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BackgroundThe cellular mechanisms involved in the lack of protective antibody response after hepatitis B vaccination are still rather unclear. Regulatory B cells (Breg) known as modulators of B-and T-cell responses may contribute to poor vaccine responsiveness. The current study aimed to investigate the role of regulatory B cells (Breg) in hepatitis B vaccine non-responsiveness after immunization with second- or third-generation hepatitis B vaccines.MethodWe performed comparative phenotypic and frequency analysis of Breg subsets (CD24+CD27+ and CD24highCD38high Breg) in second-generation hepatitis B vaccine non-responders (2nd HBvac NR, n = 11) and responders (2nd HBvac R, n = 8) before (d0), on day 7 (d7), and 28 (d28) after booster vaccination. Cryopreserved peripheral blood mononuclear cells were stimulated ex vivo with a combination of CpG, PMA, and Ionomycin (CpG+P/I) and analyzed for numbers and IL-10 expression levels of Breg by flow cytometry-based analyses.ResultsFlow cytometry-based analyses revealed elevated frequencies of CD24+CD27+ Breg at all time points and significantly higher frequencies of CD24highCD38high Breg on d0 (p = 0.004) and 28 (p = 0.012) in 2nd HBvac NR compared to 2nd HBvac R. In parallel, we observed significantly lower levels of CpG+P/I-induced IL-10 expression levels of CD24+CD27+ and CD24highCD38high Breg (d0: p < 0.0001; d7: p = 0.0004; d28: p = 0.0003 and d0: p = 0.016; d7: p = 0.016, respectively) in 2nd HBvac NR compared to 2nd HBvac R before and after booster immunization. Frequencies of CD24+CD27+ and CD24highCD38high Breg significantly decreased after third-generation hepatitis B booster vaccination (d7: p = 0.014; d28: p = 0.032 and d7: p = 0.045, respectively), whereas IL-10 expression levels of both Breg subsets remained stable.ConclusionHere we report significantly higher frequencies of CD24highCD38high Breg in parallel with significantly lower IL-10 expression levels of CD24+CD27+ and CD24highCD38high Breg in 2nd HBvac NR compared to 2nd HBvac R. Anti-HBs seroconversion accompanied by a decrease of Breg numbers after booster immunization with a third-generation hepatitis B vaccine could indicate a positive effect of third-generation hepatitis B vaccines on Breg-mediated immunomodulation in hepatitis B vaccine non-responders.

The presence of circulating microbiome in blood has been reported in both physiological and pathological conditions, although its origins, identities and function remain to be elucidated. This study aimed to investigate the presence of blood microbiome by quantitative real-time PCRs targeting the 16S rRNA gene. To our knowledge, this is the first study in which the circulating microbiome has been analyzed in such a large sample of individuals since the study was carried out on 1285 Randomly recruited Age-Stratified Individuals from the General population (RASIG). The samples came from several different European countries recruited within the EU Project MARK-AGE in which a series of clinical biochemical parameters were determined. The results obtained reveal an association between microbial DNA copy number and geographic origin. By contrast, no gender and age-related difference emerged, thus demonstrating the role of the environment in influencing the above levels independent of age and gender at least until the age of 75. In addition, a significant positive association was found with Free Fatty Acids (FFA) levels, leukocyte count, insulin, and glucose levels. Since these factors play an essential role in both health and disease conditions, their association with the extent of the blood microbiome leads us to consider the blood microbiome as a potential biomarker of human health.

The peripheral T-cell pool undergoes a striking age associated remodeling which is accelerated by progressive insulin resistance. Exercise training is known to delay several aspects of T-cell senescence. The purpose of the current study was to investigate the effect of 3 weeks regular concentric or eccentric endurance exercise training on the composition of the T-cell compartment in pre-diabetic subjects. Sixteen male older adults with impaired glucose tolerance were recruited and performed either concentric exercise (CE) or eccentric exercise (EE) walking 3 times a week for 3 weeks. Fasting venous blood sampling was performed before training and after the training intervention. Various T-cell subpopulations were analyzed by flow cytometry. We did not find significant time × group effects (interaction) but found several significant time effects for cell type ratios and cell subsets proportions. There was an increase of the CD4+/CD8+ (0.55 ± 0.85%; p = 0.033) and CD4+/CD3+ ratio (5.63 ± 8.44%; p = 0.018) and a decrease of the CD8+/CD3+ ratio (-0.95 ± 1.64%; p = 0.049) after training. We found proportional increases of CD4+/CCR7+/CD45RO+ central memory cells (5.02 ± 7.68%; p = 0.030), naïve CD8+/CCR7+/CD45RO- (3.00 ± 6.68%; p = 0.047) and CD8+/CCR7+/CD45RO+ central memory cells (3.01 ± 3.70%; p = 0.009), while proportions of CD4+/CCR7-/CD45RO- TEMRA cells (-2.17 ± 4.66%; p = 0.012), CD8+/CCR7-/CD45RO- TEMRA cells (-5.11 ± 7.02%; p = 0.018) and CD16+ cells (-4.67 ± 6.45%; p = 0.016) decreased after training. 3 weeks of either CE or EE were effective in reversing hallmarks of T-cell senescence in pre-diabetic subjects. It is suggested that exercise stimulates production and mobilization of naïve T-cells, while differentiated TEMRA cells might disappear by apoptosis.

