Direct TLR-2 Costimulation Unmasks the Proinflammatory Potential of Neonatal CD4+ T Cells.

Neonatal CD4(+) T cells have traditionally been viewed as deficient in their capacity to produce Th1 cytokines in response to polyclonal or Ag-specific stimuli. Thus, defining unique aspects of CD4(+) T cell activation and development into Th1 effector cells in neonates is essential to the successful development of novel vaccines and immunotherapies to protect infants from intracellular pathogens. Using highly purified naive CD4(+) T cells derived from cord and adult peripheral blood, we compared the impact of anti-CD3 stimulation plus costimulation through TLR-2 performed in the absence of APC on CD4(+) T cell cytokine production, proliferation, and expression of activation markers. In both age groups, TLR-2 costimulation elicited activation of naive CD4(+) T cells, characterized by robust production of IL-2 as well as key Th1-type cytokines IFN-γ and TNF-α. TLR-2 costimulation also dramatically reduced naive T cell production of the immunosuppressive cytokine IL-10. We observed that neonatal naive CD4(+) T cells are uniquely sensitive to TLR-2-mediated costimulation, which enabled them to produce equivalent amounts of IFN-γ and more IL-2 when compared with adult responses. Thus, neonatal CD4(+) T cells have a distinctive propensity to use TLR-2-mediated costimulation for development into proinflammatory Th1 effectors, and interventions that target CD4(+) T cell TLR-2-mediated responses may be exploited to enhance neonatal adaptive immunity.

CD39 is involved in mediating suppression by Mycobacterium bovis BCG-activated human CD8(+) CD39(+) regulatory T cells.

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4(+) Treg cells, relatively little is known about the phenotype and function of CD8(+) Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E-NTPDase1) on Mycobacterium-activated human CD8(+) T cells. These CD8(+) CD39(+) T cells significantly co-expressed the Treg markers CD25, Foxp3, lymphocyte activation gene-3 (LAG-3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen-specific CD4(+) T helper-1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8(+) T cells and indicate that CD39 is functionally involved in suppression by CD8(+) Treg cells.

Distinct T cell responsiveness to different COVID-19 vaccines and cross-reactivity to SARS-CoV-2 variants with age and CMV status.

Introduction: Accumulating evidence indicates the importance of T cell immunity in vaccination-induced protection against severe COVID-19 disease, especially against SARS-CoV-2 Variants-of-Concern (VOCs) that more readily escape from recognition by neutralizing antibodies. However, there is limited knowledge on the T cell responses across different age groups and the impact of CMV status after primary and booster vaccination with different vaccine combinations. Moreover, it remains unclear whether age has an effect on the ability of T cells to cross-react against VOCs. Methods: Therefore, we interrogated the Spike-specific T cell responses in healthy adults of the Dutch population across different ages, whom received different vaccine types for the primary series and/or booster vaccination, using IFNÉ£ ELISpot. Cells were stimulated with overlapping peptide pools of the ancestral Spike protein and different VOCs. Results: Robust Spike-specific T cell responses were detected in the vast majority of participants upon the primary vaccination series, regardless of the vaccine type (i.e. BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, or Ad26.COV2.S). Clearly, in the 70+ age group, responses were overall lower and showed more variation compared to younger age groups. Only in CMV-seropositive older adults (>70y) there was a significant inverse relation of age with T cell responses. Although T cell responses increased in all age groups after booster vaccination, Spike-specific T cell frequencies remained lower in the 70+ age group. Regardless of age or CMV status, primary mRNA-1273 vaccination followed by BNT162b2 booster vaccination showed limited booster effect compared to the BNT162b2/BNT162b2 or BNT162b2/mRNA-1273 primary-booster regimen. A modest reduction in cross-reactivity to the Alpha, Delta and Omicron BA.1, but not the Beta or Gamma variant, was observed after primary vaccination. Discussion: Together, this study shows that age, CMV status, but also the primary-booster vaccination regimen influence the height of the vaccination-induced Spike-specific T cell response, but did not impact the VOC cross-reactivity.

