Transcriptional and Metabolic Control of Memory B Cells and Plasma Cells.

For many infections and almost all vaccines, neutralizing-antibody-mediated immunity is the primary basis and best functional correlate of immunological protection. Durable long-term humoral immunity is mediated by antibodies secreted by plasma cells that preexist subsequent exposures and by memory B cells that rapidly respond to infections once they have occurred. In the midst of the current pandemic of coronavirus disease 2019, it is important to define our current understanding of the unique roles of memory B cells and plasma cells in immunity and the factors that control the formation and persistence of these cell types. This fundamental knowledge is the basis to interpret findings from natural infections and vaccines. Here, we review transcriptional and metabolic programs that promote and support B cell fates and functions, suggesting points at which these pathways do and do not intersect.

Locally produced autoantibodies in cancer.

The function and antigen-specificities of tumor-infiltrating B cells are mostly unknown. In a new study by Mazor et al., matrix metalloproteinase 14 (MMP14), a self-antigen that is overexpressed by ovarian cancers, is shown to drive B cell activation and autoantibody production in tertiary lymphoid structures (Mazor et al., 2022).

Unraveling the diversity and functions of tissue-resident plasma cells.

Antibody-secreting plasma cells (PCs) are generated in secondary lymphoid organs but are reported to reside in an emerging range of anatomical sites. Analysis of the transcriptome of different tissue-resident (Tr)PC populations revealed that they each have their own transcriptional signature indicative of functional adaptation to the host tissue environment. In contrast to expectation, all TrPCs were extremely long-lived, regardless of their organ of residence, with longevity influenced by intrinsic factors like the immunoglobulin isotype. Analysis at single-cell resolution revealed that the bone marrow is unique in housing a compendium of PCs generated all over the body that retain aspects of the transcriptional program indicative of their tissue of origin. This study reveals that extreme longevity is an intrinsic property of TrPCs whose transcriptome is imprinted by signals received both at the site of induction and within the tissue of residence.

Instructing durable humoral immunity for COVID-19 and other vaccinable diseases.

Many aspects of SARS-CoV-2 have fully conformed with the principles established by decades of viral immunology research, ultimately leading to the crowning achievement of highly effective COVID-19 vaccines. Nonetheless, the pandemic has also exposed areas where our fundamental knowledge is thinner. Some key unknowns are the duration of humoral immunity post-primary infection or vaccination and how long booster shots confer protection. As a corollary, if protection does not last as long as desired, what are some ways it can be improved? Here, I discuss lessons from other infections and vaccines that point to several key features that influence durable antibody production and the perseverance of immunity. These include (1) the specific innate sensors that are initially triggered, (2) the kinetics of antigen delivery and persistence, (3) the starting B cell receptor (BCR) avidity and antigen valency, and (4) the memory B cell subsets that are recalled by boosters. I further highlight the fundamental B cell-intrinsic and B cell-extrinsic pathways that, if understood better, would provide a rational framework for vaccines to reliably provide durable immunity.

Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses.

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity.

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.

The Transcription Factor AP4 Mediates Resolution of Chronic Viral Infection through Amplification of Germinal Center B Cell Responses.

B cells diversify and affinity mature their antigen receptor repertoire in germinal centers (GCs). GC B cells receive help signals during transient interaction with T cells, yet it remains unknown how these transient T-B interactions in the light zone sustain the subsequent proliferative program of selected B cells that occurs in the anatomically distant dark zone. Here, we show that the transcription factor AP4 was required for sustained GC B cell proliferation and subsequent establishment of a diverse and protective antibody repertoire. AP4 was induced by c-MYC during the T-B interactions, was maintained by T-cell-derived interleukin-21 (IL-21), and promoted repeated rounds of divisions of selected GC B cells. B-cell-specific deletion of AP4 resulted in reduced GC sizes and reduced somatic hypermutation coupled with a failure to control chronic viral infection. These results indicate that AP4 integrates T-cell-mediated selection and sustained expansion of GC B cells for humoral immunity.

Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells.

Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Plasma cells: You are what you eat.

Plasma cells are terminally differentiated B lymphocytes that constitutively secrete antibodies. These antibodies can provide protection against pathogens, and their quantity and quality are the best clinical correlates of vaccine efficacy. As such, plasma cell lifespan is the primary determinant of the duration of humoral immunity. Yet dysregulation of plasma cell function can cause autoimmunity or multiple myeloma. The longevity of plasma cells is primarily dictated by nutrient uptake and non-transcriptionally regulated metabolic pathways. We have previously shown a positive effect of glucose uptake and catabolism on plasma cell longevity and function. In this review, we discuss these findings with an emphasis on nutrient uptake and its effects on respiratory capacity, lifespan, endoplasmic reticulum stress, and antibody secretion in plasma cells. We further discuss how some of these pathways may be dysregulated in multiple myeloma, potentially providing new therapeutic targets. Finally, we speculate on the connection between plasma cell intrinsic metabolism and systemic changes in nutrient availability and metabolic diseases.

Antibody Responses to SARS-CoV-2: Let's Stick to Known Knowns.

