meningeal lymphatic system. Next, we consider age-related changes of the immune system and possible deleterious influences of immunosenescence and low-grade inflammation (inflammaging) on neurodegenerative processes in the normally aging brain. We survey the major immunomodulators and neuroregulators in the aging brain and their highly tuned dynamic and reciprocal interactions. Finally, we consider our current understanding of how physical activity, as well as a combination of physical and cognitive interventions, may mediate anti-inflammatory effects and thus positively impact brain aging.

International audience; The role of the assessment of peripheral T-cell phenotypes in predicting overall survival (OS) after ipilimumab treatment is unclear. Here, we analysed mononuclear cells in the blood before and at different time points during treatment with ipilimumab in 137 late-stage melanoma patients. The proportions of baseline naïve and memory T-cells were measured by flow cytometry and correlated with OS, with an emphasis on PD-1 expression. High frequencies (>13%) of CD8 effector-memory type 1 (EM1) T-cells at baseline correlated with longer OS (p = 0.029) and higher clinical response rates (p = 0.01). The frequency of these EM1 cells and the M category had independent impacts on OS (hazard ratio = 1.5, p = 0.033; and hazard ratio = 1.9, p = 0.007). In contrast, high baseline frequencies of late stage-differentiated effector memory CD8 cells (>23.8%) were negatively associated with OS (p = 0.034) but did not correlate with clinical response. Following treatment, a decrease of CD8 cells from baseline to the time of the second drug dose and at later time points was strongly and consistently correlated with a high clinical response rate. Our observations thus suggest an important predictive role of baseline CD8 EM1 cells and changes in CD8 cells for clinical response of ipilimumab. Further validation of these biomarker candidates is warranted.

The challenge of distinguishing between changes attributable to ageing and those attributable to pathology is even greater for the immune system than for many other organs, and this is especially true for myeloid-derived suppressor cells (MDSCs). Hematopoiesis is different in older adults with a bias towards myelopoiesis, and older adults also manifest "inflammageing" exacerbated by disease and contributing to MDSC induction. Hence, at least in humans, one can only investigate MDSCs in the context of ageing and disease states, and not in the context of ageing processes per se. This contribution provides a brief overview of the literature on MDSCs and ageing in humans.

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.

Parkinson's disease (PD) is characterised by low-level systemic inflammation, which may be at least partly due to pathophysiological activation of immunity. Here, the frequencies of different types of circulating dendritic cells (DCs) with and without a pro-inflammatory phenotype were determined in PD patients and controls. A high proportion of older people is infected with cytomegalovirus (CMV), which acts as a chronic antigenic stressor that could also contribute to increased inflammation. Following this idea, we found higher frequencies of myeloid DCs with a pro-inflammatory CD16+ILT2(high) phenotype in CMV-positive PD patients than controls, suggesting the potential involvement of CMV in exacerbating PD.

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.

Aging of an individual entails a progressive decline of functional reserves and loss of homeostasis that eventually lead to mortality. This process is highly individualized and is influenced by multiple genetic, epigenetic and environmental factors. This individualization and the diversity of factors influencing aging result in a significant heterogeneity among people with the same chronological age, representing a major challenge in daily oncology practice. Thus, many factors other than mere chronological age will contribute to treatment tolerance and outcome in the older patients with cancer. Clinical/comprehensive geriatric assessment can provide information on the general health status of individuals, but is far from perfect as a prognostic/predictive tool for individual patients. On the other hand, aging can also be assessed in terms of biological changes in certain tissues like the blood compartment which result from adaptive alterations due to past history of exposures, as well as intrinsic aging processes. There are major signs of 'aging' in lymphocytes (e.g. lymphocyte subset distribution, telomere length, p16INK4A expression), and also in (inflammatory) cytokine expression and gene expression patterns. These result from a combination of the above two processes, overlaying genetic predispositions which contribute significantly to the aging phenotype. These potential "aging biomarkers" might provide additional prognostic/predictive information supplementing clinical evaluation. The purpose of the current paper is to describe the most relevant potential "aging biomarkers" (markers that indicate the biological functional age of patients) which focus on the biological background, the (limited) available clinical data, and technical challenges. Despite their great potential interest, there is a need for much more (validated) clinical data before these biomarkers could be used in a routine clinical setting. This manuscript tries to provide a guideline on how these markers can be integrated in future research aimed at providing such data.

