Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

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.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Sec22b is a critical and nonredundant regulator of plasma cell maintenance.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

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.

Long-lived plasma cells accumulate in the bone marrow at a constant rate from early in an immune response.

Vaccines work largely by generating long-lived plasma cells (LLPCs), but knowledge of how such cells are recruited is sparse. Although it is clear that LLPCs preferentially originate in germinal centers (GCs) and relocate to survival niches in bone marrow where they can persist for decades, the issues of the timing of LLPC recruitment and the basis of their retention remain uncertain. Here, using a genetic timestamping system in mice, we show that persistent PCs accrue in bone marrow at an approximately constant rate of one cell per hour over a period spanning several weeks after a single immunization with a model antigen. Affinity-based selection was evident in persisting PCs, reflecting a relative and dynamic rather than absolute affinity threshold as evidenced by the changing pattern of VH gene somatic mutations conveying increased affinity for antigen. We conclude that the life span of persistent, antigen-specific PCs is in part intrinsic, preprogrammed, and varied and that their final number is related to the duration of the response in a predictable way. This implies that modulating vaccines to extend the duration of the GC reaction will enhance antibody-mediated protective immunity.

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.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

Targeting TLR4 during vaccination boosts MAdCAM-1+ lymphoid stromal cell activation and promotes the aged germinal center response.

The failure to generate enduring humoral immunity after vaccination is a hallmark of advancing age. This can be attributed to a reduction in the germinal center (GC) response, which generates long-lived antibody-secreting cells that protect against (re)infection. Despite intensive investigation, the primary cellular defect underlying impaired GCs in aging has not been identified. Here, we used heterochronic parabiosis to demonstrate that GC formation was dictated by the age of the lymph node (LN) microenvironment rather than the age of the immune cells. Lymphoid stromal cells are a key determinant of the LN microenvironment and are also an essential component underpinning GC structure and function. Using mouse models, we demonstrated that mucosal adressin cell adhesion molecule-1 (MAdCAM-1)-expressing lymphoid stromal cells were among the first cells to respond to NP-KLH + Alum immunization, proliferating and up-regulating cell surface proteins such as podoplanin and cell adhesion molecules. This response was essentially abrogated in aged mice. By targeting TLR4 using adjuvants, we improved the MAdCAM-1+ stromal cell response to immunization. This correlated with improved GC responses in both younger adult and aged mice, suggesting a link between stromal cell responses to immunization and GC initiation. Using bone marrow chimeras, we also found that MAdCAM-1+ stromal cells could respond directly to TLR4 ligands. Thus, the age-associated defect in GC and stromal cell responses to immunization can be targeted to improve vaccines in older people.

T follicular helper cells and their impact on humoral responses during pathogen and vaccine challenge.

T follicular helper (Tfh) cells are essential for the establishment, maintenance and output of the germinal centre (GC) response. The transient nature of this response, and its location within secondary lymphoid tissues have hampered our understanding of this critical cell type, particularly in humans. A counterpart of GC Tfh cells in peripheral blood has enabled recent discoveries in disease and vaccination settings, while direct sampling of lymph nodes provides exciting new avenues to study GC responses directly in vivo. Tfh differentiation is shaped by the cytokine milieu during inflammation, vaccination and with age, and disease-specific patterns are emerging. An improved understanding of how to support a Tfh response remains key to enhancing vaccine immunity across the lifespan.

Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans.

Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us to better understand the cellular and molecular mechanisms that underpin successful vaccination in order to improve vaccine design for at-risk groups. Here, by studying the breadth of anti-haemagglutinin (HA) IgG, serum cytokines, and B and T cell responses by flow cytometry before and after influenza vaccination, we show that formation of circulating T follicular helper (cTfh) cells was associated with high-titre antibody responses. Using Major Histocompatability Complex (MHC) class II tetramers, we demonstrate that HA-specific cTfh cells can derive from pre-existing memory CD4+ T cells and have a diverse T cell receptor (TCR) repertoire. In older people, the differentiation of HA-specific cells into cTfh cells was impaired. This age-dependent defect in cTfh cell formation was not due to a contraction of the TCR repertoire, but rather was linked with an increased inflammatory gene signature in cTfh cells. Together, this suggests that strategies that temporarily dampen inflammation at the time of vaccination may be a viable strategy to boost optimal antibody generation upon immunisation of older people.

