Carmen Campos

Immunosenescence of human natural killer cells.

Natural killer (NK) cells are a key component of innate immunity involved not only in the elimination of virus-infected or tumor cells but also in the regulation of the immune response by producing cytokines and chemokines that can activate other cellular components of innate and adaptive immunity. NK cell subsets are differentially affected by aging. Whereas CD56bright cells are decreased in healthy elderly individuals, the CD56dim subset is expanded. The expression of CD57, a marker of highly differentiated NK cells, is increased in the elderly; this supports the notion that a remodeling process of NK cell subsets occurs in aging with a gradual decrease in more immature CD56bright NK cells and an increase in highly differentiated CD56dim CD57+ NK cells. This NK cell redistribution can explain many of the phenotypic and functional changes in NK cells associated with healthy aging such as decreased proliferation and the maintenance of CD16-dependent cytotoxicity.

CMV and Immunosenescence: from basics to clinics

Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates “immunosenescence”. This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop.

Immunosenescence: Implications for response to infection and vaccination in older people

People aged 60 and older represent over 11% of the world population and it is expected to rise 22% by 2050. Population aging is associated to an increased frequency of age-related diseases including higher susceptibility to infections, cancer, cardiovascular and neurodegenerative diseases. Immunosenescence refers to the decline of the immune system associated to aging. It affects both, innate and adaptive immunity limiting the response to pathogens and to vaccines. The analyses of the immune system in elderly individuals determined several immune signatures constituting an immune risk phenotype that predicts mortality. An inverse CD4/CD8 ratio, loss of naïve T cells, increased numbers of terminally-differentiated T cells and oligoclonal expansions of virus-specific T cells constitute hallmarks of immunosenescence. Natural killer (NK) cells are also found severely altered in the elderly. The contribution of latent cytomegalovirus infection to immunosenescence of T and NK cells has been shown. Considering the worldwide ageing of the population in the next decades, the impact of infections will be a real health problem for older individuals requiring preventive strategies. Thus, further studies are required to analyse the bases of immunosenescence and to establish protocols to overcome the age-associated alterations of the immune response in order to define effective vaccines against those pathogens, such as influenza, contributing to increased morbidity and mortality in the elderly.

Shaping of NK cell subsets by aging

NK cells are key players in the innate immune response against virus infection and tumors. Here we describe the current knowledge on age-associated changes in NK cells and the role of persistent CMV infection in configuring NK cell compartment in the elderly. Aging but not CMV causes a redistribution of NK cell subsets as shown by a decrease of CD56bright cells and an increase of CD56-CD16+ NK cells. On the contrary the changes in CD56dimCD16+ NK cells are compatible with the accumulation of CD57+ long-lived NK cells that can also be observed in young CMV-seropositive individuals. NK cell function and dynamics in the elderly will be related not only with age but also with exposure to pathogens, especially CMV.

Cytokine profiles in acute myeloid leukemia patients at diagnosis: survival is inversely correlated with IL-6 and directly correlated with IL-10 levels.

BACKGROUND Several evidences support the existence of cytokine deregulation in acute myeloid leukemia (AML) patients that may be associated with pathogenesis, disease progression and patient survival. METHODS In the present study, we analyzed plasma levels of pro- and anti-inflammatory cytokines in AML patients and age-matched healthy donors. TNF-α, IL-6, IL-1β, IL-2, IFN-γ, IL-17A, IL-12p70, IL-8, IL-10, IL-4 and IL-5 were analyzed using fluorescent bead-based technology and TGF-β by ELISA technique. Because age-associated differences in cytokine profiles have been described, patients and healthy individuals were divided into two age groups: up to 65 years and over 65 years. RESULTS Our results showed that plasma TNF-α, IL-6 and IL-10 levels were higher in AML patients from both groups of age. IL-8 was increased in AML patients less than 65 years while the plasma concentrations of IL-4, IL-5 and IL-12p70 were significantly higher only in elderly AML patients compared with aged-matched healthy controls. Moreover, plasma levels of IL-6 and IL-10 were associated with patient survival and event-free survival. CONCLUSIONS An aberrant production of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 is observed in AML patients. Low levels of IL-6 and high levels of IL-10 represent favorable prognostic factors for survival in AML patients. These results support the idea that cytokine deregulation may be useful as a marker for predicting clinical evolution in AML patients.

CMV Latent Infection Improves CD8+ T Response to SEB Due to Expansion of Polyfunctional CD57+ Cells in Young Individuals

Cytomegalovirus (CMV) latent infection has a deleterious effect on the efficacy of influenza vaccination in the elderly, suggesting that CMV restricts immunological diversity impairing the immune system functionality in old age. Polyfunctional T cells produce multiple cytokines and higher amounts than mono-functional T cells. High number of polyfunctional T cells correlates with better prognosis during infection. Thus, the efficiency of T cell response associates with quality (polyfunctionality) rather than with quantity (percentage of T cells). We analyze the effect of CMV infection on CD8+ T cells polyfunctionality ―degranulation (CD107a), IFN-gamma and TNF-alpha production―, from young CMV-seropositive and CMV-seronegative individuals and in middle age CMV-seropositive donors, in response to Staphylococcal Enterotoxin B (SEB). Our results show a higher percentage of polyfunctional CD8+ T cells in young CMV-seropositive individuals compared to CMV-seronegative. Also, we find an expansion of CD8+CD57+ T cells in CMV-seropositive individuals, which are more polyfunctional than CD8+CD57− cells. In middle age individuals there is a higher frequency of SEB-responding CD8+ T cells, mainly TNF-alpha or TNF-alpha/IFN-gamma producers, whereas the percentage of polyfunctional cells (IFN-gamma/TNF-alpha/CD107a) is similar to the percentages found in young CMV-seropositive. Therefore, whereas it has been shown that CMV latent infection can be detrimental for immune response in old individuals, our results indicate that CMV-seropositivity is associated to higher levels of polyfunctional CD8+ T cells in young and middle age donors. This increase in polyfunctionality, which can provide an immunological advantage in the response to other pathogens, is due to a CD8+CD57+ T cell expansion in CMV-seropositive individuals and it is independent of age. Conversely, age could contribute to the inflammation found in old individuals by increasing the percentage of cells producing pro-inflammatory cytokines. These findings highlight the necessity of further studies on the benefits/detrimental effects of CMV infection in the response to vaccination and other infections.

