The Shaping of a B Cell Pool Maximally Responsive to Infections.

B cell subsets differ in development, tissue distribution, and mechanisms of activation. In response to infections, however, all can differentiate into extrafollicular plasmablasts that rapidly provide highly protective antibodies, indicating that these plasmablasts are the main humoral immune response effectors. Yet, the effectiveness of this response type depends on the presence of antigen-specific precursors in the circulating mature B cell pool, a pool that is generated initially through the stochastic processes of B cell receptor assembly. Importantly, germinal centers then mold the repertoire of this B cell pool to be increasingly responsive to pathogens by generating a broad array of antimicrobial memory B cells that act as highly effective precursors of extrafollicular plasmablasts. Such B cell repertoire molding occurs in two ways: continuously via the chronic germinal centers of mucosal lymphoid tissues, driven by the presence of the microbiome, and via de novo generated germinal centers following acute infections. For effectively evaluating humoral immunity as a correlate of immune protection, it might be critical to measure memory B cell pools in addition to antibody titers.

The IgM receptor FcµR limits tonic BCR signaling by regulating expression of the IgM BCR.

The FcµR receptor for the crystallizable fragment (Fc) of immunoglobulin M (IgM) can function as a cell-surface receptor for secreted IgM on a variety of cell types. We found here that FcµR was also expressed in the trans-Golgi network of developing B cells, where it constrained transport of the IgM-isotype BCR (IgM-BCR) but not of the IgD-isotype BCR (IgD-BCR). In the absence of FcµR, the surface expression of IgM-BCR was increased, which resulted in enhanced tonic BCR signaling. B-cell-specific deficiency in FcµR enhanced the spontaneous differentiation of B-1 cells, which resulted in increased serum concentrations of natural IgM and dysregulated homeostasis of B-2 cells; this caused the spontaneous formation of germinal centers, increased titers of serum autoantibodies and excessive accumulation of B cells. Thus, FcµR serves as a critical regulator of B cell biology by constraining the transport and cell-surface expression of IgM-BCR.

Memory Lapses-Winning the Slow Race.

Pre-existing humoral immunity can impact immune responses to heterologous infections. In this issue of Immunity, Wong et al. reveal that memory B cells, while failing to re-enter the germinal center, create an affinity-restricted, diversified B cell repertoire for rapid plasma blast responses. Their findings have important implications to vaccine design.

Original antigenic sin: not so sinful after all.

'Original antigenic sin' predicts that antibody responses to secondary infections with escape mutants are dominated by specificities to the original pathogen. Using transgenic mice in which antibodies are tagged based on cellular origins and kinetics, Schiepers et al. support this prediction, and reveal accumulation of cross-reactive specificities chiefly among long-lived responses.

Innate B Cells Tell ILC How It's Done.

Innate lymphoid cells (ILCs) are known as first responders to infections and as instructors of subsequent CD4(+) T cell cytokine profiles. In this issue of Immunity, Fan and colleagues now demonstrate that even earlier responding innate-like B cells (NKB) induce these protective ILC responses.

Antigen-Specific CD4 T Cell and B Cell Responses to Borrelia burgdorferi.

Long-lived T-dependent B cell responses fail to develop during persistent infection of mice with Borrelia burgdorferi, the causative agent of Lyme disease, raising questions about the induction and/or functionality of anti-B. burgdorferi adaptive immune responses. Yet, a lack of reagents has limited investigations into B. burgdorferi-specific T and B cells. We attempted two approaches to track B. burgdorferi-induced CD4 T cells. First, a B. burgdorferi mutant was generated with an influenza hemagglutinin (HA) peptide, HA111-119, inserted into the B. burgdorferi arthritis-related protein (Arp) locus. Although this B. burgdorferi arp::HA strain remained infectious, peptide-specific TCR transgenic CD4 T cells in vitro, or adoptively transferred into B. burgdorferi arp::HA-infected BALB/c mice, did not clonally expand above those of recipients infected with the parental B. burgdorferi strain or a B. burgdorferi mutant containing an irrelevant peptide. Some expansion, however, occurred in B. burgdorferi arp::HA-infected BALB/c SCID mice. Second, a (to our knowledge) newly identified I-Ab-restricted CD4 T cell epitope, Arp152-166, was used to generate Arp MHC class II tetramers. Flow cytometry showed small numbers of Arp-specific CD4 T cells emerging in mice infected with B. burgdorferi but not with Arp-deficient Borrelia afzelii. Although up to 30% of Arp-specific CD4 T cells were ICOS+PD-1+CXCR5+BCL6+ T follicular helper cells, their numbers declined after day 12, before germinal centers (GCs) are prominent. Although some Arp-specific B cells, identified using fluorochrome-labeled rArp proteins, had the phenotype of GC B cells, their frequencies did not correlate with anti-Arp serum IgG. The data suggest a failure not in the induction, but in the maintenance of GC T follicular helper and/or B cells to B. burgdorferi.

