Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging.

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.

Tissue CD14+CD8+ T cells reprogrammed by myeloid cells and modulated by LPS.

The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours1-4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability.

The memory B cell response to influenza vaccination is impaired in older persons.

Influenza infection imparts an age-related increase in mortality and morbidity. The most effective countermeasure is vaccination; however, vaccines offer modest protection in older adults. To investigate how aging impacts the memory B cell response, we track hemagglutinin-specific B cells by indexed flow sorting and single-cell RNA sequencing (scRNA-seq) in 20 healthy adults that were administered the trivalent influenza vaccine. We demonstrate age-related skewing in the memory B cell compartment 6 weeks after vaccination, with younger adults developing hemagglutinin-specific memory B cells with an FcRL5+ "atypical" phenotype, showing evidence of somatic hypermutation and positive selection, which happened to a lesser extent in older persons. We use publicly available scRNA-seq from paired human lymph node and blood samples to corroborate that FcRL5+ atypical memory B cells can derive from germinal center (GC) precursors. Together, this study shows that the aged human GC reaction and memory B cell response following vaccination is defective.

B cell-intrinsic changes with age do not impact antibody-secreting cell formation but delay B cell participation in the germinal centre reaction.

Vaccines typically protect against (re)infections by generating pathogen-neutralising antibodies. However, as we age, antibody-secreting cell formation and vaccine-induced antibody titres are reduced. Antibody-secreting plasma cells differentiate from B cells either early post-vaccination through the extrafollicular response or from the germinal centre (GC) reaction, which generates long-lived antibody-secreting cells. As the formation of both the extrafollicular antibody response and the GC requires the interaction of multiple cell types, the impaired antibody response in ageing could be caused by B cell intrinsic or extrinsic factors, or a combination of the two. Here, we show that B cells from older people do not have intrinsic defects in their proliferation and differentiation into antibody-secreting cells in vitro compared to those from the younger donors. However, adoptive transfer of B cells from aged mice to young recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the GC during the early stages of GC formation. In contrast, by the peak of the GC response, GC B cells derived from the donor cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age-related intrinsic B cell changes delay the GC response but are not responsible for the impaired antibody-secreting response or smaller peak GC response in ageing. Collectively, this study shows that B cells from aged individuals are not intrinsically defective in responding to stimulation and becoming antibody-secreting cells, implicating B cell-extrinsic factors as the primary cause of age-associated impairment in the humoral immunity.

Isolation of human intrahepatic leukocytes for phenotypic and functional characterization by flow cytometry.

With the growing appreciation of tissue-resident immunity, studying tissue-specific immune cells contributing to both homeostasis and disease is imperative. Here, we provide a protocol for the isolation of human intrahepatic leukocytes (IHL) maximizing viability, purity, and yield. Our protocol is scalable by tissue weight, allowing for reproducible and efficient IHL liberation suitable for functional characterization, cell isolation, and profiling by flow (or mass) cytometry. Furthermore, we provide a "guide" to determine an expected IHL yield per gram of tissue processed. For complete details on the use and execution of this protocol, please refer to Stegmann et al. (2016), Pallett et al. (2017), Easom et al. (2018), Swadling et al. (2020), Pallett et al. (2020), and Zakeri et al. (2022).

Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections.

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines.

Attributable factors for the rising cesarean delivery rate over 3 decades: an observational cohort study.

BACKGROUND: Cesarean delivery rates continue to rise globally, the reasons for which are incompletely understood. OBJECTIVE: We aimed to characterize the attributable factors for the increasing cesarean delivery rates over a 30-year period within our health network. STUDY DESIGN: This was a planned observational cohort study across 2 hospitals (a large tertiary referral hospital and a metropolitan hospital) in Sydney, Australia using data from a previously published study. The following 2 time periods were compared: 1989-99 and 2009-16, between which the cesarean delivery rate increased from 19% to 30%. The participants were all women who had a cesarean delivery after 24 weeks' gestational age. The data were analyzed using multiple imputation and robust Poisson regression to calculate the differences in the adjusted and unadjusted relative risk of cesarean delivery and estimate the changes in the cesarean delivery rate attributable to maternal and clinical factors. The primary outcome was cesarean delivery. RESULTS: After 576 exclusions, 102,589 births were included in the analysis. Fifty-six percent of the increase in the rate of cesarean delivery was attributed to changes in the distribution of the maternal age, body mass index, and parity and to a history of previous cesarean delivery. An additional 10% of the increase was attributed to changes in the obstetrical management of the following high-risk pregnancies: multiple gestation, malpresentation (mainly breech), and preterm singleton birth. When prelabor cesarean deliveries for maternal choice, suspected fetal compromise, previous pregnancy issues, and suspected large fetus were excluded, 78% of the increase was attributed to either maternal factors or changes in the obstetrical management of these high-risk pregnancies. CONCLUSION: Most of the steep rise in the cesarean delivery rate from 19% to 30% was attributed to changes in the maternal demographic and clinical factors. This observation is relevant to developing preventative strategies that account for nulliparity, age, body mass index, and management of high-risk pregnancies.

