Perforin, CD28 and CD95 expression in circulating CD4 and CD8 cells as predictors of head and neck (H&N) cancer patient survival.

Long-term survival of H&N cancer patients has not improved significantly over the last 30 years. The possibility of using circulating blood cell phenotypes as a prognostic biomarker of H&N cancer patient was investigated in this study. Pre-treatment, circulating T lymphocyte subpopulations as well as the survival time of the patients in question were studied. Upregulated CD4+ perforin+ and CD8+ CD95+ but downregulated CD4+ CD28+ (p < 0.001) were detected in H&N cancer patients. With 3 years of follow-up time, an increase in the frequency of the pre-treatment, circulating CD4+ perforin+ cells and CD8+ perforin+ cells was showed to have reverse effects on the survival time in H&N cancer patients (p < 0.01). Detection of perforin+ frequency in CD4+ and CD8+ lymphocyte by FACS is fast, simple and cost-effective. A potential role of perforin expression in CD4+ and CD8+ cells as a prognostic biomarker for H&N cancer patient in the clinical setting was suggested.

Independent skewing of the T cell and NK cell compartments associated with cytomegalovirus infection suggests division of labor between innate and adaptive immunity.

Cytomegalovirus (CMV) infection induces profound changes in different subsets of the cellular immune system. We have previously identified an immune risk profile (IRP) where CMV-associated changes in the T cell compartment, defined as a CD4/CD8 ratio < 1, are associated with increased mortality in elderly people. Since natural killer (NK) cells have an important role in the defense against viral infections, we examined whether the expansion of CD8 + T cells seen in individuals with CD4/CD8 ratio < 1 is coupled to a parallel skewing of the NK cell compartment. A number of 151 subjects were examined with CMV serology and a flow cytometry panel for assessment of T cell and NK cell subsets. CMV-seropositive individuals had higher frequencies of CD57 + and NKG2C + NK cells and lower frequencies of NKG2A + NK cells, in line with a more differentiated NK cell compartment. Intriguingly, however, there was no correlation between CD4/CD8 ratio and NK cell repertoires among CMV-seropositive donors, despite the profound skewing of the T cell compartment in the group with CD4/CD8 ratio < 1. Conversely, donors with profound expansion of NK cells, defined as NKG2C + NK cells with high expression of CD57 and ILT-2, did not display more common changes in their T cell repertoire, suggesting that NK cell expansion is independent of the T cell-defined IRP. Altogether, these results indicate that the effect of CMV on CD8 T cells and NK cells is largely nonoverlapping and independent.

B-cell diversity decreases in old age and is correlated with poor health status.

Older people suffer from a decline in immune system, which affects their ability to respond to infections and to raise efficient responses to vaccines. Effective and specific antibodies in responses from older individuals are decreased in favour of non-specific antibody production. We investigated the B-cell repertoire in DNA samples from peripheral blood of individuals aged 86-94 years, and a control group aged 19-54 years, using spectratype analysis of the IGHV complementarity determining region (CDR)3. We found that a proportion of older individuals had a dramatic collapse in their B-cell repertoire diversity. Sequencing of polymerase chain reaction products from a selection of samples indicated that this loss of diversity was characterized by clonal expansions of B cells in vivo. Statistical analysis of the spectratypes enabled objective comparisons and showed that loss of diversity correlated very strongly with the general health status of the individuals; a distorted spectratype can be used to predict frailty. Correlations with survival and vitamin B12 status were also seen. We conclude that B-cell diversity can decrease dramatically with age and may have important implications for the immune health of older people. B-cell immune frailty is also a marker of general frailty.

Cytomegalovirus and human immunosenescence.