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.

Alzheimer's disease (AD) is characterized by extracellular senile plaques in the brain, containing amyloid-β peptide (Aβ). We identify immunological differences between AD patients and age-matched controls greater than those related to age itself. The biggest differences were in the CD4 + rather than the CD8 + T cell compartment resulting in lower proportions of naive cells, more late-differentiated cells and higher percentages of activated CD4 + CD25 + T cells without a Treg phenotype in AD patients. Changes to CD4 + cells might be the result of chronic stimulation by Aβ present in the blood. These findings have implications for diagnosis and understanding the aetiology of the disease.

The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation.

Cytomegalovirus (CMV) has been implicated as a factor in immunosenescence, including poor antibody response to vaccination and higher immune activation and inflammation. Some people may be more or less vulnerable to the negative effects of CMV. The present investigation tested the effects of beta-blocker use and chronological age on the associations between CMV and immunity in adults aged 60–91 (N=98; 69% CMV seropositive) who were administered the trivalent influenza vaccine for up to 5 years. Peak antibody response, corrected for baseline, and spring (persistent) antibody response, corrected for peak, were assessed, as well as beta-2 microglobulin (β2μ) and interleukin-6 (IL-6). In multi-level models with years at Level 1 and people at Level 2, CMV serostatus did not predict peak antibody response, but there was a 3-way interaction between CMV serostatus, age, and beta-blockers. Age was negatively associated with peak antibody, but only among adults who were CMV seropositive and taking beta-blockers. CMV seronegative adults who were not taking beta-blockers had the highest antibody persistence. CMV serostatus was not associated with β2μ or IL-6. Results suggest that CMV+ serostatus may negatively compromise antibody response to a greater degree than inflammatory markers in older adults. Furthermore, older adults who take beta-blockers may be more vulnerable to negative effects of age and CMV on peak antibody response, perhaps by virtue of their underlying health condition.

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.

"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.

Age is a major risk factor for solid cancers, hitherto conventionally viewed in the context of the cell biology of carcinogenesis. However, if cancers are immunogenic, the immune system could protect against tumorigenesis ("immunosurveillance"), and vaccination or other immunomodulatory treatments should be therapeutically beneficial, as has been recently dramatically documented in a fraction of patients with certain tumors responding to immune checkpoint blockade. This begs the question that if immunity decreases with age ("immunosenescence"), this may contribute to increased cancer disease in the elderly and a lower likelihood of clinical benefit from immunotherapy. This brief review weighs up the evidence for and against a negative impact of immunosenescence on cancer control.

The umbrella term 'immunosenescence' is applied to describe age-associated failing systemic immunity and is believed to contribute to the increased incidence and severity of infectious disease in old animals and people. Very limited studies in man have begun to reveal biomarkers of immune ageing ('immune signatures') increasingly recognized as an 'immune risk profile' (IRP) predicting mortality in the elderly. Even more limited studies in companion animals seem consistent with the idea that most or all other mammals may also show an IRP. It is of practical and scientific interest to more accurately determine the IRP and to devise interventions to modulate immune ageing. In man, cytomegalovirus (CMV) infection has an enormous impact on biomarkers associated with immunosenescence; it is not clear whether the same is true for a persistent viral infection in other animals. A significant fraction of the human immune system is committed to controlling CMV; this commitment increases with age and may itself cause pathology as a result of maintaining higher systemic levels of inflammatory mediators. It will be interesting to test whether similar phenomena occur in relatively long-lived animals, often sharing a human environment, like cats and dogs, and whether interventions to restore appropriate immunity in companion animals might also be applicable to people.

Prothymosin alpha (ProTalpha) is a small acidic polypeptide with important immunostimulatory properties, which we have previously shown to be exerted by its carboxyl (C)-terminus. It exerts immunoenhancing effects through stimulation of monocytes via toll-like receptor (TLR) triggering. Here, we assayed the activity of synthetic peptides homologous to ProTalpha's C-terminus to stimulate lymphocyte functions, in particular natural killer cell cytotoxicity of peripheral blood mononuclear cells isolated from healthy donors. A synthetic decapeptide TKKQKTDEDD was identified as the most potent lymphocyte stimulator. The activity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProTalpha's immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProTalpha stimulates immune responses in a monocyte-dependent manner, we further investigated whether the entire molecule and its peptide TKKQKTDEDD specifically act on monocytes and show that both can promote maturation of monocyte-derived dendritic cells (DC). Finally, knowing that, under specific conditions, ProTalpha forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative staining). Although the peptide TKKQKTDEDD adopts an antiparallel beta-sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in conjunction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProTalpha during apoptosis, strengthen our hypothesis that immune response stimulation by ProTalpha is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific beta-sheet conformation and induce DC maturation.

