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Abstract Background Seasonal influenza causes significant morbidity and mortality with a disproportionately high disease burden in older adults. Strain-specific hemagglutination-inhibition (HAI) antibody titer is a well-established measure of humoral immunity against influenza and pre-vaccination HAI titer is a valuable indicator of pre-existing humoral immunity at the beginning of each influenza season in highly vaccinated older adults. While vaccine-induced HAI antibody titers are known to wane over time, accurate assessment of their interseason waning has been challenging. This is because pre-vaccination HAI titers are routinely measured using current season vaccine strain antigens instead of the prior season vaccines with which individuals were immunized; as such, they do not accurately represent residual antibody titers from prior season vaccination. This study took advantage of available pre-vaccination HAI titers measured using both current and prior season vaccine strain antigens in a longitudinal influenza immunization study with participants enrolled for multiple consecutive influenza seasons from 2014 through 2017. Influenza A virus (IAV) H3N2 and influenza B virus (IBV) strains in the vaccine formula changed in 2015 and again in 2016 season. IAV H1N1 vaccine strain remained the same from 2014 through 2016 seasons, but changed in 2017. We also investigated factors contributing to pre-existing humoral immunity. Results Interseason waning of HAI titers was evident, but rates of waning varied among vaccine strains and study seasons, from 18% (p = .43) to 61% (p

Abstract Aging is a gradual, continuous series of natural changes in biological, physiological, immunological, environmental, psychological, behavioral, and social processes. Aging entails changes in the immune system characterized by a decrease in thymic output of naïve lymphocytes, an accumulated chronic antigenic stress notably caused by chronic infections such as cytomegalovirus (CMV), and immune cell senescence with acquisition of an inflammatory senescence-associated secretory phenotype (SASP). For this reason, and due to the SASP originating from other tissues, aging is commonly accompanied by low-grade chronic inflammation, termed “inflammaging”. After decades of accumulating evidence regarding age-related processes and chronic inflammation, the domain now appears mature enough to allow an integrative reinterpretation of old data. Here, we provide an overview of the topics discussed in a recent workshop “Aging and Chronic Inflammation” to which many of the major players in the field contributed. We highlight advances in systematic measurement and interpretation of biological markers of aging, as well as their implications for human health and longevity and the interventions that can be envisaged to maintain or improve immune function in older people.

BackgroundHuman cytomegalovirus (HCMV) can reactivate in the mammary gland during lactation and is shed into breast milk of nearly every HCMV-IgG-seropositive mother of a preterm infant. Dynamics of breast milk leukocytes during lactation, as well as blood leukocytes and the comparison between both in the context of HCMV reactivation is not well understood.MethodsHere, we present the BlooMil study that aimed at comparing changes of immune cells in blood and breast milk from HCMV-seropositive- vs -seronegative mothers, collected at four time ranges up to two months post-partum. Viral load was monitored by qPCR and nested PCR. Multiparameter flow cytometry was used to identify leukocyte subsets.ResultsCD3+ T cell frequencies were found to increase rapidly in HCMV-seropositive mothers’ milk, while they remained unchanged in matched blood samples, and in both blood and breast milk of HCMV-seronegatives. The activation marker HLA-DR was more strongly expressed on CD4+ and CD8+ T cells in all breast milk samples than matched blood samples, but HCMV-seropositive mothers displayed a significant increase of HLA-DR+ CD4+ and HLA-DR+ CD8+ T cells during lactation. The CD4+/CD8+ T cell ratio was lower in breast milk of HCMV-seropositive mothers than in the blood. HCMV-specific CD8+ T cell frequencies (recognizing pp65 or IE1) were elevated in breast milk relative to blood, which might be due to clonal expansion of these cells during local HCMV reactivation. Breast milk contained very low frequencies of naïve T cells with no significant differences depending on serostatus.ConclusionTaken together, we conclude that the distribution of breast milk leukocyte populations is different from blood leukocytes and may contribute to the decrease of breast milk viral load in the late phase of HCMV reactivation in the mammary gland.

Abstract Natural antibodies (nAbs) against aggregation-prone proteins have been found in healthy normal subjects. These proteins likely have a pathogenetic role in neurodegenerative diseases of ageing. They include the amyloid β (Aβ) protein which may play an important role in Alzheimer’s dementia (AD), and α-synuclein, a major determinant of Parkinson’s disease (PD). We measured nAbs to Aβ in a group of Italian patients with AD, vascular dementia, non-demented PD patients and healthy elderly controls. We found that Aβ antibody levels in AD were similar to age- and sex-matched controls, but contrary to our expectations, they were significantly reduced in PD. This may identify patients that could be more prone to amyloid aggregation.