Borrelia burgdorferi Infection-Induced Persistent IgM Secretion Controls Bacteremia, but Not Bacterial Dissemination or Tissue Burden.

Infection with Borrelia burgdorferi causes Lyme disease in humans. In small rodents, the natural reservoir species of this spirochete, infections lead to only modest disease manifestations, despite causing persistence infection. Although B cell responses are central for controlling bacterial tissue burden and disease manifestations, they lack classical aspects of T-dependent responses, such as sustained IgG affinity maturation and longevity, corresponding with a rapid collapse of germinal centers. Instead, the Ab response is characterized by strong and ongoing secretion of IgM, whose origins and impact on protective immunity to B. burgdorferi remain unknown. In this article, we demonstrate that B. burgdorferi infection-induced IgM in mice was produced continuously, mainly by conventional B, not B-1 cells, in a T-independent manner. Although IgM was passively protective and restricted early bacteremia, its production had no effects on bacterial dissemination into solid tissues, nor did it affect Borrelia tissue burden. The latter was controlled by the induction of bactericidal IgG, as shown comparing infections in wild type mice with those of mice lacking exclusively secreted IgM-/-, all class-switched Abs via deletion of aicda (AID-/-), and all secreted Abs (secreted IgM-/- × AID-/-). Consistent with the notion that B. burgdorferi infection drives production of IgM over more tissue-penetrable IgG, we demonstrated increased short- and long-term IgM Ab responses also to a coadministered, unrelated Ag. Thus, the continued production of IgM may explain the absence of B. burgdorferi in the blood.

Enhanced Mott cell formation linked with IgM Fc receptor (FcµR) deficiency.

In previous studies, Mott cells, an unusual form of plasma cells containing Ig-inclusion bodies, were frequently observed in peripheral lymphoid tissues in our IgM Fc receptor (FcµR)-deficient (KO) mouse strain. Because of discrepancies in the reported phenotypes of different Fcmr KO mouse strains, we here examined two additional available mutant strains and confirmed that such enhanced Mott-cell formation was a general phenomenon associated with FcµR deficiency. Splenic B cells from Fcmr KO mice clearly generated more Mott cells than those from WT mice when stimulated in vitro with LPS alone or a B-1, but not B-2, activation cocktail. Nucleotide sequence analysis of the Ig variable regions of a single IgMλ+ Mott-hybridoma clone developed from splenic B-1 B cells of Fcmr KO mice revealed the near (VH) or complete (Vλ) identity with the corresponding germline gene segments and the addition of six or five nucleotides at the VH/DH and DH/JH junctions, respectively. Transduction of an FcµR cDNA into the Mott hybridoma significantly reduced cells containing IgM-inclusion bodies with a concomitant increase in IgM secretion, leading to secreted IgM binding to FcµR expressed on Mott transductants. These findings suggest a regulatory role of FcµR in the formation of Mott cells and IgM-inclusion bodies.

Genetic mapping reveals Nfkbid as a central regulator of humoral immunity to Toxoplasma gondii.

Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice ("bumble") did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.

Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity.

Patients with asthma, a major public health problem, are at high risk for serious disease from influenza virus infection, but the pathogenic mechanisms by which influenza A causes airway disease and asthma are not fully known. We show here in a mouse model that influenza infection acutely induced airway hyper-reactivity (AHR), a cardinal feature of asthma, independently of T helper type 2 (T(H)2) cells and adaptive immunity. Instead, influenza infection induced AHR through a previously unknown pathway that required the interleukin 13 (IL-13)-IL-33 axis and cells of the non-T cell, non-B cell innate lymphoid type called 'natural helper cells'. Infection with influenza A virus, which activates the NLRP3 inflammasome, resulted in much more production of IL-33 by alveolar macrophages, which in turn activated natural helper cells producing substantial IL-13.

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.

Immune Response to Borrelia: Lessons from Lyme Disease Spirochetes.

The mammalian host responds to infection with Borrelia spirochetes through a highly orchestrated immune defense involving innate and adaptive effector functions aimed toward limiting pathogen burdens, minimizing tissue injury, and preventing subsequent reinfection. The evolutionary adaptation of Borrelia spirochetes to their reservoir mammalian hosts may allow for its persistence despite this immune defense. This review summarizes our current understanding of the host immune response to B. burgdorferi sensu lato, the most widely studied Borrelia spp. and etiologic agent of Lyme borreliosis. Pertinent literature will be reviewed with emphasis on in vitro, ex vivo and animal studies that influenced our understanding of both the earliest responses to B. burgdorferi as it enters the mammalian host and those that evolve as spirochetes disseminate and establish infection in multiple tissues. Our focus is on the immune response of inbred mice, the most commonly studied animal model of B. burgdorferi infection and surrogate for one of this pathogen's principle natural reservoir hosts, the white-footed deer mouse. Comparison will be made to the immune responses of humans with Lyme borreliosis. Our goal is to provide an understanding of the dynamics of the mammalian immune response during infection with B. burgdorferi and its relation to the outcomes in reservoir (mouse) and non-reservoir (human) hosts.

