Marco Antonio Moro-García

Relationship between functional ability in older people, immune system status, and intensity of response to CMV.

Shorter survival in the elderly has been associated with deterioration of the immune system and also with functional disability. To analyze the relationship between functional and immune impairment in older individuals, we studied 100 elderly who lived in a nursing home, were age matched, and grouped according to their functional status. We characterized cell subpopulations by flow cytometry, quantified TREC by RT–PCR, and measured the T-cell proliferation and activation response (IFN-γ by ELISPOT, CD69) against anti-CD3 and CMV. Specific antibody titers against influenza virus and CMV were determined by ELISA. Individuals with worse functional status had significantly higher levels of NK cells and fewer B cells. These poorly functioning elders also had a significantly lower proportion of CD4+ T cells, increased CD8+ T cells, and a decreased CD4/CD8 ratio. TREC levels in CD4+ T cells were significantly lower in individuals with a high disability. Lower TREC levels correlated with a lower frequency of naïve T-cell subpopulations (CD45RA+CCR7+) and higher percentages of effector cells (CD45RA−CCR7−). The functionally impaired group had lower anti-CD3 responses, but gradually increased responses against CMV. Similarly, the higher CMV titers were found in elderly with worse functional status. On the contrary, the functional response in vivo, and the titer of antibodies generated after vaccination against influenza virus, was higher in individuals with better performance status. In summary, we concluded that the functional decline of elderly individuals was clearly associated with the aging of their immune system, and the intensity of the response to CMV.

When Aging Reaches CD4+ T-Cells: Phenotypic and Functional Changes.

Beyond midlife, the immune system shows aging features and its defensive capability becomes impaired, by a process known as immunosenescence that involves many changes in the innate and adaptive responses. Innate immunity seems to be better preserved globally, while the adaptive immune response exhibits profound age-dependent modifications. Elderly people display a decline in numbers of naïve T-cells in peripheral blood and lymphoid tissues, while, in contrast, their proportion of highly differentiated effector and memory T-cells, such as the CD28null T-cells, increases markedly. Naïve and memory CD4+ T-cells constitute a highly dynamic system with constant homeostatic and antigen-driven proliferation, influx, and loss of T-cells. Thymic activity dwindles with age and essentially ceases in the later decades of life, severely constraining the generation of new T-cells. Homeostatic control mechanisms are very effective at maintaining a large and diverse subset of naïve CD4+ T-cells throughout life, but although later than in CD8 + T-cell compartment, these mechanisms ultimately fail with age.

Molecular Mechanisms Involved in the Aging of the T-cell Immune Response

T-lymphocytes play a central role in the effector and regulatory mechanisms of the adaptive immune response. Upon exiting the thymus they begin to undergo a series of phenotypic and functional changes that continue throughout the lifetime and being most pronounced in the elderly. The reason postulated for this is that the dynamic processes of repeated interaction with cognate antigens lead to multiple division cycles involving a high degree of cell differentiation, senescence, restriction of the T-cell receptor (TCR) repertoire, and cell cycle arrest. This cell cycle arrest is associated with the loss of telomere sequences from the ends of chromosomes. Telomere length is reduced at each cell cycle, and critically short telomeres recruit components of the DNA repair machinery and trigger replicative senescence or apoptosis. Repetitively stimulated T-cells become refractory to telomerase induction, suffer telomere erosion and enter replicative senescence. The latter is characterized by the accumulation of highly differentiated T-cells with new acquired functional capabilities, which can be caused by aberrant expression of genes normally suppressed by epigenetic mechanisms in CD4+ or CD8+ T-cells. Age-dependent demethylation and overexpression of genes normally suppressed by DNA methylation have been demonstrated in senescent subsets of T-lymphocytes. Thus, T-cells, principally CD4+CD28null T-cells, aberrantly express genes, including those of the KIR gene family and cytotoxic proteins such as perforin, and overexpress CD70, IFN-γ, LFA-1 and others. In summary, owing to a lifetime of exposure to and proliferation against a variety of pathogens, highly differentiated T-cells suffer molecular modifications that alter their cellular homeostasis mechanisms.

NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

Human aging is characterized by changes in the immune system which have a profound impact on the T-cell compartment. These changes are more frequently found in CD8+ T cells, and there are not well-defined markers of differentiation in the CD4+ subset. Typical features of cell immunosenescence are characteristics of pathologies in which the aberrant expression of NKG2D in CD4+ T cells has been described. To evaluate a possible age-related expression of NKG2D in CD4+ T cells, we compared their percentage in peripheral blood from 100 elderly and 50 young adults. The median percentage of CD4+ NKG2D+ in elders was 5.3% (interquartile range (IR): 8.74%) versus 1.4% (IR: 1.7%) in young subjects (p < 0.3 × 10−10). CD28 expression distinguished two subsets of CD4+ NKG2D+ cells with distinct functional properties and differentiation status. CD28+ cells showed an immature phenotype associated with high frequencies of CD45RA and CD31. However, most of the NKG2D+ cells belonged to the CD28null compartment and shared their phenotypical properties. NKG2D+ cells represented a more advanced stage of maturation and exhibited greater response to CMV (5.3 ± 3.1% versus 3.4 ± 2%, p = 0.037), higher production of IFN-γ (40.56 ± 13.7% versus 24 ± 8.8%, p = 0.015), lower activation threshold and reduced TREC content. Moreover, the frequency of the CD4+ NKG2D+ subset was clearly related to the status of the T cells. Higher frequencies of the NKG2D+ subset were accompanied with a gradual decrease of NAIVE and central memory cells, but also with a higher level of more differentiated subsets of CD4+ T cells. In conclusion, CD4+ NKG2D+ represent a subset of highly differentiated T cells which characterizes the senescence of the immune system.

