Immunologic Memory to Polysaccharide Antigens

Immunologic memory is the ability of the immune system to generate an enhanced antibody response against previously encountered antigens. The clonal selection theory of acquired immunity attributes immunologic memory to the formation of an expanded compartment of quiescent antigen-specific memory B cells that are capable of rapid activation upon secondary antigen encounter. Whereas protein antigens are known to elicit memory B cells, it is not known whether polysaccharide antigens do. Here we report that polysaccharide antigens elicit memory B cells that are phenotypically distinct from those elicited by protein antigens. We show that antigen affinity of the B cell receptor regulates the development of memory B cells and that antigen-specific IgG antibodies suppress recall responses against polysaccharides via a mechanism that does not depend on epitope masking or involve known Fey receptors

Early appearance of germinal center–derived memory B cells and plasma cells in blood after primary immunization

Immunization with a T cell–dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated VH genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1–expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220+ memory B cells, and this is sufficient to account for the process of affinity maturation

The activation of memory B cells to generate high affinity antibody responses in vitro and in vivo

Immunological memory is the hallmark of the adaptive immune system. The humoral branch of the immunological memory is mediated by memory B-cells (mB). Memory B cells are marked by longevity, expression of antibodies with high affinity, and ability to generate robust antibody responses upon reencountering pathogens. However, requirements for the activation of mB cells and the induction of humoral memory responses are not well understood. This thesis examines the role of Toll-like receptors (TLRs) in mB activation using an immunized mouse model. TLRs are a family of receptors that recognize common molecular patterns of microbial pathogens and stimulate innate immune responses. Our study found that mouse mB expressed TLR9 and 4, and responded to their agonists in vitro by differentiating into high affinity IgG secreting plasma cells. However, TLR agonists alone were not sufficient to activate memory B cells in vivo. Antigen was required for the clonal expansion of antigen-specific memory B cells, the differentiation of mB cells to high affinity IgG secreting plasma cells, and the recall of high affinity antibody responses. The Ag- specific B cells that had not yet undergone isotype switching showed a relatively higher expression of TLR4 than memory B cells, which was reflected in a heightened response to its agonist, but in both cases of TLR4 and 9 yielded mostly low affinity IgM secreting plasma cells. When immunized together with the antigen, TLR agonists not only boosted the antigen-specific titers, but also increased affinity and isotype switching of the immunoglobulin. Thus, while TLR agonists alone are unable to activate mB in vivo, they can enhance humoral memory responses induced by the antigen

Long-Lived Antibody and B Cell Memory Responses to the Human Malaria Parasites, Plasmodium falciparum and Plasmodium vivax

Antibodies constitute a critical component of the naturally acquired immunity that develops following frequent exposure to malaria. However, specific antibody titres have been reported to decline rapidly in the absence of reinfection, supporting the widely perceived notion that malaria infections fail to induce durable immunological memory responses. Currently, direct evidence for the presence or absence of immune memory to malaria is limited. In this study, we analysed the longevity of both antibody and B cell memory responses to malaria antigens among individuals who were living in an area of extremely low malaria transmission in northern Thailand, and who were known either to be malaria naïve or to have had a documented clinical attack of P. falciparum and/or P. vivax in the past 6 years. We found that exposure to malaria results in the generation of relatively avid antigen-specific antibodies and the establishment of populations of antigen-specific memory B cells in a significant proportion of malaria-exposed individuals. Both antibody and memory B cell responses to malaria antigens were stably maintained over time in the absence of reinfection. In a number of cases where antigen-specific antibodies were not detected in plasma, stable frequencies of antigen-specific memory B cells were nonetheless observed, suggesting that circulating memory B cells may be maintained independently of long-lived plasma cells. We conclude that infrequent malaria infections are capable of inducing long-lived antibody and memory B cell responses

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity

B cells require DOCK8 to elicit and integrate T cell help when antigen is limiting

Dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome is characterized by a failure of the germinal center response, a process involving the proliferation and positive selection of antigen-specific B cells. Here, we describe how DOCK8-deficient B cells are blocked at a light-zone checkpoint in the germinal centers of immunized mice, where they are unable to respond to T cell–dependent survival and selection signals and consequently differentiate into plasma cells or memory B cells. Although DOCK8-deficient B cells can acquire and present antigen to initiate activation of cognate T cells, integrin up-regulation, B cell–T cell conjugate formation, and costimulation are insufficient for sustained B cell and T cell activation when antigen availability is limited. Our findings provide an explanation for the failure of the humoral response in DOCK8 immunodeficiency syndrome and insight into how the level of available antigen modulates B cell–T cell cross-talk to fine-tune humoral immune responses and immunological memory

CD21(int) CD23(+) follicular B cells express antigen-specific secretory IgM mRNA as primary and memory responses

This is the final version of the article. Available from Wiley via the DOI in this record.CD21(int) CD23(+) IgM(+) mouse follicular B cells comprise the bulk of the mature B-cell compartment, but it is not known whether these cells contribute to the humoral antibody response. We show using a direct RT-PCR method for antigen-specific VH, that FACS-sorted mouse CD21(int) CD23(+) B cells express specific secretory IgM VH transcripts in response to immunization and also exhibit a memory response. The secretory IgM expressed is distinct from the IgG expressed by cells of this phenotype, which we also analyse here, having a distinct broader distribution of CDR-H3 sequences and zero or low levels of somatic mutation in the region analysed. These results imply that cells of the CD21(int) CD23(+) phenotype have distinct IgM(+) and IgG(+) populations that contribute directly to the humoral antibody and memory responses by expressing antigen-specific secretory immunoglobulin. We also argue that the more diverse CDR-H3 sequences expressed by antigen-experienced IgM(+) CD21(int) CD23(+) follicular B cells would place them at the bottom of a recently hypothesized memory B-cell hierarchy.This work was funded by Wellcome Trust grant 062578

Human germinal center B cells differ from naive and memory B cells by their aggregated MHC class II‐rich compartments lacking HLA‐DO

To generate memory B cells bearing high‐affinity antibodies, naive B cells first encounter antigen in the T cell‐rich areas of secondary lymphoid organs. There, they are activated by antigen‐specific T cells and become germinal center (GC) founder B cells. GC founders enter the GC to become centroblasts that proliferate and mutate their BCR. Centroblasts differentiate into centrocytes that undergo selection, which requires both the recognition/capture of antigen on follicular dendritic cells and the presentation of processed antigen to GC T cells. Because at each stage of differentiation B cells act as antigen‐presenting cells, we analyzed their content of HLA‐DR+‐rich compartments (MIIC), as well as their expression of HLA‐DM, which catalyzes peptide loading of class II molecules, and HLA‐DO, which interacts with HLA‐DM and focuses MHC class II peptide loading on antigens internalized by the BCR. Naive and memory B cells concentrate HLA‐DR, ‐DM and ‐DO into compartments dispersed under the cell surface, which are identified by their expression of lysosome‐associated membrane protein (Lamp)‐1 as late endosomes/lysosomes. GC founders and GC B cells express larger Lamp‐1+DR+ compartments that are concentrated in the juxta‐nuclear region. These compartments express lower levels of HLA‐DM and virtually no HLA‐DO. Upon induction of a GC founder phenotype through the prolonged (days) co‐ligation of BCR and CD40, the naive B cell's peripheral DR+DM+Lamp‐1+ compartments aggregate in a polar fashion close to the nucleus. Furthermore, HLA‐DO expression virtually disappears, whereas low levels of HLA‐DM remain co‐localized with HLA‐DR. Anti‐κ/λ antibodies, used as surrogate antigens, are promptly (minutes) endocytosed in naive, memory and GC B cells. Then, naive and memory B cells target the surrogate antigen to their peripheral HLA‐DO+ MIIC, while GC B cells target it to their HLA‐DO- MIIC aggregates. Taken together, our results show that human GC B cells differ from naive and memory B cells by their aggregated MIIC that lack HLA‐D