[Improved immunity and mean lifespan in mature mice genetically deficient in catecholamine synthesis after living with wild type for two months]. / Mejoría de la inmunidad y la esperanza de vida en ratones maduros, genéticamente deficientes en la síntesis de catecolaminas, tras convivir con controles durante dos meses.

INTRODUCTION: Mice hemizygous in tyrosine hydroxylase (TH-HZ), the limiting enzyme in catecholamine synthesis, show premature immunosenescence, which in females is associated with a shorter lifespan than the corresponding controls (WT). The coexistence of TH-Hz with WT improves the immune function in both males and females in adulthood. OBJECTIVE: To test whether cohabitation for two months of mature male TH-HZ with WT improves the immune function of the former and whether this impacts the lifespan. MATERIAL AND METHODS: Mature male ICR-CD1 mice (13 ± 1 months) TH-HZ coexisted with WT (2:4 ratio in each cage) for two months. Peritoneal leukocytes were extracted from all animals at baseline, one month, and two months after cohabitation, and macrophage phagocytic capacity, macrophage and lymphocyte chemotaxis, natural killer (NK) antitumor activity, and lymphoproliferative capacity in response to the mitogens concanavalin A and lipopolysaccharide (LPS) were assessed. The animals were maintained under these conditions until their natural death. RESULTS: The TH-HZ, which start, in general, with lower values than the WT in the immune functions studied, improved them after two months of cohabitation, becoming similar to those of the controls. This improvement was already observed in NK activity after one month of cohabitation. The TH-HZ presented lower mean longevity than WT, but when they cohabited with WT, it was similar to the latter. CONCLUSION: The coexistence of TH-HZ male mice with WT mice for two months at mature age improves these genetically modified animals' immune response and longevity.

An Immunosenescence-Related Gene Signature to Evaluate the Prognosis, Immunotherapeutic Response, and Cisplatin Sensitivity of Bladder Cancer.

Immunosenescence refers to the immune system undergoing a series of degenerative changes with advancing age and is tightly associated with the initiation and progression of cancers. However, the immunosenescence-related genes as critical biomarkers for bladder cancer (BLCA) have not been systematically analyzed. We retrieved the immunosenescence-related genes from the public database and verified their association with hallmarks of immunosenescence based on The Cancer Genome Atlas (TCGA) cohort. Through gene pairing, Lasso, and univariate Cox regression, an 8-gene pair model was constructed to evaluate the overall survival of BLCA, which was then validated in the training cohort (P < 0.001, n = 396), two external validation cohorts (P < 0.05, n = 165; P < 0.001, n = 224), and local samples (P < 0.05, n = 10). We also downloaded the clinical information and gene expression matrices of other 32 different cancers from TCGA. The established model showed significant predictive value for the prognosis in 15 cancers (P < 0.05). The risk model could also serve as a promising predictor for immunotherapeutic response, which has been verified by the TIDE algorithm (P < 0.05), IMvigor210 dataset (P < 0.01, n = 298), and other two datasets correlated with immunotherapy (P < 0.05, n = 56; P = 0.17, n = 27). The TCGA dataset, in vitro cell experiments, and pan-cancer analysis displayed that the gene signature was associated with cisplatin sensitivity (P < 0.05). Overall, we proposed a novel immunosenescence-related gene signature to predict prognosis, immunotherapeutic response, and cisplatin sensitivity of BLCA, which were validated in different independent cohorts, local samples, and pan-cancer analyses.

Epigenetic networks driving T cell identity and plasticity during immunosenescence.

The aging process is associated with the accumulation of epigenetic alterations in immune cells, although the origin of these changes is not clear. Understanding this epigenetic drift in the immune system can provide essential information about the progression of the aging process and the immune history of each individual.

Unlocking the Role of Exercise on CD4+ T Cell Plasticity.

A hallmark of T cell ageing is a loss of effector plasticity. Exercise delays T cell ageing, yet the mechanisms driving the effects of exercise on T cell biology are not well elucidated. T cell plasticity is closely linked with metabolism, and consequently sensitive to metabolic changes induced by exercise. Mitochondrial function is essential for providing the intermediate metabolites necessary to generate and modify epigenetic marks in the nucleus, thus metabolic activity and epigenetic mechanisms are intertwined. In this perspective we propose a role for exercise in CD4+ T cell plasticity, exploring links between exercise, metabolism and epigenetic reprogramming.

Naïve Regulatory T Cell Subset Is Altered in X-Linked Agammaglobulinemia.