'Immunosenescence' is an imprecise term used to describe deleterious age-associated changes to immune parameters observed in all mammals studied so far. Primarily anecdotal evidence implies that failing immunity is responsible for the increased incidence and severity of infectious disease in old people. However, there is a serious dearth of accurate hard data concerning the actual cause of death in the elderly and the contribution thereto of the multitude of age-associated alterations measured in the immune system. Cross-sectional studies comparing those currently young with those currently old reveal a large number of differences in the distribution of immune cell types in the blood, and to some extent the functional integrity of those cells. Many of these parameters differ markedly between individuals infected with CMV and uninfected people, regardless of infection with other persistent herpesviruses. The adaptive arm of immunity appears to be more seriously affected than the innate arm, particularly the T lymphocytes. However, cross-sectional studies suffer the disadvantage that like is not being compared with like, because the conditions applied during the entire life course of the currently elderly were different from those applied now to the young. These differences in environment, nutrition, pathology and possibly genetics, rather than merely age, may be expected to influence the parameters studied. Moreover, pathogen exposure of the currently elderly was also different from contemporary exposure, probably including CMV. Some of the problems associated with cross-sectional studies can be overcome by performing longitudinal studies, as pointed out in an earlier analysis of the Baltimore Longitudinal Ageing study looking at lymphocyte numbers. However, longitudinal studies are challenging in humans. Nonetheless, the pioneering Swedish OCTO/NONA studies of the very elderly which for the first time included a range of immune parameters, have identified a set of immune parameters predicting mortality at 2, 4 and 6 year follow-up; CMV infection makes a material contribution to this so-called 'immune risk profile (IRP)'. Whether the IRP is informative in younger individuals and the mechanism of the CMV effect is discussed in this review.

Aging of the immune system as a prognostic factor for human longevity.

Accumulating data are documenting an inverse relationship between immune status, response to vaccination, health, and longevity, suggesting that the immune system becomes less effective with advancing age and that this is clinically relevant. The mechanisms and consequences of age-associated immune alterations, designated immunosenescence, are briefly reviewed here.

The immune risk profile is associated with age and gender: findings from three Swedish population studies of individuals 20-100 years of age.

Earlier we identified an Immune Risk Profile (IRP) of very old individuals, 86-94 years of age, characterised by an inverted CD4/CD8 ratio and associated with persistent cytomegalovirus infection and an increase in the numbers of CD3+CD8+CD28- cells. In the present study we included data from a population-based sample in the age range of 20-79 years to examine the prevalence of individuals with an inverted CD4/CD8 ratio relative to age and gender across the entire adult lifespan. Immunological monitoring that was conducted included analysis of the numbers of T-cells in the subsets CD3+, CD3+CD4+, and CD3+CD8+ as well as CD3+CD8+CD28+, CD3+CD8+CD28-, and CD8+CD45RA+CCR7+. There was found to be a significant lowering of the numbers of CD3+, CD3+CD4+, and CD3+CD8+, and of the CD8+CD45RA+CCR7+ cells across the adult life-span. Notably, the prevalence of individuals with an inverted CD4/CD8 ratio increased from about 8% in the age range of 20-59 years to about 16% in the age range of 60-94 years. The mortality rate in individuals with an inverted CD4/CD8 ratio also increased significantly above the age of 60. Interestingly, the proportion of individuals with an inverted CD4/CD8 ratio was found to be significantly higher in men, whereas the numbers of CD3+CD4+ helper and CD8+CD45RA+CCR7+ naïve cells and the CD4/CD8 ratio were found to be significantly higher in women. These results highlight the importance of functioning of the thymus in the development of IRP and may partly account for the differences between sexes in terms of longevity.

Cytomegalovirus infection: a driving force in human T cell immunosenescence.

The human immune system evolved to defend the organism against pathogens, but is clearly less well able to do so in the elderly, resulting in greater morbidity and mortality due to infectious disease in old people, and higher healthcare costs. Many age-associated immune alterations have been reported over the years, of which probably the changes in T cell immunity, often manifested dramatically as large clonal expansions of cells of limited antigen specificity together with a marked shrinkage of the T cell antigen receptor repertoire, are the most notable. It has recently emerged that the common herpesvirus, cytomegalovirus (CMV), which establishes persistent, life-long infection, usually asymptomatically, may well be the driving force behind clonal expansions and altered phenotypes and functions of CD8 cells seen in most old people. In those few who are not CMV-infected, another even more common herpesvirus, the Epstein-Barr virus, appears to have the same effect. These virus-driven changes are less marked in "successfully aged" centenarians, but most marked in people whom longitudinal studies have shown to be at higher risk of death, that is, those possessing an "immune risk profile" (IRP) characterized by an inverted CD4:8 ratio (caused by the accumulation primarily of CD8(+) CD28(-) cells). These findings support the hypothesis that persistent herpesviruses, especially CMV, act as chronic antigenic stressors and play a major causative role in immunosenescence and associated mortality.