The Multifaceted B Cell Response to Influenza Virus.

Protection from yearly recurring, highly acute infections with a pathogen that rapidly and continuously evades previously induced protective neutralizing Abs, as seen during seasonal influenza virus infections, can be expected to require a B cell response that is too highly variable, able to adapt rapidly, and able to reduce morbidity and death when sterile immunity cannot be garnered quickly enough. As we outline in this Brief Review, the influenza-specific B cell response is exactly that: it is multifaceted, involves both innate-like and conventional B cells, provides early and later immune protection, employs B cells with distinct BCR repertoires and distinct modes of activation, and continuously adapts to the ever-changing virus while enhancing overall protection. A formidable response to a formidable pathogen.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

A natural killer T-cell subset that protects against airway hyperreactivity.

BACKGROUND: Infection of suckling mice with influenza virus expands a CD4-CD8- double-negative (DN) natural killer T (NKT) cell subpopulation that protects the mice as adults against allergen-induced airway hyperreactivity (AHR). However, this NKT cell subset has not been characterized, and the underlying mechanisms of protection remain unknown. OBJECTIVE: We characterized this specific NKT cell subpopulation that developed during influenza infection in neonatal mice and that suppressed the subsequent development of AHR. METHODS: A cell-surface marker was identified by comparing the mRNA expression profile of wild-type CD4+ NKT cells with that of suppressive Vα14 DN NKT cells. The marker-enriched NKT cell subset was then analyzed for its cytokine profile and its suppressive in vitro and in vivo abilities. RESULTS: We showed that DN NKT cells with high CD38 expression produced IFN-γ, but not IL-17, IL-4, or IL-13, and inhibited development of AHR through contact-dependent suppression of helper CD4 T-cell proliferation. The NKT subset expanded in the lungs of neonatal mice after infection with influenza and also after treatment of neonatal mice with Nu-α-GalCer, which effectively increased DN CD38hi NKT cell numbers. CONCLUSION: These results suggest that early/neonatal exposure to infection or antigen challenge affects subsequent lung immunity by altering the cellular composition of cells in the lung and that some subsets of NKT cells suppress AHR. These results provide a possible mechanism by which prior infections can protect against the development of allergic asthma and might be further explored as a protective measure for young children.

A Hard(y) Look at B-1 Cell Development and Function.

A small population of B cells exists in lymphoid tissues and body cavities of mice that is distinct in development, phenotype, and function from the majority (B-2) B cell population. This population, originally termed "Ly-1" and now "B-1," has received renewed interest as an innate-like B cell population of fetal-derived hematopoiesis, responsible for natural Ab production and rapid immune responses. Molecular analyses have begun to define fetal and adult hematopoiesis, while cell-fate mapping studies have revealed complex developmental origins of B-1 cells. Together the studies provide a more detailed understanding of B-1 cell regulation and function. This review outlines studies that defined B-1 cells as natural Ab- and cytokine-producing B cells of fetal origin, with a focus on work conducted by R.R. Hardy, an early pioneer and codiscoverer of B-1 cells, whose seminal contributions enhanced our understanding of this enigmatic B cell population.

sIgM-FcµR Interactions Regulate Early B Cell Activation and Plasma Cell Development after Influenza Virus Infection.

Previous studies with mice lacking secreted IgM (sIgM) due to a deletion of the µs splice region (µs-/- ) had shown sIgM involvement in normal B cell development and in support of maximal Ag-specific IgG responses. Because of the changes to B cell development, it remains unclear to which extent and how sIgM directly affects B cell responses. In this study, we aimed to explore the underlying mechanisms of sIgM-mediated IgG response regulation during influenza virus infection. Generating mice with normally developed µs-deficient B cells, we demonstrate that sIgM supports IgG responses by enhancing early Ag-specific B cell expansion, not by altering B cell development. Lack of FcµR expression on B cells, but not lack of Fcα/µR expression or complement activation, reduced antiviral IgG responses to the same extent as observed in µs-/- mice. B cell-specific Fcmr-/- mice lacked robust clonal expansion of influenza hemagglutinin-specific B cells early after infection and developed fewer spleen and bone marrow IgG plasma cells and memory B cells, compared with controls. However, germinal center responses appeared unaffected. Provision of sIgM rescued plasma cell development from µs-/- but not Fcmr-/- B cells, as demonstrated with mixed bone marrow chimeric mice. Taken together, the data suggest that sIgM interacts with FcµR on B cells to support early B cell activation and the development of long-lived humoral immunity.