SARS-CoV-2-specific memory B cells can persist in the elderly who have lost detectable neutralizing antibodies.

Memory B cells (MBCs) can provide a recall response able to supplement waning antibodies (Abs) with an affinity-matured response better able to neutralize variant viruses. We studied a cohort of elderly care home residents and younger staff (median age of 87 years and 56 years, respectively), who had survived COVID-19 outbreaks with only mild or asymptomatic infection. The cohort was selected because of its high proportion of individuals who had lost neutralizing antibodies (nAbs), thus allowing us to specifically investigate the reserve immunity from SARS-CoV-2-specific MBCs in this setting. Class-switched spike and receptor-binding domain (RBD) tetramer-binding MBCs persisted 5 months after mild or asymptomatic SARS-CoV-2 infection, irrespective of age. The majority of spike- and RBD-specific MBCs had a classical phenotype, but we found that activated MBCs, indicating possible ongoing antigenic stimulation or inflammation, were expanded in the elderly group. Spike- and RBD-specific MBCs remained detectable in the majority of individuals who had lost nAbs, although at lower frequencies and with a reduced IgG/IgA isotype ratio. Functional spike-, S1 subunit of the spike protein- (S1-), and RBD-specific recall was also detectable by enzyme-linked immune absorbent spot (ELISPOT) assay in some individuals who had lost nAbs, but was significantly impaired in the elderly. Our findings demonstrate that a reserve of SARS-CoV-2-specific MBCs persists beyond the loss of nAbs but highlight the need for careful monitoring of functional defects in spike- and RBD-specific B cell immunity in the elderly.

Factors affecting cord blood leptin levels in a consecutive birth cohort.

INTRODUCTION: The fetus that fails to meet its ideal growth trajectory has increased risks of poor health outcomes throughout life. "Gold standard" methods of anthropometric assessment such as measurement of percentage body fat can be difficult to apply across populations and other biomarkers such as serum concentration of umbilical cord blood leptin may be more effective for screening. This study reports cord blood leptin levels in a large prospective consecutive birth cohort and assesses the relationship between leptin and neonatal and maternal factors. METHODS: Venous umbilical cord blood samples were collected from a prospective consecutive cohort of pregnancies at the time of delivery. Maternal and neonatal characteristics and details of delivery were collated. Serum leptin levels were measured, associations with demographic features were identified, and a normal range was established. The association between cord leptin level and neonatal outcome was tested. RESULTS: Umbilical cord leptin and maternal and neonatal characteristics were collected at 1275 births. The median leptin value was 10.8 ng/ml (IQR: 6.4, 17.8 ng/ml). Log10 leptin was significantly associated with gestation at delivery, birthweight (BWt), infant sex, plurality, and maternal body mass index (BMI) (p < .001). Observed leptin values were expressed as multiples of the median (MoM). The mean leptin MoM was significantly lower in infants admitted to NICU following delivery (0.85; 95% confidence interval [CI]: 0.78-0.91 versus 1.05; 95% CI: 1.03-1.06 (controls), p < .001). There was no significant association between leptin MoM values and 5-min Apgar scores. CONCLUSIONS: Neonatal cord leptin levels are influenced by a number of maternal and fetal characteristics. Absolute levels can be adjusted to account for normal population variation. Infants requiring admission to NICU have lower mean leptin MoM levels. Further studies are needed to see whether the identification of fetuses with polarized leptin levels (<5th or >95th centile) will benefit from further surveillance or intervention in infancy.

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint.

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

Human antiviral B cell responses: Emerging lessons from hepatitis B and COVID-19.