Booster Immunization Improves Memory B Cell Responses in Older Adults Unresponsive to Primary SARS-CoV-2 Immunization.

The generation of a specific long-term immune response to SARS-CoV-2 is considered important for protection against COVID-19 infection and disease. Memory B cells, responsible for the generation of antibody-producing plasmablasts upon a new antigen encounter, play an important role in this process. Therefore, the induction of memory B cell responses after primary and booster SARS-CoV-2 immunizations was investigated in the general population with an emphasis on older adults. Participants, 20-99 years of age, due to receive the mRNA-1273 or BNT162b2 SARS-CoV-2 vaccine were included in the current study. Specific memory B cells were determined by ex vivo ELISpot assays. In a subset of participants, antibody levels, avidity, and virus neutralization capacity were compared to memory B cell responses. Memory B cells specific for both Spike S1 and receptor-binding domain (RBD) were detected in the majority of participants following the primary immunization series. However, a proportion of predominantly older adults showed low frequencies of specific memory B cells. Booster vaccination resulted in a large increase in the frequencies of S1- and RBD-specific memory B cells also for those in which low memory B cell frequencies were detected after the primary series. These data show that booster immunization is important for the generation of a memory B cell response, as a subset of older adults shows a suboptimal response to the primary SARS-CoV-2 immunization series. It is anticipated that these memory B cells will play a significant role in the immune response following viral re-exposure.

Contribution of SARS-CoV-2 infection preceding COVID-19 mRNA vaccination to generation of cellular and humoral immune responses in children.

Primary COVID-19 vaccination for children, 5-17 years of age, was offered in the Netherlands at a time when a substantial part of this population had already experienced a SARS-CoV-2 infection. While vaccination has been shown effective, underlying immune responses have not been extensively studied. We studied immune responsiveness to one and/or two doses of primary BNT162b2 mRNA vaccination and compared the humoral and cellular immune response in children with and without a preceding infection. Antibodies targeting the original SARS-CoV-2 Spike or Omicron Spike were measured by multiplex immunoassay. B-cell and T-cell responses were investigated using enzyme-linked immunosorbent spot (ELISpot) assays. The activation of CD4+ and CD8+ T cells was studied by flowcytometry. Primary vaccination induced both a humoral and cellular adaptive response in naive children. These responses were stronger in those with a history of infection prior to vaccination. A second vaccine dose did not further boost antibody levels in those who previously experienced an infection. Infection-induced responsiveness prior to vaccination was mainly detected in CD8+ T cells, while vaccine-induced T-cell responses were mostly by CD4+ T cells. Thus, SARS-CoV-2 infection prior to vaccination enhances adaptive cellular and humoral immune responses to primary COVID-19 vaccination in children. As most children are now expected to contract infection before the age of five, the impact of infection-induced immunity in children is of high relevance. Therefore, considering natural infection as a priming immunogen that enhances subsequent vaccine-responsiveness may help decision-making on the number and timing of vaccine doses.

The adaptive immune system in early life: The shift makes it count.

Respiratory infectious diseases encountered early in life may result in life-threatening disease in neonates, which is primarily explained by the relatively naive neonatal immune system. Whereas vaccines are not readily available for all infectious diseases, vaccinations have greatly reduced childhood mortality. However, repeated vaccinations are required to reach protective immunity in infants and not all vaccinations are effective at young age. Moreover, protective adaptive immunity elicited by vaccination wanes more rapidly at young age compared to adulthood. The infant adaptive immune system has previously been considered immature but this paradigm has changed during the past years. Recent evidence shows that the early life adaptive immune system is equipped with a strong innate-like effector function to eliminate acute pathogenic threats. These strong innate-like effector capacities are in turn kept in check by a tolerogenic counterpart of the adaptive system that may have evolved to maintain balance and to reduce collateral damage. In this review, we provide insight into these aspects of the early life's adaptive immune system by addressing recent literature. Moreover, we speculate that this shift from innate-like and tolerogenic adaptive immune features towards formation of immune memory may underlie different efficacy of infant vaccination in these different phases of immune development. Therefore, presence of innate-like and tolerogenic features of the adaptive immune system may be used as a biomarker to improve vaccination strategies against respiratory and other infections in early life.