The interplay between T- and B-cell compartments during naïve, effector and memory T cell maturation is critical for a balanced immune response. Primary B-cell immunodeficiency arising from X-linked agammaglobulinemia (XLA) offers a model to explore B cell impact on T cell subsets, starting from the thymic selection. Here we investigated characteristics of naïve and effector T cell subsets in XLA patients, revealing prominent alterations in the corresponding T-cell receptor (TCR) repertoires. We observed immunosenescence in terms of decreased diversity of naïve CD4+ and CD8+ TCR repertoires in XLA donors. The most substantial alterations were found within naïve CD4+ subsets, and we have investigated these in greater detail. In particular, increased clonality and convergence, along with shorter CDR3 regions, suggested narrower focused antigen-specific maturation of thymus-derived naïve Treg (CD4+CD45RA+CD27+CD25+) in the absence of B cells - normally presenting diverse self and commensal antigens. The naïve Treg proportion among naïve CD4 T cells was decreased in XLA patients, supporting the concept of impaired thymic naïve Treg selection. Furthermore, the naïve Treg subset showed prominent differences at the transcriptome level, including increased expression of genes specific for antigen-presenting and myeloid cells. Altogether, our findings suggest active B cell involvement in CD4 T cell subsets maturation, including B cell-dependent expansion of the naïve Treg TCR repertoire that enables better control of self-reactive T cells.

Immunosuppressive network promotes immunosenescence associated with aging and chronic inflammatory conditions.

The functional competence of the immune system gradually declines with aging, a process called immunosenescence. The age-related remodelling of the immune system affects both adaptive and innate immunity. In particular, a chronic low-grade inflammation, termed inflammaging, is associated with the aging process. Immunosenescence not only is present in inflammaging state, but it also occurs in several pathological conditions in conjunction with chronic inflammation. It is known that persistent inflammation stimulates a counteracting compensatory immunosuppression intended to protect host tissues. Inflammatory mediators enhance myelopoiesis and induce the generation of immature myeloid-derived suppressor cells (MDSC) which in mutual cooperation stimulates the immunosuppressive network. Immunosuppressive cells, especially MDSCs, regulatory T cells (Treg), and M2 macrophages produce immunosuppressive factors, e.g., TGF-ß, IL-10, ROS, arginase-1 (ARG1), and indoleamine 2,3-dioxygenase (IDO), which suppress the functions of CD4/CD8T and B cells as well as macrophages, natural killer (NK) cells, and dendritic cells. The immunosuppressive armament (i) inhibits the development and proliferation of immune cells, (ii) decreases the cytotoxic activity of CD8T and NK cells, (iii) prevents antigen presentation and antibody production, and (iv) suppresses responsiveness to inflammatory mediators. These phenotypes are the hallmarks of immunosenescence. Immunosuppressive factors are able to control the chromatin landscape, and thus, it seems that the immunosenescence state is epigenetically regulated.

Transcriptomic landscape of skin lesions in cutaneous leishmaniasis reveals a strong CD8+ T cell immunosenescence signature linked to immunopathology.

The severity of lesions that develop in patients infected by Leishmania braziliensis is mainly associated with a highly cytotoxic and inflammatory cutaneous environment. Recently, we demonstrated that senescent T and NK cells play a role in the establishment and maintenance of this tissue inflammation. Here, we extended those findings using transcriptomic analyses that demonstrate a strong co-induction of senescence and pro-inflammatory gene signatures in cutaneous leishmaniasis (CL) lesions. The senescence-associated signature was characterized by marked expression of key genes such as ATM, Sestrin 2, p16, p21 and p38. The cell type identification from deconvolution of bulk sequencing data showed that the senescence signature was linked with CD8+ effector memory and TEMRA subsets and also senescent NK cells. A key observation was that the senescence markers in the skin lesions are age-independent of patients and were correlated with lesion size. Moreover, a striking expression of the senescence-associated secretory phenotype (SASP), pro-inflammatory cytokine and chemokines genes was found within lesions that were most strongly associated with the senescent CD8 TEMRA subset. Collectively, our results confirm that there is a senescence transcriptomic signature in CL lesions and supports the hypothesis that lesional senescent cells have a major role in mediating immunopathology of the disease.

Potential of Mesenchymal Stem Cells in the Rejuvenation of the Aging Immune System.