Humoral immunity is a critical component of the coordinated response required to resolve viral infections and mediate protection following pathogen clearance or vaccination. A better understanding of factors shaping the memory B cell response will allow tailored development of efficient preventative vaccines against emerging acute viral infections, therapeutic vaccines, and immunotherapies for chronic viral infections. Here, we use recent data obtained by profiling antigen-specific B cell responses in hepatitis B as a framework to explore lessons that can be learnt from different viral infections about the diverse influences on humoral immunity. Hepatitis B provides a paradigm where successful B cell responses in resolved or vaccinated individuals can be contrasted to the failed response in chronic infection, while also exemplifying the degree to which B cell responses within infected individuals can differ to two antigens from the same virus. Drawing on studies in other human and murine infections, including emerging data from COVID-19, we consider the influence of antigen quantity and structure on the quality of the B cell response, the role of differential CD4 help, the importance of germinal center vs extrafollicular responses and the emerging concept that responses residing in non-lymphoid organs can participate in B cell memory.

A hospital-based cohort study of gender and gestational age-specific body fat percentage at birth.

BACKGROUND: Birthweight is the most commonly used proxy marker but does not adequately define true nutritional status. Modalities like DXA (dual energy x-ray absorptiometry) and TOtal Body Electric Conductivity (TOBEC) have been validated to assess body composition but their accuracy in neonates has not been established. The PEAPOD (COSMED, Rome Italy) has been validated as an accurate tool for measuring percentage body fat (%BF) in newborns. The study aim was to determine the gender-specific %BF percentiles at different gestations (35-41 weeks) for a healthy population of newborn infants. A secondary aim was to determine whether there is any relationship between %BF and neonatal condition at birth (cord gas measurement). METHODS: %BF was measured using air displacement plethysmography (PEAPOD) within 6 h of birth. RESULTS: There is an increase in the mean %BF with increasing gestation for female and males from 36 weeks' gestation in the 7667 infants who underwent assessment. Females have a higher %BF than their male equivalents. There was no correlation between %BF and cord pH. CONCLUSION: Gender and gestation are both important in determining the quantiles and mean %BF at birth. There was no correlation between low cord pH and %BF. IMPACT: Measuring the percentage body fat (PEAPOD) at birth is a useful marker of an infant's nutritional status. This is the largest hospital-based cohort of gestational age and gender-specific %BF in healthy newborns. The normative graphs from this study will help to accurately determine high-risk infants with low %BF so they can be monitored appropriately.

Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes.

The human liver contains specialized subsets of mononuclear phagocytes (MNPs) and T cells, but whether these have definitive features of tissue residence (long-term retention, lack of egress) and/or can be replenished from the circulation remains unclear. Here we addressed these questions using HLA-mismatched liver allografts to discriminate the liver-resident (donor) from the infiltrating (recipient) immune composition. Allografts were rapidly infiltrated by recipient leukocytes, which recapitulated the liver myeloid and lymphoid composition, and underwent partial reprogramming with acquisition of CD68/CD206 on MNPs and CD69/CD103 on T cells. The small residual pool of donor cells persisting in allografts for over a decade contained CX3CR1hi/CD163hi/CD206hi Kupffer cells (KCs) and CXCR3hi tissue-resident memory T cells (TRM). CD8+ TRM were found in the local lymph nodes but were not detected egressing into the hepatic vein. Our findings inform organ transplantation and hepatic immunotherapy, revealing remarkably long-lived populations of KCs and TRM in human liver, which can be additionally supplemented by their circulating counterparts.

Human Liver Memory CD8+ T Cells Use Autophagy for Tissue Residence.

Tissue-resident memory T cells have critical roles in long-term pathogen and tumor immune surveillance in the liver. We investigate the role of autophagy in equipping human memory T cells to acquire tissue residence and maintain functionality in the immunosuppressive liver environment. By performing ex vivo staining of freshly isolated cells from human liver tissue, we find that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells. CD8+ T cells with increased autophagy are those best able to proliferate and mediate cytotoxicity and cytokine production. Conversely, blocking autophagy induction results in the accumulation of depolarized mitochondria, a feature of exhausted T cells. Primary hepatic stellate cells or the prototypic hepatic cytokine interleukin (IL)-15 induce autophagy in parallel with tissue-homing/retention markers. Inhibition of T cell autophagy abrogates tissue-residence programming. Thus, upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence.

Cesarean delivery: Trend in indications over three decades within a major city hospital network.

