Beatriz Sanchez-Correa

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.

Decreased expression of DNAM-1 on NK cells from acute myeloid leukemia patients.

This study tested the hypothesis that the expression of CD112 and CD155 (DNAM‐1 ligands) on leukemic blasts induces a decreased expression of the activating receptor DNAM‐1 on natural killer (NK) cells from acute myeloid leukemia (AML) patients. DNAM‐1 is a co‐receptor involved in the activation of NK cell cytotoxicity after its interaction with its ligands CD112 and CD155 on target cells. Here we study the expression of DNAM‐1 on NK cells and DNAM‐1 ligands on blasts from AML patients stratified by age. The results demonstrate that NK cells from AML patients younger than 65 years have a reduced expression of DNAM‐1 compared with age‐matched controls. The analysis of DNAM‐1 ligands showed a high expression of CD112 and CD155 on leukemic blasts. An inverse correlation between CD112 expression on leukemic blasts and DNAM‐1 expression on NK cells was found. Furthermore, downregulation of DNAM‐1 was induced on healthy donors’ NK cells after in vitro culture with leukemic blasts expressing DNAM‐1 ligands. In conclusion, these results support the hypothesis that receptor–ligand crosslinking downregulates DNAM‐1 expression on NK cells from patients <65 years of age. Considering the relevance of DNAM‐1 in NK recognition and killing of leukemic cells, the reduced expression of this receptor on NK cells from AML patients can represent an additional mechanism of tumor escape.

Human NK cells in acute myeloid leukaemia patients: analysis of NK cell-activating receptors and their ligands

Natural killer (NK) cell activation is strictly regulated to ensure that healthy cells are preserved, but tumour-transformed or virus-infected cells are recognized and eliminated. To carry out this selective killing, NK cells have an ample repertoire of receptors on their surface. Signalling by inhibitory and activating receptors by interaction with their ligands will determine whether the NK cell becomes activated and kills the target cell. Here, we show reduced expression of NKp46, NKp30, DNAM-1, CD244 and CD94/NKG2C activating receptors on NK cells from acute myeloid leukaemia patients. This reduction may be induced by chronic exposure to their ligands on leukaemic blasts. The analysis of ligands for NK cell-activating receptors showed that leukaemic blasts from the majority of patients express ligands for NK cell-activating receptors. DNAM-1 ligands are frequently expressed on blasts, whereas the expression of the NKG2D ligand MICA/B is found in half of the patients and CD48, a ligand for CD244, in only one-fourth of the patients. The decreased expression of NK cell-activating receptors and/or the heterogeneous expression of ligands for major receptors on leukaemic blasts can lead to an inadequate tumour immunosurveillance by NK cells. A better knowledge of the activating receptor repertoire on NK cells and their putative ligands on blasts together with the possibility to modulate their expression will open new possibilities for the use of NK cells in immunotherapy against leukaemia.

Human Adipose-Derived Stem Cells Impair Natural Killer Cell Function and Exhibit Low Susceptibility to Natural Killer-Mediated Lysis

Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared.

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the “European Searchable Tumour Cell Line and Data Bank” (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.

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.

Human prostasomes express CD48 and interfere with NK cell function.

Prostasomes are small vesicles secreted by the prostate gland to the seminal fluid. In male fertility, the fusion of prostasomes to sperm has been associated with an enhancement of sperm function. Prostasomes also contribute to sperm protection within the female reproductive tract and immunologically relevant proteins have been identified on the prostasomal membrane that may prevent immune-mediated destruction of the sperm.The female genital tract is a potentially hostile environment for spermatozoa and a large number of NK cells have been found in the female reproductive tract. Since it has been suggested that NK cells may represent an important component of innate immunity in the female reproductive tract, we have analyzed the role of prostasomes in the regulation of NK cell activity.Flow cytometric analysis revealed that prostasomes expressed high levels of CD48, the ligand for the activating receptor CD244. The interactions between NK cells and purified prostasomes resulted in a decrease of CD244 expression. Moreover, the decreased NK cell activity observed in NK cells cultured in the presence of prostasomes suggests that prostasomes may immunomodulate the local environment within the female reproductive tract preventing immune-mediated sperm destruction and prolonging their survival rate.