Rapid growth of the geriatric population has been made possible with advancements in pharmaceutical and health sciences. Hence, age-associated diseases are becoming more common. Aging encompasses deterioration of the immune system, known as immunosenescence. Dysregulation of the immune cell production, differentiation, and functioning lead to a chronic subclinical inflammatory state termed inflammaging. The hallmarks of the aging immune system are decreased naïve cells, increased memory cells, and increased serum levels of pro-inflammatory cytokines. Mesenchymal stem cell (MSC) transplantation is a promising solution to halt immunosenescence as the cells have excellent immunomodulatory functions and low immunogenicity. This review compiles the present knowledge of the causes and changes of the aging immune system and the potential of MSC transplantation as a regenerative therapy for immunosenescence.

Premature Senescence and Increased Oxidative Stress in the Thymus of Down Syndrome Patients.

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues. Based on previous studies suggesting immune senescence in DS, we hypothesized that induction of cellular senescence may contribute to early thymic involution and immune dysregulation. Immunohistochemical analysis of thymic tissue showed signs of accelerated thymic aging in DS patients, normally seen in older healthy subjects. Moreover, our whole transcriptomic analysis on human Epcam-enriched thymic epithelial cells (hTEC), isolated from three DS children, which revealed disease-specific transcriptomic alterations. Gene set enrichment analysis (GSEA) of DS TEC revealed an enrichment in genes involved in cellular response to stress, epigenetic histone DNA modifications and senescence. Analysis of senescent markers and oxidative stress in hTEC and thymocytes confirmed these findings. We detected senescence features in DS TEC, thymocytes and peripheral T cells, such as increased ß-galactosidase activity, increased levels of the cell cycle inhibitor p16, telomere length and integrity markers and increased levels of reactive oxygen species (ROS), all factors contributing to cellular damage. In conclusion, our findings support the key role of cellular senescence in the pathogenesis of immune defect in DS while adding new players, such as epigenetic regulation and increased oxidative stress, to the pathogenesis of immune dysregulation.

Methods for Characterization of Senescent Circulating and Tumor-Infiltrating T-Cells: An Overview from Multicolor Flow Cytometry to Single-Cell RNA Sequencing.

Immunosenescence is the general term used to describe the aging-associated decline of immunological function that explains the higher susceptibility to infectious diseases and cancer, increased autoimmunity, or the reduced effectiveness of vaccinations. Senescence of CD8+ T-cells has been described in all these conditions.The most important classical markers of T senescent cells are the cell cycle inhibitors p16ink4a, p21, and p53, together with positivity for SA-ßgal expression and the acquirement of a peculiar IFNγ -based secretory phenotype commonly defined SASP (Senescence Associated Secretory Phenotype). Other surface markers are the CD28 and CD27 loss together with gain of expression of CD45RA, CD57, TIGIT, and/or KLRG1. However, this characterization could not be sufficient to distinguish from truly senescent cells and exhausted T-cells. Furthermore, more complexity is added by the wide heterogeneity of T-cells subset in aged individuals or in the tumor microenvironment. A combined analysis by multicolor flow cytometry for surface and intracellular markers integrated with gene-expression arrays and single-cell RNA sequencing is required to develop effective interventions for therapeutic modulation of specific T-cell subsets. The RNASeq offers the great possibility to reveal at single-cell resolution the exact molecular hallmarks of senescent CD8+ T-cells without the limitations of bulk analysis. Furthermore, the comprehensive integration of multidimensional approaches (genomics, epigenomics, proteomics, metabolomics) will increase our global understanding of how immunosenescence of T-cells is interlinked to human aging.

Obesity Increases Gene Expression of Markers Associated With Immunosenescence in Obese Middle-Aged Individuals.

Recently, it has been argued that obesity leads to a chronic pro-inflammatory state that can accelerate immunosenescence, predisposing to the early acquisition of an immune risk profile and health problems related to immunity in adulthood. In this sense, the present study aimed to verify, in circulating leukocytes, the gene expression of markers related to early immunosenescence associated with obesity and its possible relationships with the physical fitness in obese adults with type 2 diabetes or without associated comorbidities. The sample consisted of middle-aged obese individuals (body mass index (BMI) between 30-35 kg/m²) with type 2 diabetes mellitus (OBD; n = 17) or without associated comorbidity (OB; n = 18), and a control group of eutrophic healthy individuals (BMI: 20 - 25 kg/m²) of same ages (E; n = 18). All groups (OBD, OB and E) performed the functional analyses [muscle strength (1RM) and cardiorespiratory fitness (VO2max)], anthropometry, body composition (Air Displacement Plethysmograph), blood collections for biochemical (anti-CMV) and molecular (gene expression of leptin, IL-2, IL-4, IL-6, IL-10, TNF-α, PD-1, P16ink4a, CCR7, CD28 and CD27) analyses of markers related to immunosenescence. Increased gene expression of leptin, IL-2, IL-4, IL-10, TNF-α, PD-1, P16ink4a, CCR7 and CD27 was found for the OBD and OB groups compared to the E group. Moreover, VO2max for the OBD and OB groups was significantly lower compared to E. In conclusion, obesity, regardless of associated disease, induces increased gene expression of markers associated with inflammation and immunosenescence in circulating leukocytes in obese middle-aged individuals compared to a eutrophic group of the same age. Additionally, increased adipose tissue and markers of chronic inflammation and immunosenescence were associated to impairments in the cardiorespiratory capacity of obese middle-aged individuals.