The scale of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has thrust immunology into the public spotlight in unprecedented ways. In this article, which is part opinion piece and part review, we argue that the normal cadence by which we discuss science with our colleagues failed to properly convey likelihoods of the immune response to SARS-CoV-2 to the public and the media. As a result, biologically implausible outcomes were given equal weight as the principles set by decades of viral immunology. Unsurprisingly, questionable results and alarmist news media articles have filled the void. We suggest an emphasis on setting expectations based on prior findings while avoiding the overused approach of assuming nothing. After reviewing Ab-mediated immunity after coronavirus and other acute viral infections, we posit that, with few exceptions, the development of protective humoral immunity of more than a year is the norm. Immunity to SARS-CoV-2 is likely to follow the same pattern.

Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

Metabolic Links between Plasma Cell Survival, Secretion, and Stress.

Humoral immunity is generated and maintained by antigen-specific antibodies that counter infectious pathogens. Plasma cells are the major producers of antibodies during and after infections, and each plasma cell produces some thousands of antibody molecules per second. This magnitude of secretion requires enormous quantities of amino acids and glycosylation sugars to properly build and fold antibodies, biosynthetic substrates to fuel endoplasmic reticulum (ER) biogenesis, and additional carbon sources to generate energy. Many of these processes are likely to be linked, thereby affording possibilities to improve vaccine design and to develop new therapies for autoimmunity. We review here aspects of plasma cell biology with an emphasis on recent studies and the relationships between intermediary metabolism, antibody production, and lifespan.

IgA natural antibodies are produced following T-cell independent B-cell activation following stroke.

Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA + cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII-/-, CD4-/-, and MyD88-/- mice to determine if B-lymphocytes mature into IgA + PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA + PCs develops independently of CD4 + helper T-lymphocytes and MyD88 signaling. Subsequent sequencing of immunoglobulin genes of individual IgA + PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA + PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.

Allorecognition in a basal chordate consists of independent activating and inhibitory pathways.

Histocompatibility in the basal chordate Botryllus schlosseri is controlled by the polymorphisms of a single gene: the fuhc. A polymorphic candidate receptor (fester) appeared to play roles in both initiating the reaction and discriminating between fuhc alleles. Here we report the characterization of a related protein, uncle fester. uncle fester is not polymorphic, and although coexpressed with fester, has different functional properties. Loss-of-function studies demonstrate that uncle fester was required for incompatible reactions but has no role in interactions between compatible individuals. Furthermore, stimulation with monoclonal antibodies could initiate a rejection phenotype on a single colony, and in both assays the severity of the rejection could be manipulated. These findings suggest that allorecognition in Botryllus consists of independent pathways that control compatible and incompatible outcomes that are integrated within the interacting cells, and may provide insight into basal processes conserved in allorecognition responses throughout the metazoa.

Basics of memory B-cell responses: lessons from and for the real world.

The production of pathogen-specific B cells and antibodies underlies protective immunity elicited by most vaccines and many infections. Humoral immunity follows a regulated process by which high-affinity antibody-secreting plasma cells and memory B cells are generated. Yet for certain pathogens, protective immunity is inefficiently generated and/or maintained. For example, Dengue virus infections lead to lasting immunity against re-infection by the same serotype. However, if infected with a different Dengue serotype, the individual is predisposed to more severe disease than if he/she was completely naive. As another example, both natural infections with or vaccination against malaria do not necessarily lead to lasting immunity, as the same individual can be re-infected many times over the course of a lifetime. In this review, we discuss how these real-world problems can both instruct and be informed by recent basic studies using model organisms and antigens. An emphasis is placed on protective epitopes and functional distinctions between memory B-cell subsets in both mice and humans. Using flavivirus and Plasmodium infections as examples, we also speculate on the differences between ineffective B-cell responses that actually occur in the real world, and perfect-world responses that would generate lasting immunity.

ZBTB32 Restricts the Duration of Memory B Cell Recall Responses.

Memory B cell responses are more rapid and of greater magnitude than are primary Ab responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of Ab recall responses. ZBTB32 is highly expressed by mouse and human memory B cells but not by their naive counterparts. Zbtb32(-/-) mice mount normal primary Ab responses to T-dependent Ags. However, Zbtb32(-/-) memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32(-/-) secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32(-/-) secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of Ab recall responses.

Basophils take a slice of IRF8 pie.

In this issue of Blood, Sasaki and colleagues demonstrate a strict requirement for the transcription factor interferon regulator factor 8 (IRF8) in the development of mouse basophils.

Granulocyte colony-stimulating factor reprograms bone marrow stromal cells to actively suppress B lymphopoiesis in mice.

The mechanisms that mediate the shift from lymphopoiesis to myelopoiesis in response to infectious stress are largely unknown. We show that treatment with granulocyte colony-stimulating factor (G-CSF), which is often induced during infection, results in marked suppression of B lymphopoiesis at multiple stages of B-cell development. Mesenchymal-lineage stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells and osteoblasts, constitutively support B lymphopoiesis through the production of multiple B trophic factors. G-CSF acting through a monocytic cell intermediate reprograms these stromal cells, altering their capacity to support B lymphopoiesis. G-CSF treatment is associated with an expansion of CAR cells and a shift toward osteogenic lineage commitment. It markedly suppresses the production of multiple B-cell trophic factors by CAR cells and osteoblasts, including CXCL12, kit ligand, interleukin-6, interleukin-7, and insulin-like growth factor-1. Targeting bone marrow stromal cells is one mechanism by which inflammatory cytokines such as G-CSF actively suppress lymphopoiesis.