Immunosupportive therapies in aging.

The primary role of the immune system is to protect the organism against pathogens, but age-associated alterations to immunity increase the susceptibility of the elderly to infectious disease. The exact nature of these changes is still controversial, but the use of screening procedures, such as the SENIEUR protocol to exclude underlying illness, helped to better characterize the changes actually related to physiological aging rather than pathology. It is generally agreed that the most marked changes occur in the cellular immune response reflecting profound alterations in T cells. Much of this is due to thymic involution as well as changes in the proportions of T cell subpopulations resulting from antigen exposure, and altered T cell activation pathways. However, a body of data indicates that innate immune responses, including the critical bridge between innate and adaptive immunity, and antigen presenting capacity are not completely resistant to senescence processes. The consequences of all these alterations are an increased incidence of infections, as well as possibly cancers, autoimmune disorders, and chronic inflammatory diseases. The leading question is what, if anything, can we do to prevent these deleterious changes without dangerously dysregulating the precarious balance of productive immunity versus immunopathology? There are many potential new therapeutic means now available to modulate immunosenescence and many others are expected to be available shortly. One main problem in applying these experimental therapies is ethical: there is a common feeling that as ageing is not a disease; the elderly are not sick and therefore do not require adventurous therapies with unpredictable side-effects in mostly frail individuals. Animal models are not helpful in this context. In this chapter we will first briefly review what we think we know about human immunosenescence and its consequences for the health status of elderly individuals. We will then discuss possible interventions that might one day become applicable in an appropriate ethical environment.

No Immune Risk Profile among individuals who reach 100 years of age: findings from the Swedish NONA immune longitudinal study.

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.

Role of persistent CMV infection in configuring T cell immunity in the elderly.

Ageing is associated with declines in many physiological parameters, including multiple immune system functions. The rate of acceleration of the frequency of death due to cardiovascular disease or cancer seems to increase with age from middle age up to around 80 years, plateauing thereafter. Mortality due to infectious disease, however, does not plateau, but continues to accelerate indefinitely. The elderly commonly possess oligoclonal expansions of T cells, especially of CD8 cells, which, surprisingly, are often associated with cytomegalovirus (CMV) seropositivity. This in turn is associated with many of the same phenotypic and functional alterations to T cell immunity that have been suggested as biomarkers of immune system aging. Thus, the manner in which CMV and the host immune system interact is critical in determining the "age" of specific immunity. We may therefore consider immunosenescence in some respects as an infectious state. This implies that interventions aimed at the pathogen may improve the organ system affected. Hence, CMV-directed anti-virals or vaccination may have beneficial effects on immunity in later life.

Effects of simvastatin on human T cells in vivo.

OBJECTIVE: The use of statins has shown several anti-inflammatory actions, including modulatory effects on T cells in vitro. Since the effects on human T cells in vivo are less clarified, our aim was to investigate the effects of simvastatin on human T cells in vivo and ex vivo. METHODS AND RESULTS: A randomized, double-blind, placebo-controlled study design was applied. Eighty volunteers with mild to moderate hypercholesterolemia received either simvastatin 40 mg or placebo for 6 weeks. The serum levels of C-reactive protein (CRP) were significantly reduced by simvastatin. The proportions of CD4+ and CD8+ T cell subsets expressing early (CD25) or late (HLA-DR) activation markers, as assessed by flow cytometry, were not changed by simvastatin. However, simvastatin tended to increase the density of HLA-DR and L-selectin per CD8+ T cell. The T helper(h)1/Th2 response was evaluated by stimulatory assays followed by intra-cellular staining of interferon-gamma and interleukin-4. Simvastatin treatment did not affect the Th1 response but the results indicated a potential to suppress Th2. CONCLUSION: Simvastatin treatment resulted in a few discrete changes as regards peripheral T cells. However, the findings do not provide evidence that simvastatin-induced anti-inflammatory actions are related to any significant modulatory effects on human T cells in clinically healthy men with hypercholesterolemia.

Antinuclear antibodies in the oldest-old women and men.