Immune cells are thought to be involved in a destructive cycle of sterile cerebral inflammatory responses in neurodegenerative diseases such as Parkinson’s Disease (PD). Despite their peripheral origin, immune cells may enter the CNS due to impaired blood–brain barrier function and may potentially contribute to neuronal damage. Hence, specific characteristics of peripherally activated immune cells could help in understanding neurodegeneration in PD and could potentially serve as accessible disease markers. To investigate immune cell activation status, the expression of receptors for cell surface molecules CD161, NKG2A, NKG2C and NKG2D as well as chemokine receptors CCR6, CXCR2, CXCR3 and CCR5 associated with neurodegenerative diseases was investigated. The frequencies of peripheral CD8+ T-cells expressing the inhibitory and activating receptors NKG2A and NKG2C, and the activating receptor NKG2D were higher in PD patients than in healthy matched controls. The frequencies of NKG2C+CD8− cells were also higher, whereas the frequencies of CD161+ cells were not significantly different. Of the chemokine receptor-expressing cells, only the proportion of CD4−CD56+CCR5+ T-cells was higher in PD patients than in the controls. These observations support the hypothesis that an imbalance in the activation state of T-cells plays a role in the pathological processes of PD and suggest that peripheral blood immune cell phenotypes could be specific early markers for inflammation in PD.

Abstract Background Biological aging represents a loss of integrity and functionality of physiological systems over time. While associated with an enhanced risk of adverse outcomes such as hospitalization, disability and death following infection, its role in perceived age-related declines in vaccine responses has yet to be fully elucidated. Using data and biosamples from a 4-year clinical trial comparing immune responses of standard- and high-dose influenza vaccination, we quantified biological age (BA) prior to vaccination in adults over 65 years old (n = 292) using a panel of ten serological biomarkers (albumin, alanine aminotransferase, creatinine, ferritin, free thyroxine, cholesterol, high-density lipoprotein, triglycerides, tumour necrosis factor, interleukin-6) as implemented in the BioAge R package. Hemagglutination inhibition antibody titres against influenza A/H1N1, A/H3N2 and B were quantified prior to vaccination and 4-, 10- and 20- weeks post-vaccination. Results Counter to our hypothesis, advanced BA was associated with improved post-vaccination antibody titres against the different viral types and subtypes. However, this was dependent on both vaccine dose and CMV serostatus, as associations were only apparent for high-dose recipients (d = 0.16–0.26), and were largely diminished for CMV positive high-dose recipients. Conclusions These findings emphasize two important points: first, the loss of physiological integrity related to biological aging may not be a ubiquitous driver of immune decline in older adults; and second, latent factors such as CMV infection (prevalent in up to 90% of older adults worldwide) may contribute to the heterogeneity in vaccine responses of older adults more than previously thought.

Background The need for reliable clinical biomarkers to predict which patients with melanoma will benefit from immune checkpoint blockade (ICB) remains unmet. Several different parameters have been considered in the past, including routine differential blood counts, T cell subset distribution patterns and quantification of peripheral myeloid-derived suppressor cells (MDSC), but none has yet achieved sufficient accuracy for clinical utility.Methods Here, we investigated potential cellular biomarkers from clinical routine blood counts as well as several myeloid and T cell subsets, using flow cytometry, in two independent cohorts of a total of 141 patients with stage IV M1c melanoma before and during ICB.Results Elevated baseline frequencies of monocytic MDSCs (M-MDSC) in the blood were confirmed to predict shorter overall survival (OS) (HR 2.086, p=0.030) and progression-free survival (HR 2.425, p=0.001) in the whole patient cohort. However, we identified a subgroup of patients with highly elevated baseline M-MDSC frequencies that fell below a defined cut-off during therapy and found that these patients had a longer OS that was similar to that of patients with low baseline M-MDSC frequencies. Importantly, patients with high M-MDSC frequencies exhibited a skewed baseline distribution of certain other immune cells but these did not influence patient survival, illustrating the paramount utility of MDSC assessment.Conclusion We confirmed that in general, highly elevated frequencies of peripheral M-MDSC are associated with poorer outcomes of ICB in metastatic melanoma. However, one reason for an imperfect correlation between high baseline MDSCs and outcome for individual patients may be the subgroup of patients identified here, with rapidly decreasing M-MDSCs on therapy, in whom the negative effect of high M-MDSC frequencies was lost. These findings might contribute to developing more reliable predictors of late-stage melanoma response to ICB at the individual patient level. A multifactorial model seeking such markers yielded only MDSC behavior and serum lactate dehydrogenase as predictors of treatment outcome.