IL-15 preferentially enhances functional properties and antigen-specific responses of CD4+CD28null compared to CD4+CD28+ T cells

One of the most prominent changes during T‐cell aging in humans is the accumulation of CD28null T cells, mainly CD8+ and also CD4+ T cells. Enhancing the functional properties of these cells may be important as they provide an antigen‐specific defense against chronic infections. Recent studies have shown that IL‐15 does in fact play an appreciable role in CD4 memory T cells under physiological conditions. We found that treatment with IL‐15 increased the frequency of elderly CD4+CD28null T cells by the preferential proliferation of these cells compared to CD4+CD28+ T cells. IL‐15 induced an activated phenotype in CD4+CD28null T cells. Although the surface expression of IL‐15R α‐chain was not increased, the transcription factor STAT‐5 was preferentially activated. IL‐15 augmented the cytotoxic properties of CD4+CD28null T cells by increasing both the mRNA transcription and storage of granzyme B and perforin for the cytolytic effector functions. Moreover, pretreatment of CD4+CD28null T cells with IL‐15 displayed a synergistic effect on the IFN‐γ production in CMV‐specific responses, which was not observed in CD4+CD28+ T cells. IL‐15 could play a role enhancing the effector response of CD4+CD28null T cells against their specific chronic antigens.

Immunosenescence and inflammation characterize chronic heart failure patients with more advanced disease

BACKGROUND Chronic heart failure (CHF) is characterized by an inflammatory status with high levels of cytokines such as IL-6. We hypothesized that patients with CHF may develop immunosenescence due to inflammation and that this may be associated with a worse stage of the disease. METHODS AND RESULTS We compared the immunological features of 58 elderly CHF patients (ECHF), 40 young CHF patients (YCHF), 60 healthy elderly controls (HEC) and 40 healthy young controls (HYC). We characterized leukocyte and lymphocyte subpopulations by flow cytometry, and IL-6 concentration by ELISA. The extent of CHF was classified according to functional and/or morphological criteria: New York Heart Association functional class, AHA/ACC heart failure stages, left ventricular ejection fraction, and left ventricular hypertrophy. CHF patients showed an increased number of leukocytes, neutrophils and monocytes, but a decreased number of lymphocytes. CHF patients had significantly lower levels of B-cells and CD4+ T-cells, increased NK-cells in YCHF, and increased CD8+ T-cells only in ECHF. CHF was associated with high differentiation in CD4+ and CD8+ T-lymphocyte subsets. Aging of T-lymphocyte subpopulations and high IL-6 levels were associated with a worse clinical status. IL-6 also correlated positively with the number of highly differentiated T-lymphocytes and with their accelerated aging. CONCLUSIONS We conclude that CHF patients show a higher degree of immunosenescence than age-matched healthy controls. T-lymphocyte differentiation and IL-6 levels are increased in patients with an advanced clinical status and may contribute to disease impairment through a compromised adaptive immune response due to accelerated aging of their immune system.

Frequent participation in high volume exercise throughout life is associated with a more differentiated adaptive immune response.

Exercise induces changes in the immune system depending on its intensity and duration. For example, transient states of immunodepression can be induced after acute intense physical activity whereas beneficial anti-inflammatory effects of moderate chronic exercise on many diseases and longevity have been described. To study the impact of high volume exercise over a lifetime on aspects of immunity we compared immunological features of 27 young and 12 elderly athletes with 30 young and 26 elderly non-athletes stratified by their CMV serostatus. We characterized blood leukocyte and lymphocyte subpopulations by flow cytometry, quantified TREC content, and measured activation and proliferation ability of T-lymphocytes in the presence of anti-CD3. NK-cells functionality was determined in response to K-562, 721.221 and 721.221-AEH cell-lines. High volume physical activity reduced the total number of circulating leukocytes, neutrophils, and lymphocytes. In the lymphocyte compartment, athletes had higher frequencies of NK-cells and CD8+ T-lymphocytes, whereas CD4+ T-lymphocytes were present at significantly lower levels in CMV-seropositive athletes. We found, in the high volume physical activity individuals, a higher degree of differentiation in CD4+ T-lymphocytes. CD8+ T-lymphocytes from young athletes had reduced TREC content and lower frequencies of recent thymic emigrants. Furthermore, the functional ability of CD4+ and CD8+ T-lymphocytes was significantly impaired in young but not in elderly athletes, and may be compensated for significantly higher activation and degranulation of NK-cells. In conclusion, high volume exercise throughout life appears to be associated with increased levels of biomarkers that are associated with an aging immune system, which are partially reduced with physiological aging.