Omicron BA.1 Mutations in SARS-CoV-2 Spike Lead to Reduced T-Cell Response in Vaccinated and Convalescent Individuals.

Omicron BA.1 variant can readily infect people with vaccine-induced or naturally acquired SARS-CoV-2 immunity facilitated by escape from neutralizing antibodies. In contrast, T-cell reactivity against the Omicron BA.1 variant seems relatively well preserved. Here, we studied the preexisting T cells elicited by either vaccination with the mRNA-based BNT162b2 vaccine or by natural infection with ancestral SARS-CoV-2 for their cross-reactive potential to 20 selected CD4+ T-cell epitopes of spike-protein-harboring Omicron BA.1 mutations. Although the overall memory CD4+ T-cell responses primed by the ancestral spike protein was still preserved generally, we show here that there is also a clear loss of memory CD4+ T-cell cross-reactivity to immunodominant epitopes across the spike protein due to Omicron BA.1 mutations. Complete or partial loss of preexisting T-cell responsiveness was observed against 60% of 20 nonconserved CD4+ T-cell epitopes predicted to be presented by a broad set of common HLA class II alleles. Monitoring such mutations in circulating strains helps predict which virus variants may escape previously induced cellular immunity and could be of concern.

SARS-CoV-2 Omicron BA.4/BA.5 Mutations in Spike Leading to T Cell Escape in Recently Vaccinated Individuals.

SARS-CoV-2 Omicron (B.1.1.529) lineages rapidly became dominant in various countries reflecting its enhanced transmissibility and ability to escape neutralizing antibodies. Although T cells induced by ancestral SARS-CoV-2-based vaccines also recognize Omicron variants, we showed in our previous study that there was a marked loss of T cell cross-reactivity to spike epitopes harboring Omicron BA.1 mutations. The emerging BA.4/BA.5 subvariants carry other spike mutations than the BA.1 variant. The present study aims to investigate the impact of BA.4/BA.5 spike mutations on T cell cross-reactivity at the epitope level. Here, we focused on universal T-helper epitopes predicted to be presented by multiple common HLA class II molecules for broad population coverage. Fifteen universal T-helper epitopes of ancestral spike, which contain mutations in the Omicron BA.4/BA.5 variants, were identified utilizing a bioinformatic tool. T cells isolated from 10 subjects, who were recently vaccinated with mRNA-based BNT162b2, were tested for functional cross-reactivity between epitopes of ancestral SARS-CoV-2 spike and the Omicron BA.4/BA.5 spike counterparts. Reduced T cell cross-reactivity in one or more vaccinees was observed against 87% of the tested 15 non-conserved CD4+ T cell epitopes. These results should be considered for vaccine boosting strategies to protect against Omicron BA.4/BA.5 and future SARS-CoV-2 variants.

Reduced Antibody Acquisition with Increasing Age following Vaccination with BNT162b2: Results from Two Longitudinal Cohort Studies in The Netherlands.

Vaccine-induced protection against severe COVID-19, hospitalization, and death is of the utmost importance, especially in the elderly. However, limited data are available on humoral immune responses following COVID-19 vaccination in the general population across a broad age range. We performed an integrated analysis of the effect of age, sex, and prior SARS-CoV-2 infection on Spike S1-specific (S1) IgG concentrations up to three months post-BNT162b2 (Pfizer/BioNTech; Comirnaty) vaccination. In total, 1735 persons, eligible for COVID-19 vaccination through the national program, were recruited from the general population (12 to 92 years old). Sixty percent were female, and the median vaccination interval was 35 days (interquartile range, IQR: 35−35). All participants had seroconverted to S1 one month after two vaccine doses. S1 IgG was higher in participants with a history of SARS-CoV-2 infection (median: 4535 BAU/mL, IQR: 2341−7205) compared to infection-naive persons (1842 BAU/mL, 1019−3116), p < 0.001. In infection-naive persons, linear mixed effects regression showed a strong negative association between age and S1 IgG (p < 0.001) across the entire age range. Females had higher S1 IgG than males (p < 0.001). In persons with an infection history, age nor sex was associated with S1 IgG concentrations. The lower magnitude of S1 antibodies in older persons following COVID-19 vaccination will affect long-term protection.