INTRODUCTION: The cesarean delivery rate has been increasing globally in recent decades. The reasons for this are complex and subject to ongoing debate. Investigation of the indications for cesarean delivery and how these have changed over an extended period of time could provide insight into the reasons for changing obstetric practice. Our objective was to explore contributing factors to the increasing rate of cesarean delivery by examining the incidence of and indications for cesarean delivery over the past three decades at our institutions. MATERIAL AND METHODS: We conducted a retrospective observational study of all cesarean deliveries, from 24 weeks' gestational age onwards, within an inner-city hospital network in Sydney, Australia, between August 1989 and December 2016. The primary outcome measures were the rates of and indications for emergency and planned cesarean delivery. We also examined our data within the Robson 10-Group Classification system. RESULTS: There were 147 722 births over the study period, with 37 309 cesarean deliveries for an overall rate of 25.3%. The rate of cesarean delivery increased from 18.7% in 1989-1994 (8.7% emergency, 10% planned) to 30.4% in 2010-2016 (11.4% emergency, 19% planned). Emergency cesarean delivery for slow progress increased from 3.4% to 5.5% of all births (a relative increase of 62%) and other emergency cesareans mainly performed for suspected intrapartum fetal compromise increased from 5.2% to 5.6% (a relative increase of 8%). Previous uterine surgery (predominantly cesarean section) was the largest contributor to the increase in planned procedures from 3.8% to 9.0% of all births, and 29% of all cesarean deliveries. Primary cesarean delivery for planned antenatal fetal indications, previous pregnancy problems, multiple gestation and maternal choice all increased substantially in combined rate from 0.7% to 4.9%. Cesarean rates in Robson groups 6, 7 and 8 (term breech and multiple gestations) increased most over time. CONCLUSIONS: The increased rate of cesarean delivery is mainly attributable to a greater number of procedures performed for slow progress in labor, breech presentation or repeat cesarean section.

Restoring, releasing or replacing adaptive immunity in chronic hepatitis B.

Multiple new therapeutic approaches are currently being developed to achieve sustained, off-treatment suppression of HBV, a persistent hepatotropic infection that kills ~2,000 people a day. A fundamental therapeutic goal is the restoration of robust HBV-specific adaptive immune responses that are able to maintain prolonged immunosurveillance of residual infection. Here, we provide insight into key components of successful T cell and B cell responses to HBV, discussing the importance of different specificities and effector functions, local intrahepatic immunity and pathogenic potential. We focus on the parallels and interactions between T cell and B cell responses, highlighting emerging areas for future investigation. We review the potential for different immunotherapies in development to restore or release endogenous adaptive immunity by direct or indirect approaches, including limitations and risks. Finally, we consider an alternative HBV treatment strategy of replacing failed endogenous immunity with infusions of highly targeted T cells or antibodies.

Fine needle aspirates comprehensively sample intrahepatic immunity.

OBJECTIVE: In order to refine new therapeutic strategies in the pipeline for HBV cure, evaluation of virological and immunological changes compartmentalised at the site of infection will be required. We therefore investigated if liver fine needle aspirates (FNAs) could comprehensively sample the local immune landscape in parallel with viable hepatocytes. DESIGN: Matched blood, liver biopsy and FNAs from 28 patients with HBV and 15 without viral infection were analysed using 16-colour multiparameter flow cytometry. RESULTS: The proportion of CD4 T, CD8 T, Mucosal Associated Invariant T cell (MAIT), Natural Killer (NK) and B cells identified by FNA correlated with that in liver biopsies from the same donors. Populations of Programmed Death-1 (PD-1)hiCD39hi tissue-resident memory CD8 T cells (CD69+CD103+) and liver-resident NK cells (CXCR6+T-betloEomeshi), were identified by both FNA and liver biopsy, and not seen in the blood. Crucially, HBV-specific T cells could be identified by FNAs at similar frequencies to biopsies and enriched compared with blood. FNAs could simultaneously identify populations of myeloid cells and live hepatocytes expressing albumin, Scavenger Receptor class B type 1 (SR-B1), Programmed Death-Ligand 1 (PD-L1), whereas hepatocytes were poorly viable after the processing required for liver biopsies. CONCLUSION: We demonstrate for the first time that FNAs identify a range of intrahepatic immune cells including locally resident sentinel HBV-specific T cells and NK cells, together with PD-L1-expressing hepatocytes. In addition, we provide a scoring tool to estimate the extent to which an individual FNA has reliably sampled intrahepatic populations rather than contaminating blood. The broad profiling achieved by this less invasive, rapid technique makes it suitable for longitudinal monitoring of the liver to optimise new therapies for HBV.