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.

In the present NONA immune longitudinal study, we further examine the previously identified T cell immune risk phenotype (IRP), relative inflammatory activity, morbidity and 2-year mortality in very old individuals >90 years. T-cell subsets as well as the inflammatory markers IL-6, IL-10, C-reactive protein, transthyretin and albumin were evaluated. IRP and low-grade inflammation predicted 57% of observed deaths and 97% of survival over 2 years, and was not significantly affected by individuals' health status, suggesting that the physiological ageing processes of T-cell immunosenescence and low-grade inflammation are of primary importance in late life survival. IRP non-survivors showed only a minor inflammatory activity at baseline, but had in contrast to survivors developed increased activity at follow-up. The results suggest a sequence of stages for IRP individuals that begin with acquisition of CMV infection in earlier life, followed by generation of CD8+CD28- cells to control persistent CMV infection and eventually the development of an IRP. Intriguingly, we also found that rare individuals moved out of the IRP category by a process of immune suppression, including increases in IL-6 and IL-10 and decreases in the number of CD3+CD8+CD28- cells. The further characterisation of these exceptional individuals may allow insight into remedial approaches for those who remain in the IRP category until death.

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.

Herpes viruses are endemic. Once established, the virus is never eliminated but persists throughout life. The fraction of infected individuals gradually increases with age, such that the majority of elderly people are cytomegalovirus (CMV) + , Epstein–Barr virus (EBV) + and Varicella + . Clinically relevant reactivation of Varicella causes painful shingles; CMV reactivation can cause fatal pneumonia. Overt reactivation, even in the very elderly, occurs only in immunocompromised individuals; however, the necessity for maintaining immunity to these viruses is costly. We argue that this cost is not only reflected in the requirement for continuous immunosurveillance against these viruses but, more importantly, results in a re-configuration of T-cell immunity due to the accumulation of dysfunctional virus-specific cells, which fail to be eliminated from the system. Thus, we hypothesize that it is the chronic antigenic stimulation by CMV (and possibly other persisting antigens) that leads to an increasing prevalence of senescent, dysfunctional T cells, and therefore contributes to more general alterations in the immune system, which are associated with earlier mortality.

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 α 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.

Human leukocyte antigen (HLA)-bound peptides are central for recognition of infected/transformed cells by T cells, and have formed the basis for many immunotherapy strategies. Epitopes from a given protein sequence (e.g. from viral proteins or oncoproteins) can be predicted by algorithms, as individual HLA receptors bind peptides through defined binding motifs. Peptides with the highest predicted binding score are then normally tested for their binding ability in binding assays. However, with the assays already established, only one peptide can be tested for binding per assay. This is certainly not a reflection of the in vivo situation, where several peptides generated via the major histocompatability complex (MHC)-class I processing pathway compete for HLA-receptor binding. Here, we describe the development of a method that can mimic the competition between multiple peptides for binding to a single HLA receptor molecule. We used silica nanoparticles with immobilised HLA-A2 complexes to screen HLA-A2 binder-peptides out of a known peptide mixture. The washed beads were analysed for selectively bound peptides by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. The advantage of the system is that the bound peptides can be unambiguously identified without any prior modification (e.g. radioactive or fluorescence labelling), even from complex peptide mixtures.

The aim of this study was to provide a basis for investigating the effects of one very common environmental factor, Epstein-Barr virus (EBV), on age-related changes in the immune system. To this end, the frequency of CD8(+) T cells carrying receptors for an immunodominant EBV lytic epitope was assessed by direct staining with HLA-peptide tetrameric complexes in 19 very old (>87 years) and 12 young (20-40 years) EBV carriers. The frequency of EBV-tetramer-positive cells within the CD8(+) subset was significantly greater in the old compared to the young group (P=0.001). However, the frequency of EBV antigen-specific IFN-gamma producing T cells, as determined by ELISPOT, was significantly lower in the old (P=0.001). Therefore, the absolute number of functional EBV-specific T cells in the elderly and the young was probably similar. These data suggest CD8 clonal expansions in the elderly, resulting in an accumulation of dysfunctional EBV-specific cells which possibly fill the 'immunological space' and could lead to a shrinking of the T cell repertoire for other novel antigens. This may help to explain the increased incidence and case-fatality caused by viruses and intracellular pathogens in the elderly.