A predictive model of treatment outcome in patients with chronic HCV infection using IL28B and PD-1 genotyping

BACKGROUND & AIMS The advent of new chronic hepatitis C virus (HCV) therapies requires characterization of patients in order to predict adequate treatment. A good candidate marker is Programmed Cell Death-1 (PD-1) which is involved in progression of HCV infection. The aim of this study was to analyse the effect of several single nucleotide polymorphisms of PD-1 gene and several previously associated factors (IL28B and KIR receptors) on treatment responses. METHODS 407 HCV chronic infected patients treated with PEG-IFN-α and ribavirin were recruited and classified according to their response to treatment. They were genotyped for PD-1 and IL28B polymorphisms, killer immunoglobulin-like receptors (KIR) and HLA genes. A multivariate logistic regression analysis and a Chi-squared Automatic Interaction Detector (CHAID) prediction model of response included these and other clinical parameters. RESULTS Our results showed that PD-1.3/A allele was significantly associated with sustained virological response (SVR) in a multivariate logistic regression analysis (p<0.01, OR=2.57). Additionally, IL28B C/C genotype was the most significant predictor of an SVR to treatment in all HCV genotypes (74.5%). In IL28B C/C patients, the presence of PD-1.3/A allele increased the probability of an SVR to 93.3%. Moreover, when this analysis was made only with patients infected by HCV-1, the predictive value of IL28B C/C genotype with PD-1.3/A allele was 90%. CONCLUSIONS PD-1.3/A allele is associated with SVR to treatment and notably increases the predictive value of IL28B C/C genotype. Both markers in conjunction could be a useful tool, more relevant than HCV genotype in some cases, in clinical practice.

Significant association of the KIR2DL3/HLA-C1 genotype with susceptibility to Crohn's disease.

We aimed to analyze the possible association of KIR/HLA-C genotypes with the susceptibility to Crohns disease (CD) in a Spanish population. A total of 125 patients with CD and 339 healthy controls were selected for this study. KIR and HLA-C typing were developed by sequence-specific oligonucleotide probing. We found that the centromeric A/A genotype and HLA-C1 combination was significantly increased in CD patients (P<10(-3)). The KIR2DL3/2DL3 genotype was significantly increased in CD patients (P<0.0005). Moreover, we also observed a highly significant increase of KIR2DL3-HLA-C1 homozygosis in CD patients (P<0.0005). Our results confirm the relevance of the KIR2DL2/KIR2DL3 genes and their interaction with HLA-C to CD. We show that the contribution of the KIR genes to CD susceptibility extends beyond the association with individual KIRs, with an imbalance between activating and inhibitory KIR genes seeming to influence the susceptibility to CD.

CD4+CD28null T lymphocytes resemble CD8+CD28null T lymphocytes in their responses to IL-15 and IL-21 in HIV-infected patients

HIV‐infected individuals suffer from accelerated immunologic aging. One of the most prominent changes during T lymphocyte aging is the accumulation of CD28null T lymphocytes, mainly CD8+ but also CD4+ T lymphocytes. Enhancing the functional properties of these cells may be important because they provide antigen‐specific defense against chronic infections. The objective of this study was to compare the responses of CD4+CD28null and CD8+CD28null T lymphocytes from HIV‐infected patients to the immunomodulatory effects of cytokines IL‐15 and IL‐21. We quantified the frequencies of CD4+CD28null and CD8+CD28null T lymphocytes in peripheral blood from 110 consecutive, HIV‐infected patients and 25 healthy controls. Patients showed increased frequencies of CD4+CD28null and CD8+CD28null. Both subsets were positively correlated to each other and showed an inverse correlation with the absolute counts of CD4+ T lymphocytes. Higher frequencies of HIV‐specific and CMV‐specific cells were found in CD28null than in CD28+ T lymphocytes. Activation of STAT5 by IL‐15 and STAT3 by IL‐21 was higher in CD28null compared with CD28+ T lymphocytes. Proliferation, expression of CD69, and IFN‐γ production in CD28null T lymphocytes were increased after treatment with IL‐15, and IL‐21 potentiated most of those effects. Nevertheless, IL‐21 alone reduced IFN‐γ production in response to anti‐CD3 stimulation but increased CD28 expression, even counteracting the inhibitory effect of IL‐15. Intracytoplasmic stores of granzyme B and perforin were increased by IL‐15, whereas IL‐21 and simultaneous treatment with the 2 cytokines also significantly enhanced degranulation in CD4+CD28null and CD8+CD28null T lymphocytes. IL‐15 and IL‐21 could have a role in enhancing the effector response of CD28null T lymphocytes against their specific chronic antigens in HIV‐infected patients.