The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses.

Follicular helper T (TFH) cells control antibody responses by supporting antibody affinity maturation and memory formation. Inadequate TFH function has been found in individuals with ineffective responses to vaccines, but the mechanism underlying TFH regulation in vaccination is not understood. Here, we report that lower serum levels of the metabolic hormone leptin associate with reduced vaccine responses to influenza or hepatitis B virus vaccines in healthy populations. Leptin promotes mouse and human TFH differentiation and IL-21 production via STAT3 and mTOR pathways. Leptin receptor deficiency impairs TFH generation and antibody responses in immunisation and infection. Similarly, leptin deficiency induced by fasting reduces influenza vaccination-mediated protection for the subsequent infection challenge, which is mostly rescued by leptin replacement. Our results identify leptin as a regulator of TFH cell differentiation and function and indicate low levels of leptin as a risk factor for vaccine failure.

Dairy manure as a potential source of crop nutrients and environmental contaminants.

Although animal manure is applied to agricultural fields for its nutrient value, it may also contain potential contaminants. To determine the variability in such contaminants as well as in valuable nutrients, nine uncomposted manure samples from Idaho dairies collected during 2.5 years were analyzed for macro- and micro-nutrients, hormones, phytoestrogens, antibiotics, veterinary drugs, antibiotic resistance genes, and genetic elements involved in the spread of antibiotic resistance. Total N ranged from 6.8 to 30.7 (C:N of 10 to 21), P from 2.4 to 9.0, and K from 10.2 to 47.7 g/kg manure. Zn (103 - 348 mg/kg) was more abundant than Cu (56 - 127 mg/kg) in all samples. Phytoestrogens were the most prevalent contaminants detected, with concentrations fluctuating over time, reflecting animal diets. This is the first study to document the presence of flunixin, a non-steroidal anti-inflammatory drug, in solid stacked manure from regular dairy operations. Monensin was the most frequently detected antibiotic. Progesterones and sulfonamides were regularly detected. We also investigated the relative abundance of several types of plasmids involved in the spread of antibiotic resistance in clinical settings. Plasmids belonging to the IncI, IncP, and IncQ1 incompatibility groups were found in almost all manure samples. IncQ1 plasmids, class 1 integrons, and sulfonamide resistance genes were the most widespread and abundant genetic element surveyed, emphasizing their potential role in the spread of antibiotic resistance. The benefits associated with amending agricultural soils with dairy manure must be carefully weighed against the potential negative consequences of any manure contaminants.

Rejuvenating conventional dendritic cells and T follicular helper cell formation after vaccination.

Germinal centres (GCs) are T follicular helper cell (Tfh)-dependent structures that form in response to vaccination, producing long-lived antibody secreting plasma cells and memory B cells that protect against subsequent infection. With advancing age the GC and Tfh cell response declines, resulting in impaired humoral immunity. We sought to discover what underpins the poor Tfh cell response in ageing and whether it is possible to correct it. Here, we demonstrate that older people and aged mice have impaired Tfh cell differentiation upon vaccination. This deficit is preceded by poor activation of conventional dendritic cells type 2 (cDC2) due to reduced type 1 interferon signalling. Importantly, the Tfh and cDC2 cell response can be boosted in aged mice by treatment with a TLR7 agonist. This demonstrates that age-associated defects in the cDC2 and Tfh cell response are not irreversible and can be enhanced to improve vaccine responses in older individuals.

Immune system development varies according to age, location, and anemia in African children.