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.

Telomerase activity is upregulated after stimulation of human T cells, but as they progress through their finite culture lifespans, this ability is progressively lost. Upregulation of telomerase requires T cell stimulation through the antigen receptor (TCR) and through costimulatory receptors such as CD28. A hypothesis is put forward here that T cell signalling through the TCR is maintained throughout the lifespan of the clones, and that alterations in costimulatory signals are responsible for the progressive loss of telomerase induction. A minimal model of T cell activation during progression through the lifespan is presented in which the TCR provides an unchanging signal 1, but in which there is a progressive decrease in CD28 signalling (signal 2), as well as a decrease in other costimuli, which are here designated as signal 4 (via CD134) and 5 (via CD154). Moreover, in addition, increases in negative costimulation, here designated signal minus 2 (via CD152) and possibly via ICOS (signal 3) may play a part. The balance of these positive and negative signals at each encounter with antigen will determine T cell fate by regulating activation and telomerase induction.

The meeting focussed on age-associated alterations in immunity in vivo and in vitro, but also considered other ageing models and other tissues in an interdisciplinary approach to this problem. Because the ImAginE program has been set up to examine how best to take advantage of in vitro models for the analysis of immunosenescence and how the information acquired can be extrapolated to the in vivo situation, the first part of the meeting dealt with manifestations of immunosenescence in vivo and in vitro. Next, the possible mechanisms underlying these changes were discussed, and how this knowledge might be exploited to prevent such alterations or minimise their detrimental effects. Finally, age-associated changes in immunity in vivo, their physiological impact and their possible remediation were considered.

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.

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 from25 PD up to100 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.

Aged persons and most animals studied show a significant decline in the immune response primarily caused by changes in the T cell compartment. This decline in T cell function is due to a combination of factors: decreased production of new naive T cells by the involuting thymus; extensive antigen-induced activation leading to replicative senescence and clonal exhaustion of certain T cells; and postmitotic aging of resting T cells, a phenomenon also observed in other non-dividing cells such as neurons or muscle cells. The relative importance of these processes in the T cell defective immune response observed in aged humans and animals remains to be established although it is very likely that all of them participate in immunosenescence. The cellular, molecular biological basis and the clinical consequences of the defective immune response in aging were extensively discussed and reviewed at the 5th EUCAMBIS meeting. This paper summarizes the main presentations at the meeting. This special issue contains selected presentations from the congress, in the form of peer-reviewed full papers and the abstracts of two papers previously published in the journal.

Aged persons frequently manifest declining parameters of those immune functions which protect the young against disease. Longitudinal studies are beginning to show that number, type and function of T cells may be associated with longevity, morbidity and mortality in free-living elderly humans. Multi-faceted alterations in the ability of T cells from old donors to respond to stimulation are being dissected, and pathways which are compromized in the elderly compared to the young are being defined. Successful immune responses depend upon waves of rapid and extensive clonal expansion to combat primary infection, followed by death of most T cells, survival of memory cells and their later reactivation and further expansion. This implies that the finite replicative potential of T cells might impose a critical limiting factor on the maintenance of immune responses in the face of thymic involution and drastically reduced capacity to generate new naive T cells. This type of 'clonal exhaustion' can readily be studied in vitro using human T cell clones and the findings can be applied to the in vivo situation. Understanding the processes of replicative senescence in such in vitro models may shed light not only on some of the underlying mechanisms of immunosenescence but also in situations of chronic antigenic stimulation in vivo. Moreover, it might begin to indicate how the system could be manipulated on the one hand to prevent or reverse T cell senescence without nullifying the control mechanisms of tumor suppression, and on the other hand, to reconstitute possibly faulty suppression, for example in autoimmune disease.

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.