The aim of this study was to compare the prevalence of antinuclear antibodies (ANA) in very old individuals (>or=86 years of age) with healthy younger (18-68 years) blood donors (n=200) regarding gender, health status, ratio of circulating CD4/CD8 cells and cytomegalovirus (CMV) serology. Frozen plasma was used for ANA detection in two study groups, i.e. 'OCTO' (97 persons aged 86-92 years, 65% women) and 'NONA' (136 persons aged 86-95 years, 70% women). OCTO participants were recruited on the basis that they were healthy or moderately healthy according to a selection protocol. No exclusion criteria regarding health status were applied in the NONA sample. The prevalence of ANA was significantly higher in the oldest-old samples compared to blood donors. There was no association between health status and the presence of ANA in the oldest-old. The difference across age was most pronounced in men, with low levels at younger age, whereas the prevalence among the oldest-old men reached similar levels as in women. There were no associations between the presence of ANA and CD4/CD8 ratio or with CMV status in the oldest-old. Our findings confirm an increased prevalence of ANA in the oldest-old, and emphasize the importance of taking gender and age into consideration when evaluating ANA.

Immunological biomarkers of ageing in man: changes in both innate and adaptive immunity are associated with health and longevity.

Scientific and clinical advances in the last century have led to increased numbers of individuals living to older ages. Thus a major concern is how to live these years with a high quality of life. The ageing immune system is less well able to cope with infectious diseases than the youthful immune system probably as a consequence of altered immune response to pathogens. Thus, both innate and adaptive immune responses show age-related changes that could be decisive for healthy ageing and survival. Longitudinal studies in healthy elderly have allowed the definition of the ''immune risk phenotype" (IRP) a predictor of mortality in elderly individuals that is based on several parameters of the adaptive immune response. Here, we hypothesize that failures in innate immunity observed in frail elderly are related to those alterations described in adaptive immunity defined as the IRP. It will be important to include assays of NK cell markers and functions in future longitudinal studies in order to investigate this point in detail as well as to consider the trace element zinc as an essential co-factor for optimal NK cell activity.

The immune risk phenotype is associated with IL-6 in the terminal decline stage: findings from the Swedish NONA immune longitudinal study of very late life functioning.

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.

Immunorejuvenation in the elderly.

Dysregulated T-cell-mediated immunity contributes materially to the increased susceptibility to infectious disease, and possibly cancer, in the elderly. One hallmark of this state of "immunosenescence" in humans is the predominance of large clones of peripheral T cells with limited antigen receptor heterogeneity and a corresponding contraction of diversity in the T-cell antigen recognition repertoire. Surprisingly, a major driving force for these clonal expansions in humans is cytomegalovirus. The lifelong chronic antigenic stress caused by infection with this persistent activating virus results in clonal exhaustion of specific CD8 T cells, and their acquisition of a state of anergy and apoptosis resistance similar in many respects, and, the authors believe for similar reasons, to that commonly seen in the tumor-specific T cells of cancer patients. This excess of dysfunctional cells is indirectly immunosuppressive by filling the "immunologic space" and shrinking the T-cell repertoire for new antigens, as well as directly suppressive via cytokine secretion. It is associated with the "immunologic risk profile" predicting 2- and 4-year mortality in longitudinal studies of very old people. Therefore, it is hypothesized that deletion of such accumulations of dysfunctional cells would be beneficial to the individual. It may be possible to distinguish functional CMV-specific cells (which are essential to maintain immunosurveillance) from dysfunctional ones by their expression of certain surface molecules. This, coupled with methods directed at reinvigorating the thymus (e.g., use of interleukin 7), and targeting CMV by pharmacologic and immunotherapeutic interventions might result in a degree of "immunorejuvenation" sufficient to take elderly individuals out of the risk category and thereby extend healthy longevity.

Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly.