Children from low- and middle-income countries, where there is a high incidence of infectious disease, have the greatest need for the protection afforded by vaccination, but vaccines often show reduced efficacy in these populations. An improved understanding of how age, infection, nutrition, and genetics influence immune ontogeny and function is key to informing vaccine design for this at-risk population. We sought to identify factors that shape immune development in children under 5 years of age from Tanzania and Mozambique by detailed immunophenotyping of longitudinal blood samples collected during the RTS,S malaria vaccine phase 3 trial. In these cohorts, the composition of the immune system is dynamically transformed during the first years of life, and this was further influenced by geographical location, with some immune cell types showing an altered rate of development in Tanzanian children compared to Dutch children enrolled in the Generation R population-based cohort study. High-titer antibody responses to the RTS,S/AS01E vaccine were associated with an activated immune profile at the time of vaccination, including an increased frequency of antibody-secreting plasmablasts and follicular helper T cells. Anemic children had lower frequencies of recent thymic emigrant T cells, isotype-switched memory B cells, and plasmablasts; modulating iron bioavailability in vitro could recapitulate the B cell defects observed in anemic children. Our findings demonstrate that the composition of the immune system in children varies according to age, geographical location, and anemia status.

The adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRß clonotypes.

The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE-formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRß clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines.

Peptidase inhibitor 16 identifies a human regulatory T-cell subset with reduced FOXP3 expression over the first year of recent onset type 1 diabetes.

CD4+ T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16+ Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes.

Transport of Potential Manure Hormone and Pharmaceutical Contaminants through Intact Soil Columns.

Although adding manure to agricultural soils is a commonly practiced disposal method and a means to enhance soil productivity, potential environmental contamination by any associated chemicals of emerging concern (CECs) such as hormones and pharmaceuticals is not well understood. Our objective was to provide field-relevant predictions of soil transport and attenuation of 19 potential manure CECs using undisturbed soil columns irrigated under unsaturated conditions. The CEC concentrations in leached water were monitored for 13 wk using high performance liquid chromatography-time of flight-mass spectrometry (HPLC-TOF-MS), after which time soil in the cores was removed and sampled for extractable CECs. Compounds quantified in column leachate included all four of the added sulfonamide antibiotics and the nonsteroidal, anti-inflammatory drug flunixin. Only trace amounts of several of the seven hormones, five remaining antibiotics, and two antimicrobials leached from the columns from exogenous soil additions. Soil residues of all 19 compounds were detected, with highest extractable amounts for 17α-hydroxyprogesterone > triclosan (antimicrobial) > flunixin > oxytetracycline. Those CECs with the highest recoveries as calculated by summing leached and extractable amounts were flunixin (14.5%), 17α-hydroxyprogesterone (5.3%), triclosan (4.6%), and sulfadimethoxine (4.8%). Manure management to prevent CEC contamination should consider the potential environmental problems caused by negatively charged compounds with the greatest mobility (flunixin and sulfadimethoxine) and those that have long residence times in soil (triclosan, 17α-hydroxyprogesterone, flunixin, and oxytetracycline). Flunixin is particularly important given its mobility and long residence time in soil.

The Calcineurin Inhibitor Tacrolimus Specifically Suppresses Human T Follicular Helper Cells.

Background: T follicular helper (Tfh) cells are key players in the production of antibody-producing B cells via the germinal center reaction. Therapeutic strategies targeting Tfh cells are important where antibody formation is implicated in disease, such as transplant rejection and autoimmune diseases. We investigated the impact of the immunosuppressive agent tacrolimus on human Tfh cell differentiation and function in transplant recipients. Methods: Paired blood and lymph node (LN) samples were obtained from 61 transplant recipients immediately prior to organ implantation. Living-donor recipients received a week of tacrolimus prior to kidney transplantation. Deceased-donor recipients served as controls, as tacrolimus was not administered until after the transplant operation. Flow cytometry was used to compare LN and circulating cell subsets. Results: The calcineurin inhibitor (CNIs) tacrolimus specifically suppresses both LN Tfh cells and circulating Tfh cells, but not their regulatory counterparts or other CD4 T cell subsets. Conclusion: Our findings suggest that CNIs may have a more important role in the prevention of antibody formation than previously understood and, therefore, have potential for antibody-associated conditions in which aberrant Tfh function has been implicated in disease.