Human monoclonal T lymphocyte populations maintained in long-term culture by intermittent reactivation via the antigen receptor and supplied with exogenous interleukin 2 manifest finite proliferative lifespans. T lymphocytes cloned from mature peripheral T cells of adult donors were constantly lost from the time point of their first isolation up to an estimated maximum of 80 population doublings (PD) for the longest lived. T lymphocytes cloned from T cell progenitors in bone marrow, on the other hand, survived for a maximum of ca. 100 PD. One facet of the functional capacity of cells derived from these two different sources was assessed by measuring their autocrine proliferation after mitogenic stimulation. For a majority of T cell clones (TCC), autocrine proliferative capacity decreased as a function of culture age, becoming absent by 50 PD for adult-derived-TCC and by 70 PD for bone marrow-derived TCC, thereby clearly occurring prior to the end of the proliferative life spans of the clones. Limiting dilution frequency analysis showed that the number of autocrine proliferative precursors within these monoclonal populations declined with age, paralleling loss of autocrine proliferative capacity in the 'bulk' clones. Of a variety of surface structures monitored during culture ageing of TCC, the density of expression of the coreceptor molecule CD28 was found to correlate with decreasing autocrine proliferative capacity in two-thirds of the clones. Thus, at least for a fraction of monoclonal human T lymphocytes, decreasing autocrine proliferative capacity, a measure of clonal expansion, may correlate with decreasing numbers of CD28 molecules expressed on the surface and therefore presumably with the strength of costimulatory signal delivered via this important coreceptor.

A condition of hyporesponsiveness can be induced in certain mature human αβ (TCR2) cells relatively easily by their stimulation in the absence of costimulatory signals (signal 1 alone). This state of "anergy" has been implicated in tolerance to self and transplanted organs as well as tumors and may represent an important regulatory component or immune responsiveness. Little is known about whether the same condition applies to γδ (TCR1) cells. We therefore undertook to investigate anergy induction in TCR1 cell clones using several approaches known to induce this state in TCR2 cells. First, TCR1 clones were found not to be anergized by culture on immobilized CD3 monoclonal antibody (mAb), while the majority of TCR2 clones were anergized. Second, blocking of autocrine proliferation (stimulated in TCR1 or TCR2 clones by mitogen in the presence of accessory cells) using CTLA-4-Ig, a soluble B7 family counterreceptor resulted in anergy induction in TCR2 cells but not TCR1 cells, although experiments with CHO cells transfected with B7-1 (CD80) genes confirmed that these TCR1 clones were responsive to costimulation with B7. Third, blocking mitogen-induced proliferation with anti-IL 2 receptor antibodies and anti-IL 2 antisera resulted in anergy induction in TCR2 but not TCR1 cells. Fourth, stimulation with the calcium ionophore ionomycin also anergized TCR2 but not TCR1 cells. In all four systems, but especially in the latter, stimulation by signal 1 alone resulted in high levels of cell death (>50%) which was similar for both TCR1 and TCR2 cells. Therefore, these data may reflect a high level of resistance to tolerance induction (manifested as proliferative anergy) but not to clonal deletion (manifested as stimulation-dependent cell death) on the part of TCR1 cells.

Spontaneous secretion of interleukin 1 (IL-1) alpha, IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) from acute myelogenous leukaemia (AML) blasts showed significant correlation, and detectable levels of all cytokines were seen for a majority of patients. IL-3 and granulocyte/macrophage colony-stimulating factor increased secretion of IL-1 alpha, IL-1 beta and TNF-alpha for a majority of AML patients, whereas IL-4 decreased cytokine secretion. The effect of IL-6 and stem cell factor on cytokine secretion varied between different patients. A wide variation in IL-1 alpha, IL-1 beta and TNF-alpha secretion between different patients was seen both for spontaneous secretion and in the presence of all cytokines.

The 3rd ImAginE Conference on ‘Basic Biology and Clinical Impact of Immunosenescence' was held at Palermo University, Italy from 10–13 April 2002.

Because Cyclosporine A (CsA) is often reported to inhibit only the proliferation of quiescent T-cells and to have much less or no effect on activated proliferating T-cells, the effects of CsA and FK506 on long-term in vitro T-cell proliferation were investigated for alloreactive T-cell clones and clones derived from a leukaemia patient early after allogeneic bone marrow transplantation. Drug effects were tested in the presence of exogenous IL2 or IL2 + IL4 during weekly T-cell activation/restimulation. In the presence of either drug, cloned T-cells were able to respond to a reduced number of restimulations (mitogenic activation, allostimulation) and could undergo fewer cell divisions/population doublings. These effects were seen in the presence of both excess IL2 or IL2 + IL4.

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.