Continuous germinal center invasion contributes to the diversity of the immune response.

Antibody responses are characterized by increasing affinity and diversity over time. Affinity maturation occurs in germinal centers by a mechanism that involves repeated cycles of somatic mutation and selection. How antibody responses diversify while also undergoing affinity maturation is not as well understood. Here, we examined germinal center (GC) dynamics by tracking B cell entry, division, somatic mutation, and specificity. Our experiments show that naive B cells continuously enter GCs where they compete for T cell help and undergo clonal expansion. Consistent with late entry, invaders carry fewer mutations but can contribute up to 30% or more of the cells in late-stage germinal centers. Notably, cells entering the germinal center at later stages of the reaction diversify the immune response by expressing receptors that show low affinity to the immunogen. Paradoxically, the affinity threshold for late GC entry is lowered in the presence of high-affinity antibodies.

Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection.

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year.

Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies 1,2 . Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines 3,4 . In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve individuals 2,5-8 . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern 4,9 . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Germinal center-dependent and -independent memory B cells produced throughout the immune response.

Memory B cells comprise a heterogenous group of cells that differ in origin and phenotype. During the early phases of the immune response, activated B cells can differentiate into IgM-expressing memory cells, short-lived plasma cells, or seed germinal centers (GCs). The memory compartment is subsequently enriched by B cells that have been through several rounds of division and selection in the GC. Here, we report on the use of an unbiased lineage-tracking approach to explore the origins and properties of memory B cell subsets in mice with an intact immune system. We find that activated B cells continue to differentiate into memory B cells throughout the immune response. When defined on the basis of their origins, the memory B cells originating from activated B cells or GCs differ in isotype and overall gene expression, somatic hypermutation, and their affinity for antigen.

Sequencing, cloning, and antigen binding analysis of monoclonal antibodies isolated from single mouse B cells.

The analysis of B cell receptors (BCR) from single B cells is crucial to understanding humoral immune responses. Here, we describe a protocol for the sequencing, cloning, and characterization of antibody genes that encode BCRs. We used this method to analyze the BCRs of different mouse B cell populations for somatic hypermutations, clonal and phylogenic relationships, and their affinity for cognate antigen. For complete details on the use and execution of this protocol, please refer to Viant et al. (2020).

Evolution of antibody immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Evolution of Antibody Immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Enhanced SARS-CoV-2 neutralization by dimeric IgA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

Antibody Affinity Shapes the Choice between Memory and Germinal Center B Cell Fates.

Immunological memory is required for protection against repeated infections and is the basis of all effective vaccines. Antibodies produced by memory B cells play an essential role in many of these responses. We have combined lineage tracing with antibody cloning from single B cells to examine the role of affinity in B cell selection into germinal centers (GCs) and the memory B cell compartment in mice immunized with an HIV-1 antigen. We find that contemporaneously developing memory and GC B cells differ in their affinity for antigen throughout the immune response. Whereas GC cells and their precursors are enriched in antigen binding, memory B cells are not. Thus, the polyclonal memory B cell compartment is composed of B cells that were activated during the immune response but whose antigen binding affinity failed to support further clonal expansion in the GC.

Enhanced SARS-CoV-2 Neutralization by Secretory IgA in vitro.

SARS-CoV-2 primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of the illness. Although potent IgG antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might impact the initial viral spread and transmissibility from the mucosa. Here we characterize the IgA response to SARS-CoV-2 in a cohort of 149 individuals. IgA responses in plasma generally correlate with IgG responses and clones of IgM, IgG and IgA producing B cells that are derived from common progenitors are evident. Plasma IgA monomers are 2-fold less potent than IgG equivalents. However, IgA dimers, the primary form in the nasopharynx, are on average 15 times more potent than IgA monomers. Thus, secretory IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals.

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

The microanatomic segregation of selection by apoptosis in the germinal center.

B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.

[Innate lymphoid cells]. / Les cellules lymphoïdes innées.

Innate Lymphoid Cells (ILC) are tissue-resident effector lymphocytes that differ from the B and T lymphocytes subsets since they lack specific antigen receptors derived from gene rearrangements. They can be divided into two groups, cytotoxic ILC (NK cells) which are dependent on the transcription factors Eomes and T-bet and produce IFN-γ, and helper-like ILC (ILC1, ILC2, ILC3) which are dependent on T-bet, GATA-3 or RORγT and produce IFN-γ, IL-5/IL-13 or IL-17/IL-22 respectively. Although they were described recently, ILC are the subject of a large number of studies that characterize their role as one of the first effectors of the innate immune response as well as in the development of a wide number of inflammatory disorders.

Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival.

Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.

Transforming growth factor-ß and Notch ligands act as opposing environmental cues in regulating the plasticity of type 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) are composed of subsets that are either positive or negative for the natural cytotoxicity receptor (NCR) NKp46 (encoded by Ncr1). ILC3s are located at mucosal sites, such as in the intestine and lung, where they are exposed to billions of commensal microbes and potentially harmful pathogens. Together with T cells, the various ILC3 subsets maintain the balance between homeostasis and immune activation. Through genetic mapping, we identified a previously uncharacterized subset of NCR(-) ILC3s in mice that transiently express Ncr1, demonstrating previously undescribed heterogeneity within the ILC3 population. In addition, we showed that sustained Notch signaling was required for the maintenance of the NCR(+) phenotype and that the cytokine transforming growth factor-ß (TGF-ß) impaired the development of NCR(+) ILC3s. Thus, the plasticity of ILC3s is regulated by the balance between the opposing effects of Notch and TGF-ß signaling, maintaining homeostasis in the face of continual challenges.

SHP-1-mediated inhibitory signals promote responsiveness and anti-tumour functions of natural killer cells.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are involved in immune defense. NK cell reactivity is controlled in part by MHC class I recognition by inhibitory receptors, but the underlying molecular mechanisms remain undefined. Using a mouse model of conditional deletion in NK cells, we show here that the protein tyrosine phosphatase SHP-1 is essential for the inhibitory function of NK cell MHC class I receptors. In the absence of SHP-1, NK cells are hyporesponsive to tumour cells in vitro and their early Ca(2+) signals are compromised. Mice without SHP-1 in NK cells are unable to reject MHC class I-deficient transplants and to control tumours in vivo. Thus, the inhibitory activity of SHP-1 is needed for setting the threshold of NK cell reactivity.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.

Interleukin 15 (IL-15) controls both the homeostasis and the peripheral activation of natural killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we found that the metabolic checkpoint kinase mTOR was activated and boosted bioenergetic metabolism after exposure of NK cells to high concentrations of IL-15, whereas low doses of IL-15 triggered only phosphorylation of the transcription factor STAT5. mTOR stimulated the growth and nutrient uptake of NK cells and positively fed back on the receptor for IL-15. This process was essential for sustaining NK cell proliferation during development and the acquisition of cytolytic potential during inflammation or viral infection. The mTORC1 inhibitor rapamycin inhibited NK cell cytotoxicity both in mice and humans; this probably contributes to the immunosuppressive activity of this drug in different clinical settings.