Effect of age and CMV on NK cell subpopulations

NK cells represent an important component of the innate immune response against infection and tumors. Age-associated changes in NK cell phenotype have been previously reported that can be responsible of functional NK cell deficiency. The aim of this work was to analyze the effect CMV seropositivity and aging on the distribution of NK cell subsets with a focus on the expression of cytotoxicity-related molecules and on the expression of CD94/NKG2 heterodimers and CD57 on these NK cell subsets. Our results show that CMV seropositivity in young individuals does not significantly affect peripheral blood NK cell percentage and NK cell subsets defined by the use of CD56 and CD16 markers. In contrast a significant increase in the percentage of NK cells is observed in elderly donors, all of them are CMV seropositive, when compared with young CMV seropositive subjects. A decrease in the percentage of CD56bright NK cells, either fully immature CD16 negative or CD16+ and an increase in the CD56-CD16+ subset are also found in the elderly. CMV seropositivity either in healthy young or elderly individuals is associated to the expression of CD94/NKG2C dimers and high expression of CD57on the CD56dimCD16+ NK cell subset. CD56-CD16+ NK cells, which are expanded in the elderly, show a decreased expression of granzymes A and B and an increased expression of CD94/NKG2C and CD57 in CMV seropositive young donors when compared with CMV seronegative young individuals. These results indicate that CMV and age have a different effect on NK cell phenotype and emphasize the relevance of including the determination of CMV serostatus in those studies addressed to analyze the immune response in the elderly.

Proinflammatory status influences NK cells subsets in the elderly.

Aging is associated to dysfunctional changes in the immune system, a process termed immunosenescence. Elderly individuals usually present chronic low level inflammation, likely as the consequence of continued exposure to antigens combined with poor immune function, increases in production of pro-inflammatory cytokines by effector memory and senescent T cells and macrophages. This condition not only results from, but also drives immunosenescence. Aging affects all cell components of the immune system, including NK cells and its different subsets (CD56dimCD16+, CD56brightCD16+/- and CD56-CD16+). In particular, the percentage of total NK cells is increased in healthy aging and centenarians, whereas there is a decrease in the CD56bright NK cell subset and an expansion of CD56-CD16+ NK cells. However, the causes of these alterations on NK cells in old donors are not fully understood. In this work we analyse NK cell subsets in the elderly in relation with markers of inflammation and health status. The results show that there is a positive correlation between the number of total NK cells and the body mass index (BMI), while the number of CD56bright NK cells negatively correlates with the levels of C-reactive protein (CRP), supporting that chronic inflammation is involved in the decrease of this NK cell subset.

Natural killer cell immunosenescence in acute myeloid leukaemia patients: new targets for immunotherapeutic strategies?

Several age-associated changes in natural killer (NK) cell phenotype have been reported that contribute to the defective NK cell response observed in elderly patients. A remodelling of the NK cell compartment occurs in the elderly with a reduction in the output of immature CD56bright cells and an accumulation of highly differentiated CD56dim NK cells. Acute myeloid leukaemia (AML) is generally a disease of older adults. NK cells in AML patients show diminished expression of several activating receptors that contribute to impaired NK cell function and, in consequence, to AML blast escape from NK cell immunosurveillance. In AML patients, phenotypic changes in NK cells have been correlated with disease progression and survival. NK cell-based immunotherapy has emerged as a possibility for the treatment of AML patients. The understanding of age-associated alterations in NK cells is therefore necessary to define adequate therapeutic strategies in older AML patients.

Immunosenescence: limitations of natural killer cell-based cancer immunotherapy

Cancer is primarily considered a disease of old age. Immunosenescence refers to the age-associated changes in the immune system, and its contribution to the increased risk of cancer in old individuals has been discussed for many years. Natural killer (NK) cells are cytotoxic innate immune cells specialized in defence against tumour and virus-infected cells. NK cell cytotoxicity is the result of a fine balance between activating and inhibitory receptors. Several activating receptors have been identified that recognize different ligands frequently found over-expressed on tumour cells or virus-infected cells. The most important NK cell inhibitory receptors interact with major histocompatibility complex class I molecules expressed on almost all nucleated cells preventing NK cell-mediated lysis of healthy cells. NK cell immunosenescence is characterized by a redistribution of NK cell subsets, a diminished expression of several activating receptors and lower per-cell cytotoxicity. Altered expression of activating receptors has also been described in young and elderly cancer patients probably due to chronic exposure to ligands on tumour cells. Thus, the effect of both age and cancer may act synergistically to diminish NK cell-mediated tumour immunosurveillance. Different strategies harnessing the power of NK cells to target tumour cells have been designed including adoptive therapy with autologous or allogeneic expanded NK cells. In addition, checkpoint blockade of inhibitory receptors and the use of agonist antibodies to stimulate activating receptors are emerging areas of research. In this context, the effect of immunosenescence should be considered to improve the efficiency of cancer immunotherapy.