The age-associated decrease in functionality of the human immune system is thought to have a negative impact on the capacity to provide protection against infection, in turn leading to increased incidence of mortality. In a previous longitudinal study of octogenarians, we identified an immune risk phenotype (IRP) in the very elderly defined by an inverted CD4/CD8 ratio, which was associated with increased mortality and persistent CMV infection. In this study, we analyzed the CD8 clonal composition of nonagenarians and middle-aged individuals. An increased number of CD8 T cell clones was observed in the nonagenarians, and was associated with CMV-seropositivity. Surprisingly, CMV-seropositive nonagenarians with the IRP had a significantly lower number of clones compared with non-IRP individuals. The decrease in clone numbers in IRP individuals was associated with shorter survival time. MHC/peptide multimer staining indicated that the frequency of CMV-specific T cells was higher in nonagenarians than in the middle-aged, but the ratio of functionally intact cells was significantly lower. The lowest ratio of functional CMV-specific T cells was found in an IRP individual. A thorough longitudinal analysis of the CMV-specific T cells in nonagenarians showed a stable pattern with respect to frequency, phenotype, and clonal composition. We hypothesize that the number of different CD8 T cell clonal expansions increases as the individual ages, possibly, as a compensatory mechanism to control latent infections, e.g., CMV, but eventually a point is reached where clonal exhaustion leads to shrinkage of the CD8 clonal repertoire, associated with decreased survival.

Dysregulation of T-cell function in the elderly : scientific basis and clinical implications.

The function of the immune system is to maintain body integrity by defending against infections, cancers, autoimmune diseases and inflammation-related chronic diseases. The immune response is known to become defective with aging, leading to decreased longevity and appearance of age-related disease. The most important changes occur in T-cell immunity, and are manifested particularly as altered clonal expansion of cells of limited antigen specificity. The causes of these alterations are multifactorial, and include thymic involution, T-cell subset changes and signal transduction alterations. The clinical consequences of these changes are not well defined, except for their extremely important negative impact on defence against infections, especially by new pathogens, and decreased responses to vaccination. Considering the public health consequences of decreased immune competence in old age, strategies for immune response modulation are desirable to decrease the health burden for the elderly and improve their quality of life.

An immune risk phenotype, cognitive impairment, and survival in very late life: impact of allostatic load in Swedish octogenarian and nonagenarian humans.

In the previous OCTO longitudinal study, we identified an immune risk phenotype (IRP) of high CD8 and low CD4 numbers and poor proliferative response. We also demonstrated that cognitive impairment constitutes a major predictor of nonsurvival. In the present NONA longitudinal study, we simultaneously examine in a model of allostatic load IRP and compromised cognition in 4-year survival in a population-based sample (n = 138, 86-94 years). Immune system measurements consisted of determinations of T-cell subsets, plasma interleukin 6 and cytomegalovirus and Epstein-Barr virus serology. Interleukin 2 responsiveness to concanavalin A, using data from the previous OCTO (octogenarians) immune study, hereafter OCTO immune, was also examined. Cognitive status was rated using a battery of neuropsychological tests. Logistic regression indicated that the IRP and cognitive impairment together predicted 58% of observed deaths. IRP was associated with late differentiated CD8+CD28-CD27- cells (p < .001), decreased interleukin 2 responsiveness (p < .05) and persistent viral infection (p < .01). Cognitive impairment was associated with increased plasma interleukin 6 (p < .001). IRP individuals with cognitive impairment were all deceased at the follow-up, indicating an allostatic overload.

Human immunosenescence: is it infectious?

Morbidity and mortality due to infectious disease is greater in the elderly than in the young, at least partly because of age-associated decreased immune competence, which renders individuals more susceptible to pathogens. This susceptibility is particularly evident for novel infectious agents such as in severe acute respiratory syndrome but is also all too apparent for common pathogens such as influenza. Many years ago, it was noted that the elderly possessed oligoclonal expansions of T cells, especially of CD8(+) cells. At the same time, it was established that cytomegalovirus (CMV) seropositivity was associated with many of the same phenotypic and functional alterations to T-cell immunity that were being reported as biomarkers associated with aging. It was discovered that CMV was the prime driving force behind most of the oligoclonal expansions and altered phenotypes and functions of CD8 cells. Independently, longitudinal studies of a free-living population of the very old in Sweden over the past decade have led to the emerging concept of an 'immune risk phenotype' (IRP), predicting mortality, which was itself found to be associated with CMV seropositivity. These findings support our hypothesis that the manner in which CMV and the host immune system interact is critical in determining the IRP and hence is predictive of mortality. In this sense, then, we suggest that immunosenescence is contagious.