Distinct Age-Related Epigenetic Signatures in CD4 and CD8 T Cells.

Healthy immune aging is in part determined by how well the sizes of naïve T cell compartments are being maintained with advancing age. Throughout adult life, replenishment largely derives from homeostatic proliferation of existing naïve and memory T cell populations. However, while the subpopulation composition of CD4 T cells is relatively stable, the CD8 T cell compartment undergoes more drastic changes with loss of naïve CD8 T cells and accumulation of effector T cells, suggesting that CD4 T cells are more resilient to resist age-associated changes. To determine the epigenetic basis for these differences in behaviors, we compared chromatin accessibility maps of CD4 and CD8 T cell subsets from young and old individuals and related the results to the expressed transcriptome. The dominant age-associated signatures resembled hallmarks of differentiation, which were more pronounced for CD8 naïve and memory than the corresponding CD4 T cell subsets, indicating that CD8 T cells are less able to keep cellular quiescence upon homeostatic proliferation. In parallel, CD8 T cells from old adults, irrespective of their differentiation state, displayed greater reduced accessibility to genes of basic cell biological function, including genes encoding ribosomal proteins. One possible mechanism is the reduced expression of the transcription factors YY1 and NRF1. Our data suggest that chromatin accessibility signatures can be identified that distinguish CD4 and CD8 T cells from old adults and that may confer the higher resilience of CD4 T cells to aging.

New Insights for BPIFB4 in Cardiovascular Therapy.

Aging is the most relevant risk factor for cardiovascular diseases which are the main cause of mortality in industrialized countries. In this context, there is a progressive loss of cardiovascular homeostasis that translates in illness and death. The study of long living individuals (LLIs), which show compression of morbidity toward the end of their life, is a valuable approach to find the key to delay aging and postpone associate cardiovascular events. A contribution to the age-related decline of cardiovascular system (CVS) comes from the immune system; indeed, it is dysfunctional during aging, a process described as immunosenescence and comprises the combination of several processes overpowering both innate and adaptative immune system. We have recently discovered a longevity-associated variant (LAV) in bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4), which is a secreted protein able to enhance endothelial function through endothelial nitric oxide synthase (eNOS) activation and capable to protect from hypertension, atherosclerosis, diabetic cardiopathy, frailty, and inflammaging. Here, we sum up the state of the art of the mechanisms involved in the main pathological processes related to CVD (atherosclerosis, aging, diabetic cardiopathy, and frailty) and shed light on the therapeutic effects of LAV-BPIFB4 in these contexts.

Immunosenescence profile and expression of the aging biomarker (p16INK4a) in testicular cancer survivors treated with chemotherapy.

BACKGROUND: Cytotoxic chemotherapy can cure advanced germ cell tumors. Nevertheless, cancer treatment may induce cellular senescence and accelerate molecular aging. The aging process implies an increase of cells expressing p16INK4a and changes in lymphocyte subpopulations. Our aim was to study the potential induction of premature immunosenescence in testicular cancer survivors (TCS) exposed to chemotherapy. METHODS: Case-control exploratory study of TCS treated with chemotherapy (≥3 BEP cycles, disease-free ≥3 months) compared with age matched healthy controls. Peripheral blood mononuclear cells were isolated, and lymphocyte subpopulations were analyzed by flow cytometry. CDKN2A/p16INK4a expression in T cells was measured using qPCR. The percentage of lymphocyte subpopulations and the CDKN2A/p16INK4a expression in TCS were compared with the control group using the Wilcoxon signed-rank test. RESULTS: We included 16 cases and 16 controls. The median age was 27 years (minimum 24, maximum 54) and the median time on surveillance was 26.5 months (minimum 3, maximum192). TCS had a lower percentage of total T cells and CD4+ T cells in total lymphocytes. Among the CD4+ T lymphocytes, TCS had less naïve CD4+ and increased memory CD4+ cells. Within the CD8+ T lymphocytes, TCS exhibited a decrease in the percentage of naïve cells and an increase in CD8 + CD45RA + CD57+ cells. TCS also exhibited decreased memory CD19+ B cells compared to the controls. The relative expression of CDKN2A/p16INK4a in T cells was increased in TCS (mean 1.54; 95% CI of the mean: 1.074-2.005; p = 0.048). CONCLUSION: In this exploratory study, TCS showed increased expression of CDKN2A/p16INK4a and a lymphocyte phenotype that has been associated with immunosenescence. Further studies are warranted to define the clinical implications of these alterations in TCS.