Abstract Background With increasing age, overall health declines while systemic levels of inflammatory mediators tend to increase. Although the underlying mechanisms are poorly understood, there is a wealth of data suggesting that this so-called “inflammaging” contributes to the risk of adverse outcomes in older adults. We sought to determine whether markers of systemic inflammation were associated with antibody responses to the seasonal influenza vaccine. Results Over four seasons, hemagglutination inhibition antibody titres and ex vivo bulk peripheral blood mononuclear cell (PBMC) responses to live influenza viruses assessed via interferon (IFN)-γ/interleukin (IL)-10 production, were measured pre- and 4-weeks post-vaccination in young adults (n = 79) and older adults randomized to standard- or high-dose inactivated vaccine (n = 612). Circulating tumour necrosis factor (TNF), interleukin (IL)-6 and C-reactive protein (CRP) were also measured pre-vaccination. Post-vaccination antibody titres were significantly associated with systemic inflammatory levels; specifically, IL-6 was positively associated with A/H3N2 titres in young adults (Cohen’s d = 0.36), and in older high-dose, but not standard-dose recipients, all systemic inflammatory mediators were positively associated with A/H1N1, A/H3N2 and B titres (d = 0.10–0.45). We further show that the frequency of ILT2(+)CD57(+) CD56-Dim natural killer (NK)-cells was positively associated with both plasma IL-6 and post-vaccination A/H3N2 titres in a follow-up cohort of older high-dose recipients (n = 63). Pathway analysis suggested that ILT2(+)CD57(+) Dim NK-cells mediated 40% of the association between IL-6 and A/H3N2 titres, which may be related to underlying participant frailty. Conclusions In summary, our data suggest a complex relationship amongst influenza vaccine responses, systemic inflammation and NK-cell phenotype in older adults, which depends heavily on age, vaccine dose and possibly overall health status. While our results suggest that “inflammaging” may increase vaccine immunogenicity in older adults, it is yet to be determined whether this enhancement contributes to improved protection against influenza disease.

Granzymes are a family of serine-proteases that act as critical mediators in the cytolytic and immunomodulatory activities of immune cells such as CD8+ T-cells and natural killer (NK) cells. Previous work indicates that both granzyme B (GZB) and K (GZK) are increased with age in CD8+ T-cells, and in the case of GZB, contribute to dysfunctional immune processes observed in older adults. Here, we sought to determine how GZB and GZK expression in NK-cells, and CD4+, CD8+, and gamma-delta T-cells, quantified in terms of positive cell frequency and mean fluorescence intensity (MFI), differed with age, age-related health-traits and the antibody response to high-dose influenza vaccine. We found that the frequency and MFI of GZB-expressing NK-cells, and CD8+ and Vδ1+ T-cells, and GZK-expressing CD8+ T-cells was significantly higher in older (66–97 years old; n = 75) vs. younger (24–37 years old; n = 10) adults by up to 5-fold. There were no significant associations of GZB/GZK expression with sex, frailty or plasma levels of TNF or IL-6 in older adults, but those who were seropositive for cytomegalovirus (CMV) exhibited significantly higher frequencies of GZB+ NK-cells, and CD4+, CD8+ and Vδ1+ T-cells, and GZK+ CD8+ T-cells (Cohen’s d = .5–1.5). Pre-vaccination frequencies of GZB+ NK-cells were positively correlated with vaccine antibody responses against A/H3N2 (d = .17), while the frequencies of GZK+ NK and CD8+ T-cells were inversely associated with A/H1N1 (d = −0.18 to −0.20). Interestingly, GZK+ NK-cell frequency was inversely correlated with pre-vaccination A/H1N1 antibody titres, as well as those measured over the previous 4 years, further supporting a role for this subset in influencing vaccine antibody-responses. These findings further our understanding of how granzyme expression in different lymphoid cell-types may change with age, while suggesting that they influence vaccine responsiveness in older adults.

Background: Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. Methods: We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. Results: Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. Conclusions: The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three most informative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anti-cancer mechanisms.

Older individuals are more likely to develop solid cancers, but at the same time are more sensitive to the side effects of chemotherapy. In addition, older adults are more likely to present with chronic diseases (comorbidities) and immunosenescence that may decrease immunosurveillance against cancer. Clinical outcomes for the older patient with cancer are different from the younger patient and require different research and treatment approaches. Thus, alternative therapeutic approaches tailored specifically to the older patients are required. Colorectal cancer (CRC) has a high incidence in older individuals and is the third leading cause of cancer death globally. Anti-hypertensives are used by a large proportion of older patients and some studies have pointed to a positive impact of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB) on CRC outcomes. As we have previously shown in a mouse model, lung metastases express ACE and contain many infiltrating myeloid-derived suppressor cells (MDSC); particularly high levels of MDSC are also present in the blood of older patients with CRC and other cancers, and are associated with disease severity. In this Commentary, we hypothesize that one mechanism responsible for the positive impact of ACEi or ARB on the outcome of CRC is the modulation of myeloid cells contributing to their maturation to non-suppressive neutrophils/monocytes and diverting them away from retaining an immature MDSC phenotype.

Organismal ageing is associated with many physiological changes, including differences in the immune system of most animals. These differences are often considered to be a key cause of age-associated diseases as well as decreased vaccine responses in humans. The most often cited vaccine failure is seasonal influenza, but, while it is usually the case that the efficiency of this vaccine is lower in older than younger adults, this is not always true, and the reasons for the differential responses are manifold. Undoubtedly, changes in the innate and adaptive immune response with ageing are associated with failure to respond to the influenza vaccine, but the cause is unclear. Moreover, recent advances in vaccine formulations and adjuvants, as well as in our understanding of immune changes with ageing, have contributed to the development of vaccines, such as those against herpes zoster and SARS-CoV-2, that can protect against serious disease in older adults just as well as in younger people. In the present article, we discuss the reasons why it is a myth that vaccines inevitably protect less well in older individuals, and that vaccines represent one of the most powerful means to protect the health and ensure the quality of life of older adults.