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Memory and innate-like B-cell subsets: deciphering a multi-layered B-cell response in mice and humans

Periodic Reporting for period 4 - B-response (Memory and innate-like B-cell subsets: deciphering a multi-layered B-cell response in mice and humans)

Reporting period: 2021-03-01 to 2021-08-31

Vaccination is one of the most successful advances in human health. Protection induced by vaccination is afforded by antibodies, produced by two types of long-lived memory lymphocytes: memory B cells, which remain quiescent for decades in the organism, ready to respond actively to a new encounter with the pathogen; and long-lived plasma cells, which constantly secrete protective antibodies but do not react to an infectious challenge. Vaccination is mainly an empiric procedure, and, surprisingly, little is known about the diversity of memory B cell subsets generated and/or mobilized in an immune response or on the immune conditions that dictate their longevity. These two questions are the basis of the grant "B-cell response", which addresses them through three independent approaches, both in mice and humans.
These three approaches include:
1. analyzing the diversity of memory B cell subsets in the mouse, through a reporter model in which B cells engaged in an immune response become irreversibly "marked" in a time-controlled fashion, and of the impact of the parameters of the immunization like time elapsed between immunization and recall, nature of the antigen (protein or whole inactivated pathogen) or adjuvants, on the diversity of these subsets and their persistence over time.
2. deciphering the specific subset involved in the immune defense against polysaccharide antigens (the hallmark of bacterial infections) in humans, to understand the origin of their immune diversification in the absence of collaboration with T cells.
3. studying smallpox-specific memory B cells in individuals vaccinated since several decades, to decipher the origin of their longevity, which exceeds the one of their host, in terms of transcriptional program, metabolism and cellular homeostasis.
4. a fourth approach was included in 2020, to describe the memory B cell responses against SARS-Cov-2
Studies performed in a germinal center fate mapping mouse reporter line have led to the unanticipated discovery of a large IgM+ memory subset, which arises spontaneously in the spleen in the absence of any external challenge and is maintained through constant output from gut mucosal responses. This subset present reactivity against bacterial epitopes, possibly glycan structures (Le Gallou et al., 2018). This observation makes a link with our study of human anti-polysaccharide responses, suggesting that such responses may capitalize upon previous B-cell activation by gut microbial antigens, rather than mobilizing naive B cells.
In collaboration with the group of Daniel Pinschewer (Basel University), we described that chronic viral infection in the mouse, while it leads to exhaustion of cytotoxic T cell responses, is able to generate protracted germinal centers from which B cells secreting neutralizing antibodies will be able to emerge (Fallet et al., 2020). Finally, we also identified in the mouse key factors that favor affinity maturation over plasma cell differentiation of memory cells during recall responses, notably persistent germinal center structures and waning antigen-specific serum antibodies.
Concerning long-lived memory B cells in humans, transcriptional studies performed at the single cell level did not reveal major transcriptional changes compared to the global memory population of the same individual, although telomere length clearly establish the anteriority of smallpox-specific memory B cell formation. Metabolomic studies, transcriptomic and ATAC-seq studies identified a Notch-dependent resident signature which, surprisingly is shared by a major fraction of memory B cells, clearly indicating that longevity is the hallmark of most immune memories elicited during immune responses.
A cohort of volunteers has been immunized with Pneumo23, a 23-valent pneumococcal polysaccharide vaccine, and their blood response followed up to 2 months. High throughput sequencing of the immune repertoire has been performed and revealed, as mentioned above, the surprising mobilization of pre-diversified B cells against non-protein antigens. Isolation of anti-pneumococcal antibodies allowed us to identify a fraction of the gut microbiota recognized by these antibodies, suggesting that this pre-diversification of marginal zone B cells takes place in gut-associated lymphoid tissues. In parallel, emergence of marginal zone-like human B cells was observed in a new humanized mouse model that is able to support the development of lymphoid tissue in the gut, strengthening the link between immune diversification in the gut and the capacity of Marginal-zone B cells to respond to polysaccharide antigens.
The fourth subject addressed since 2020 was the study of the B cell response against SARS-Cov-2, based on the longitudinal follow-up of a cohort of patients with Covid-19: this study described a first wave of activation and differentiation into antibody-secreting cells of naive B cells as well as crossreactive B cells that recognize seasonal coronaviruses, responsible of common cold, followed by a phase of germinal-center reactions lasting for several months during which memory B cells with better recognition for the spike protein of the SARS-Cov-2 are selected. In a second study, the response to mRNA vaccine was studied in Covid19 recovered and naive individuals, highlighting a strong difference in serum neutralization potency against virus variants between naive and recovered individuals. Memory B cells from naive individuals, in contrast, although numerically less abundant, showed a good neutralization potential, indicating that a booster dose will lead to their efficient mobilization and the production of protective serum antibodies in all cases.
Technological developments
- We developed high-throughput single B cell cultures for serial affinity measurements of the monoclonal antibody produced (developed in the context of SARS-CoV-2 variants, Sokal et al. Immunity, 2021), an approach whose novelty was acknowledged in the field and considered as a highly competitive compared to classical VH/VL cloning and re-expression.
- We distributed our mouse reporter line to more than 50 laboratories across the world. This model was instrumental in a series of high-profile studies on germinal center B cell homeostasis and memory B cell recall (Tas et al., Science 351, 1048 (2016); Degn et al., Cell 170, 913 (2017); Mesin et al., Cell 180, 92 (2020)). This mouse line is now deposited at EMMA and at Jackson laboratories.

We attained at the end of the project:
- the description of mobilization of memory B cell subsets in recall responses in the mouse
- the identification of structural and biochemical hallmarks of long-lived memory B cells in humans.
- the identification of human B-cell memory subsets mobilized in anti-polysaccharidic responses, as well as their developmental origin.
- the description of the B cell memory response against SARS-CoV-2 during infection or vaccination, and the role of affinity laturation in the protection afforded against virus variants
Persistent memory B cells and plasma cells issued from spontaneous immune responses