B Cells from Aged Mice Do Not Have Intrinsic Defects in Affinity Maturation in Response to Immunization.

Affinity maturation, the progressive increase in serum Ab affinity after vaccination, is an essential process that contributes to an effective humoral response against vaccines and infections. Germinal centers are key for affinity maturation, because they are where B cells undergo somatic hypermutation of their Ig genes in the dark zone before going through positive selection in the light zone via interactions with T follicular helper cells and follicular dendritic cells. In aged mice, affinity maturation has been shown to be impaired after immunization, but whether B cell-intrinsic factors contribute to this defect remains unclear. In this study, we show that B cells from aged BCR transgenic mice are able to become germinal center B cells, which are capable of receiving positive selection signals to a similar extent as B cells from young adult mice. Consistent with this, aging also does not impact the ability of B cells to undergo somatic hypermutation and acquire affinity-enhancing mutations. By contrast, transfer of B cells from young adult BCR mice into aged recipients resulted in the impaired acquisition of affinity-enhancing mutations, demonstrating that the aged microenvironment causes altered affinity maturation.

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.

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.

A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice.

BACKGROUND: The spread of SARS-CoV-2 has caused a worldwide pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection and may enable the relaxation of social-distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection; therefore, it is desirable that any new vaccine candidates elicit a robust immune response in older adults. METHODS: Here, we use in-depth immunophenotyping to characterize the innate and adaptive immune response induced upon intramuscular administration of the adenoviral vectored ChAdOx1 nCoV-19 (AZD-1222) COVID-19 vaccine candidate in mice. FINDINGS: A single vaccination generates spike-specific Th1 cells, Th1-like Foxp3+ regulatory T cells, polyfunctional spike-specific CD8+ T cells. and granzyme-B-producing CD8 effectors. Spike-specific IgG and IgM are generated from both the early extrafollicular antibody response and the T follicular helper cell-supported germinal center reaction, which is associated with the production of virus-neutralizing antibodies. A single dose of this vaccine generated a similar type of immune response in aged mice but of a reduced magnitude than in younger mice. We report that a second dose enhances the immune response to this vaccine in aged mice. CONCLUSIONS: This study shows that ChAdOx1 nCoV-19 induces both cellular and humoral immunity in adult and aged mice and suggests a prime-boost strategy is a rational approach to enhance immunogenicity in older persons. FUNDING: This study was supported by BBSRC, Lister institute of Preventative Medicine, EPSRC VaxHub, and Innovate UK.

Stromal cell control of conventional and ectopic germinal centre reactions.

The germinal centre (GC) is a specialized cellular structure that forms in response to antigenic stimulation. It generates long-term humoral immunity through the production of memory B cells and long-lived antibody-secreting plasma cells. Conventional GCs form within secondary lymphoid organs, where networks of specialised stromal cells that form during embryogenesis act as the stage upon which the various GC immune cell players are brought together, nurtured and co-ordinated to generate a productive response. In non-lymphoid organs, ectopic GCs can form in response to persistent antigenic and inflammatory stimuli. Unlike secondary lymphoid tissues, non-lymphoid organs do not have a developmentally programmed stromal cell network capable of supporting the germinal centre reaction; therefore, the local tissue stroma must be remodelled by inflammatory stimuli in order to host a GC reaction. These ectopic GCs produce memory B cells and plasma cells that form a critical component of the humoral immune response.

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.

Heterochronic faecal transplantation boosts gut germinal centres in aged mice.

Ageing is a complex multifactorial process associated with a plethora of disorders, which contribute significantly to morbidity worldwide. One of the organs significantly affected by age is the gut. Age-dependent changes of the gut-associated microbiome have been linked to increased frailty and systemic inflammation. This change in microbial composition with age occurs in parallel with a decline in function of the gut immune system; however, it is not clear whether there is a causal link between the two. Here we report that the defective germinal centre reaction in Peyer's patches of aged mice can be rescued by faecal transfers from younger adults into aged mice and by immunisations with cholera toxin, without affecting germinal centre reactions in peripheral lymph nodes. This demonstrates that the poor germinal centre reaction in aged animals is not irreversible, and that it is possible to improve this response in older individuals by providing appropriate stimuli.

Regulation of the Germinal Center Response.

The germinal center (GC) is a specialized microstructure that forms in secondary lymphoid tissues, producing long-lived antibody secreting plasma cells and memory B cells, which can provide protection against reinfection. Within the GC, B cells undergo somatic mutation of the genes encoding their B cell receptors which, following successful selection, can lead to the emergence of B cell clones that bind antigen with high affinity. However, this mutation process can also be dangerous, as it can create autoreactive clones that can cause autoimmunity. Because of this, regulation of GC reactions is critical to ensure high affinity antibody production and to enforce self-tolerance by avoiding emergence of autoreactive B cell clones. A productive GC response requires the collaboration of multiple cell types. The stromal cell network orchestrates GC cell dynamics by controlling antigen delivery and cell trafficking. T follicular helper (Tfh) cells provide specialized help to GC B cells through cognate T-B cell interactions while Foxp3+ T follicular regulatory (Tfr) cells are key mediators of GC regulation. However, regulation of GC responses is not a simple outcome of Tfh/Tfr balance, but also involves the contribution of other cell types to modulate the GC microenvironment and to avoid autoimmunity. Thus, the regulation of the GC is complex, and occurs at multiple levels. In this review we outline recent developments in the biology of cell subsets involved in the regulation of GC reactions, in both secondary lymphoid tissues, and Peyer's patches (PPs). We discuss the mechanisms which enable the generation of potent protective humoral immunity whilst GC-derived autoimmunity is avoided.