KLRG1 and PD-1 expression are increased on T-cells following tuberculosis-treatment and identify cells with different proliferative capacities in BCG-vaccinated adults.

In cancer and chronic infectious diseases, immune checkpoint-blockade of inhibitory receptors can enhance T-cell immunity. In tuberculosis (TB), a chronic infectious disease, prolonged antigen exposure can potentially drive terminal T-cell differentiation towards functional 'exhaustion': in human TB T-cells express PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein-4). However, in murine TB not PD-1 but rather killer cell lectin-like receptor subfamily-G1 (KLRG1) was a superior indicator of terminal T-cell differentiation. We therefore compared expression of KLRG1, PD-1 and CTLA-4 on T-cells in different stages of human TB, and also analysed their induction following BCG-vaccination. KLRG1, PD-1 and CTLA-4-expression were highest on in vitro BCG-stimulated CD4(+) T-cells following recent TB-treatment; KLRG1 and PD-1-expression on CD4(+) T-cells in active--but not latent--TB were only slightly increased compared to healthy donors. BCG-vaccination induced KLRG1-expression on BCG-stimulated CD8(+) but not CD4(+) T-cells, while neither PD-1 nor CTLA-4-expression increased. KLRG1-expressing CD8(+) T-cells exhibited markedly decreased proliferation, whereas PD-1(+) T-cells proliferated after in vitro BCG-stimulation. Thus, we demonstrate the presence of increased KLRG1-expressing T-cells in TB-treated individuals, and present KLRG1 as a marker of decreased human T-cell proliferation following BCG-vaccination. These results expand our understanding of cell-mediated immune control of mycobacterial infections.

Regulatory T-Cells at the Interface between Human Host and Pathogens in Infectious Diseases and Vaccination.

Regulatory T-cells (Tregs) act at the interface of host and pathogen interactions in human infectious diseases. Tregs are induced by a wide range of pathogens, but distinct effects of Tregs have been demonstrated for different pathogens and in different stages of infection. Moreover, Tregs that are induced by a specific pathogen may non-specifically suppress immunity against other microbes and parasites. Thus, Treg effects need to be assessed not only in homologous but also in heterologous infections and vaccinations. Though Tregs protect the human host against excessive inflammation, they probably also increase the risk of pathogen persistence and chronic disease, and the possibility of disease reactivation later in life. Mycobacterium leprae and Mycobacterium tuberculosis, causing leprosy and tuberculosis, respectively, are among the most ancient microbes known to mankind, and are master manipulators of the immune system toward tolerance and pathogen persistence. The majority of mycobacterial infections occur in settings co-endemic for viral, parasitic, and (other) bacterial coinfections. In this paper, we discuss recent insights in the activation and activity of Tregs in human infectious diseases, with emphasis on early, late, and non-specific effects in disease, coinfections, and vaccination. We highlight mycobacterial infections as important models of modulation of host responses and vaccine-induced immunity by Tregs.

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults.

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ(+)) interleukin 2-positive (IL-2(+)) tumor necrosis factor α-positive (TNF-α(+)) polyfunctional CD4(+) T cells concurrent with CD4(+) IL-17A(+) and CD8(+) IFN-γ(+) T cells or, in contrast, virtually absent cytokine responses with induction of CD8(+) regulatory T cells. Significant induction of polyfunctional CD4(+) IFN-γ(+) IL-2(+) TNF-α(+) T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8(+) T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8(+) regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4(+) T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.