CD4+ regulatory T cells have been intensively studied during aging, but little is still known about age-related changes of other regulatory T cell subsets. It was, therefore, the goal of the present study to analyze CD8+human leukocyte antigen–antigen D related (HLADR)+ T cells in old age, a cell population reported to have suppressive activity and to be connected to specific genetic variants. We demonstrate a strong increase in the number of CD8+HLADR+ T cells with age in a cohort of female Sardinians as well as in elderly male and female persons from Austria. We also show that CD8+HLADR+ T cells lack classical activation molecules, such as CD69 and CD25, but contain increased numbers of checkpoint inhibitory molecules, such as cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin and mucin protein-3, LAG-3, and PD-1, when compared with their HLADR− counterparts. They also have the capacity to inhibit the proliferation of autologous peripheral blood mononuclear cells. This suppressive activity is, however, decreased when CD8+HLADR+ T cells from elderly persons are analyzed. In accordance with this finding, CD8+HLADR+ T cells from persons of old age contain lower percentages of checkpoint inhibitory molecules than young controls. We conclude that in spite of high abundance of a CD8+ regulatory T cell subset in old age its expression of checkpoint inhibitory molecules and its suppressive function on a per cell basis are reduced. Reduction of suppressive capacity may support uncontrolled subclinical inflammatory processes referred to as “inflamm-aging.”

Many current vaccines are less immunogenic and less effective in elderly compared to younger adults due to age-related changes of the immune system. Most vaccines utilized in the elderly contain antigens, which the target population has had previous contact with due to previous vaccination or infection. Therefore, most studies investigating vaccine-induced immune responses in the elderly do not analyze responses to neo-antigens but rather booster responses. However, age-related differences in the immune response could differentially affect primary versus recall responses. We therefore investigated the impact of age on primary and recall antibody responses following hepatitis B vaccination in young and older adults. Focused gene expression profiling was performed before and 1 day after the vaccination in order to identify gene signatures predicting antibody responses. Young (20–40 years; n = 24) and elderly (>60 years; n = 17) healthy volunteers received either a primary series (no prior vaccination) or a single booster shot (documented primary vaccination more than 10 years ago). Antibody titers were determined at days 0, 7, and 28, as well as 6 months after the vaccination. After primary vaccination, antibody responses were lower and delayed in the elderly compared to young adults. Non-responders after the three-dose primary series were only observed in the elderly group. Maximum antibody concentrations after booster vaccination were similar in both age groups. Focused gene expression profiling identified 29 transcripts that correlated with age at baseline and clustered in a network centered around type I interferons and pro-inflammatory cytokines. In addition, smaller 8- and 6-gene signatures were identified at baseline that associated with vaccine responsiveness during primary and booster vaccination, respectively. When evaluating the kinetic changes in gene expression profiles before and after primary vaccination, a 33-gene signature, dominated by IFN-signaling, pro-inflammatory cytokines, inflammasome components, and immune cell subset markers, was uncovered that was associated with vaccine responsiveness. By contrast, no such transcripts were identified during booster vaccination. Our results document that primary differs from booster vaccination in old age, in regard to antibody responses as well as at the level of gene signatures.Clinical Trial Registrationwww.clinicaltrialsregister.eu, this trial was registered at the EU Clinical Trial Register (EU-CTR) with the EUDRACT-Nr. 2013-002589-38.

Cytomegalovirus (CMV) has been described as a contributor to immunosenescence, thus exacerbating age-related diseases. In persons with latent CMV infection, the CD8+ T cell compartment is irreversibly changed, leading to the accumulation of highly differentiated virus-specific CD8+ T cells in the peripheral blood. The bone marrow (BM) has been shown to play a major role in the long-term survival of antigen-experienced T cells. Effector CD8+ T cells are preferentially maintained by the cytokine IL-15, the expression of which increases in old age. However, the impact of CMV on the phenotype of effector CD8+ T cells and on the production of T cell survival molecules in the BM is not yet known. We now show, using BM samples obtained from persons who underwent hip replacement surgery because of osteoarthrosis, that senescent CD8+ TEMRA cells with a bright expression of CD45RA and a high responsiveness to IL-15 accumulate in the BM of CMV-infected persons. A negative correlation was found between CMV antibody (Ab) titers in the serum and the expression of CD28 and IL-7Rα in CD8+TEMRAbright cells. Increased IL-15 mRNA levels were observed in the BM of CMV+ compared to CMV− persons, being particularly high in old seropositive individuals. In summary, our results indicate that a BM environment rich in IL-15 may play an important role in the maintenance of highly differentiated CD8+ T cells generated after CMV infection.