Age-Related Lymphocyte Output During Disease-Modifying Therapies for Multiple Sclerosis.

BACKGROUND: Patients with multiple sclerosis exhibit the same qualitative and quantitative changes in immune system cells observed in aging. In the last 20 years, multiple sclerosis patients have shown an increase in life expectancy and average age, but clinical trial inclusion criteria typically exclude patients over the age of 55 years. Therefore, disease-modifying therapies are likely administered to patients older than those enrolled in clinical trials. OBJECTIVE: In order to investigate whether disease-modifying therapies for multiple sclerosis induce modifications to the immune system that may have (super)additive effects resulting in an acceleration of immunosenescence, we quantified the number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). These molecules are contained in new T and B lymphocytes released by the thymus and bone marrow and are considered molecular age-related markers. METHODS: The markers of aging were measured by a multiplex quantitative real-time PCR assay in 122 patients who had started therapy with interferon-beta (IFN-ß), fingolimod, alemtuzumab, or natalizumab. Samples were obtained before the therapy and at 6 and 12 months of treatment. Comparisons between the variables were performed by a non-parametric statistical analysis. RESULTS: In therapy-naive patients, a significant and direct correlation was found between a lower number of newly produced T and B cells and older age. Although disease-modifying therapies induced different changes (both increases and decreases) in the production of new T and B lymphocytes, 12 months of therapy with IFN-ß or natalizumab did not affect the correlations found at baseline between the release of lymphocytes containing TRECs or KRECs and age. On the contrary, in patients treated with alemtuzumab, both correlations were lost, while in fingolimod-treated patients, only the correlation between TRECs and age disappeared. CONCLUSIONS: This observational study indicated that different age-related changes of the new T and B lymphocyte production could be one of the reasons for the emergence, in the real-world setting, of adverse events not otherwise observed in clinical trials; thus, caution is advised when choosing disease-modifying therapies for multiple sclerosis patients.

Accelerated epigenetic age and cognitive decline among urban-dwelling adults.

OBJECTIVES: Epigenetic modifications are closely linked with aging, but their relationship with cognition remains equivocal. Given known sex differences in epigenetic aging, we explored sex-specific associations of 3 DNA methylation (DNAm)-based measures of epigenetic age acceleration (EAA) with baseline and longitudinal change in cognitive performance among middle-aged urban adults. METHODS: We used exploratory data from a subgroup of participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span study with complete DNA samples and whose baseline ages were >50.0 years (2004-2009) to estimate 3 DNAm EAA measures: (1) universal EAA (AgeAccel); (2) intrinsic EAA (IEAA); and (3) extrinsic EAA (EEAA). Cognitive performance was measured at baseline visit (2004-2009) and first follow-up (2009-2013) with 11 test scores covering global mental status and specific domains such as learning/memory, attention, visuospatial, psychomotor speed, language/verbal, and executive function. A series of mixed-effects regression models were conducted adjusting for covariates and multiple testing (n = 147-156, ∼51% men, k = 1.7-1.9 observations/participant, mean follow-up time ∼4.7 years). RESULTS: EEAA, a measure of both biological age and immunosenescence, was consistently associated with greater cognitive decline among men on tests of visual memory/visuoconstructive ability (Benton Visual Retention Test: γ11 = 0.0512 ± 0.0176, p = 0.004) and attention/processing speed (Trail-Making Test, part A: γ11 = 0.219 ± 0.080, p = 0.007). AgeAccel and IEAA were not associated with cognitive change in this sample. CONCLUSIONS: EEAA capturing immune system cell aging was associated with faster decline among men in domains of attention and visual memory. Larger longitudinal studies are needed to replicate our findings.