CD8+ regulatory T cells, and not CD4+ T cells, dominate suppressive phenotype and function after in vitro live Mycobacterium bovis-BCG activation of human cells.

Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG), the only currently available vaccine against tuberculosis, has been reported to induce regulatory T cells in humans. The activity of regulatory T cells may not only dampen immunogenicity and protective efficacy of tuberculosis-vaccines, but also hamper diagnosis of infection of tuberculosis, when using immune (e.g. IFNγ-release) assays. Still, in settings of infectious diseases and vaccination, most studies have focused on CD4+ regulatory T cells, and not CD8+ regulatory T-cells. Here, we present a comparative analysis of the suppressive phenotype and function of CD4+ versus CD8+ T cells after in vitro live BCG activation of human cells. Moreover, as BCG is administered as a (partly) live vaccine, we also compared the ability of live versus heatkilled BCG in activating CD4+ and CD8+ regulatory T cell responses. BCG-activated CD8+ T cells consistently expressed higher levels of regulatory T cell markers, and after live BCG activation, density and (co-)expression of markers were significantly higher, compared to CD4+ T cells. Furthermore, selection on CD25-expression after live BCG activation enriched for CD8+ T cells, and selection on co-expression of markers further increased CD8+ enrichment. Ultimately, only T cells activated by live BCG were functionally suppressive and this suppressive activity resided predominantly in the CD8+ T cell compartment. These data highlight the important contribution of live BCG-activated CD8+ Treg cells to immune regulation and emphasize their possible negative impact on immunity and protection against tuberculosis, following BCG vaccination.

Is fatigue in Marfan syndrome related to orthostatic intolerance?

Patients with Marfan syndrome have a tall stature, which could be associated with low orthostatic tolerance. Fatigue, a common complaint of these patients, is also related to orthostatic intolerance. Treatment with beta-blockers, to prevent aortic complications, could be a reinforcing factor of both. This study aimed to investigate (1) the relationship between symptoms of orthostatic tolerance and in patients with Marfan syndrome, and (2) whether termination of beta-blocker therapy improves orthostatic tolerance. Symptoms of fatigue and orthostatic complaints were assessed in 49 subjects using questionnaires (MFI-20 and Autonomic Symptoms Profile). Marfan patients have a high level of fatigue and orthostatic complaints when compared to the general population. Fatigue and orthostatic tolerance are significantly correlated. Orthostatic tolerance was assessed in 9 additional subjects by an active-standing test and head-up tilt for 5 minutes, and 24 hours blood pressure monitoring, once during beta-blocker therapy and once after ceasing beta-blockers for 2 weeks. During hemodynamic testing Marfan patients frequently showed Initial Orthostatic Hypotension and an abnormally high initial heart rate response. Ceasing beta-blockers did not affect the initial blood pressure response. Patients with Marfan syndrome are fatigued and have low orthostatic tolerance, which are significantly correlated. Patients could be educated in physical counterpressure maneuvers to increase orthostatic tolerance.

Daily, weekly, monthly, and seasonal patterns in the occurrence of vasovagal syncope in an older population.

AIMS: The aim of this study was to assess the frequency of vasovagal episodes over the day, week, month, and seasons. METHODS AND RESULTS: This study was part of the multi-centre International Study on Syncope of Uncertain Etiology-2 (ISSUE-2), which included patients, aged 30 years or older, with severe neurally mediated syncope between June 2002 and July 2004. The Implantable Loop Recorder (ILR) was used to document the syncope-related ECG periods. For this study patients with recorded syncopal episodes after ILR-implantation was selected. At least one episode was documented in 106 patients. A higher number of episodes were documented during the morning than during other periods of the day (P < 0.01). There was no difference between various days of the week, episodes per month, or between seasons. There was no difference between age and gender groups, although elderly patients seemed to be responsible for the peak in the morning. CONCLUSION: A circadian pattern in the frequency of vasovagal episodes exists, with a peak in the morning. This is in accordance with reports of diurnal variations in blood pressure and heart rate. No difference was observed in syncope distribution between days of the week, months, or seasons.