Altered immune system in frailty: Genetics and diet may influence inflammation.

Frailty is a complex geriatric syndrome associated with biological vulnerability to stressors and decreased physiological reserve. Its etiology and pathogenesis are not completely understood, although various causes and complex pathways have been proposed. Immune system alterations (immunosenescence and "InflammAging") have been suggested to contribute to frailty, but a precise causative role of such alterations remains to be determined. Genetic studies support the suggestion of immune system involvement in frailty: genetic variants in genes involved in immune system function have been associated with the syndrome. Interestingly, nutritional status, through its effects on cellular metabolism, may also influence the immune system, i.e. hormone and cytokine (mainly adipocytokine) levels, and immune cell populations and function, increasing inflammation and contributing to frailty. This review aims to discuss the role of immune system alterations in frailty, analyzing the role of genetic factors in frailty onset and the impact of diet on inflammation and, in turn, on frailty.

Cell-type-specific role of lamin-B1 in thymus development and its inflammation-driven reduction in thymus aging.

Cellular architectural proteins often participate in organ development and maintenance. Although functional decay of some of these proteins during aging is known, the cell-type-specific developmental role and the cause and consequence of their subsequent decay remain to be established especially in mammals. By studying lamins, the nuclear structural proteins, we demonstrate that lamin-B1 functions specifically in the thymic epithelial cells (TECs) for proper thymus organogenesis. An up-regulation of proinflammatory cytokines in the intra-thymic myeloid immune cells during aging accompanies a gradual reduction of lamin-B1 in adult TECs. We show that these cytokines can cause senescence and lamin-B1 reduction of the young adult TECs. Lamin-B1 supports the expression of TEC genes that can help maintain the adult TEC subtypes we identified by single-cell RNA-sequencing, thymic architecture, and function. Thus, structural proteins involved in organ building and maintenance can undergo inflammation-driven decay which can in turn contribute to age-associated organ degeneration.

Tasmanian devils with contagious cancer exhibit a constricted T-cell repertoire diversity.

The Tasmanian devil (Sarcophilus harrisii) is threatened by a contagious cancer, known as Devil Facial Tumour Disease (DFTD). A highly diverse T-cell receptor (TCR) repertoire is crucial for successful host defence against cancers. By investigating TCR beta chain diversity in devils of different ages, we show that the T-cell repertoire in devils constricts in their second year of life, which may explain the higher DFTD prevalence in older devils. Unexpectedly, we also observed a pronounced decline in TCR diversity and T cell clonal expansion in devils after DFTD infection. These findings overturned the previous assumption that DFTD did not directly impact host immunity.

Immunosenescence in honey bees (Apis mellifera L.) is caused by intrinsic senescence and behavioral physiology.

Young honey bee workers (0 to 2-3 weeks old) perform tasks inside the colony, including brood care (nursing), whereas older workers undergo foraging tasks during the next 3-4 weeks, when an intrinsic senescence program culminates in worker death. We hypothesized that foragers are less able to react to immune system stimulation than nurse bees and that this difference is due to an inefficient immune response in foragers. To test this hypothesis, we used an experimental design that allowed us to uncouple chronological age and behavior status (nursing/foraging). Worker bees from a normal age demography colony (where workers naturally transit from nursing to foraging tasks as they age) and of a single-cohort colony setup (composed of same-aged workers performing nursing or foraging tasks) were tested for survival and capability of activation of the immune system after bacterial injection. Expression of an antimicrobial peptide gene, defensin-1 (def-1), was used to assess immune system activation. We then checked whether the immune response includes changes in the expression of aging- and behavior-related genes, specifically vitellogenin (vg), juvenile hormone esterase (jhe), and insulin-like peptide-1 (ilp-1). We found a significant difference in survival rate between bees of different ages but carrying out the same tasks. Our results thus indicate that the bees' immune response is negatively affected by intrinsic senescence. Additionally, independent of age, foragers had a shorter lifespan than nurses after bacterial infection, although both were able to induce def-1 transcription. In the normal age demography colony, the immune system activation resulted in a reduction in the expression of vg, jhe and ilp-1 genes in foragers, but not in the nurse bees, demonstrating that age and behavior are both important influences on the bees' immune response. By disentangling the effects of age and behavior in the single-cohort colony, we found that vg, jhe and ilp-1 response to immune system stimulation was independent of behavior. Younger bees were able to mount a stronger immune response than older bees, thus highlighting age as an important factor for immunity. Taken together, our results provide new insights into how age and behavior affect the honey bee's immune response.