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Delta’s spike Understanding the molecular mechanisms of the increased transmissibility and immune evasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is critical to guiding current and future intervention strategies. Zhang et al . determined cryo–electron microscopy structures of the full-length spike protein trimers of the Delta, Kappa, and Gamma variants of SARS-CoV-2 and studied their function and antigenic properties. The Delta spike protein fused membranes more efficiently at low levels of the cellular receptor ACE2, and its pseudotyped viruses infected target cells substantially more rapidly than all other variants tested, possibly at least partly accounting for its heightened transmissibility. Mutations of each variant rearranged the antigenic surface of the N-terminal domain of the spike protein but only caused local changes in the receptor-binding domain, consistent with greater resistance to neutralizing antibodies. These findings elucidate the molecular events that have led these viruses to adapt in human communities and to evade host immunity. —VV

Severe acute respiratory syndrome coronavirus 2 (COVID)-19 has emerged as the greatest global health threat in generations. An unprecedented mobilization of researchers has generated a wealth of data on humoral responses to SARS-CoV-2 within a year of the pandemic's beginning. The rapidly developed understanding of acute-phase antibody induction and medium-term antibody durability in COVID-19 is important at an individual level to inform patient care and a population level to help predict transmission dynamics. In this brief review, we will describe the development and maintenance of antibody responses to immunization and infections generally and the specific antibody dynamics observed for COVID-19. These crucial features of the humoral response have implications for the use of antibody therapeutics against the virus and can inform the likelihood of reinfection of individuals by the virus.

Optimal immune responses furnish long-lasting (durable) antibodies protective across dynamically mutating viral variants (broad). To assess robustness of mRNA vaccine-induced immunity, we compared antibody durability and breadth after SARS-CoV-2 infection and vaccination. While vaccination delivered robust initial virus-specific antibodies with some cross-variant coverage, pre-variant SARS-CoV-2 infection-induced antibodies, while modest in magnitude, showed highly stable long-term antibody dynamics. Vaccination after infection induced maximal antibody magnitudes with enhanced longitudinal stability while infection-naïve vaccinee antibodies fell with time to post-infection-alone levels. The composition of antibody neutralizing activity to variant relative to original virus also differed between groups, with infection-induced antibodies demonstrating greater relative breadth. Differential antibody durability trajectories favored COVID-19-recovered subjects with dual memory B cell features of greater early antibody somatic mutation and cross-coronavirus reactivity. By illuminating an infection-mediated antibody breadth advantage and an anti-SARS-CoV-2 antibody durability-enhancing function conferred by recalled immunity, these findings may serve as guides for ongoing vaccine strategy improvement.

B cell immunoglobulin (Ig) repertoire composition shapes immune responses. The generation of Ig diversity begins with Ig variable region exon assembly from gene segments, random inter-segment junction sequence diversity, and combinations of Ig heavy and light chain. This generates vast preemptive sequence freedom in early developing B lineage cell Ig genes that can anticipate a great diversity of threats. This freedom is met with large restrictions that ultimately define the naïve (i.e. preimmune) Ig repertoire. Activation-induced somatic hypermutation (SHM), which further diversifies Ig V regions, is also met with strong selection that shapes Ig affinity maturation. While individual repertoire features, such as affinity for self and competition for foreign antigen, are known to drive selection, the selection filters themselves may be subject to regulation. Large sequence freedom coupled with strong selection for each diversification process provides flexibility for demand-driven regulation to dynamically balance antigen recognition capacities and associated autoimmune risks according to host needs.

SARS-CoV-2 from alpha to epsilon As battles to contain the COVID-19 pandemic continue, attention is focused on emerging variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that have been deemed variants of concern because they are resistant to antibodies elicited by infection or vaccination or they increase transmissibility or disease severity. Three papers used functional and structural studies to explore how mutations in the viral spike protein affect its ability to infect host cells and to evade host immunity. Gobeil et al . looked at a variant spike protein involved in transmission between minks and humans, as well as the B1.1.7 (alpha), B.1.351 (beta), and P1 (gamma) spike variants; Cai et al . focused on the alpha and beta variants; and McCallum et al . discuss the properties of the spike protein from the B1.1.427/B.1.429 (epsilon) variant. Together, these papers show a balance among mutations that enhance stability, those that increase binding to the human receptor ACE2, and those that confer resistance to neutralizing antibodies. —VV

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity—suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants., Graphical abstract, Unbiased charting of memory B cell receptor-encoded antibodies from COVID-19 convalescent subjects identifies seven major antibody competition groups recognizing epitopic regions with group assignment correlating with cross-CoV-reactivity breadth and neutralization potency. SARS-CoV-2 variants tend to escape antibodies from groups with the most potent neutralizers, but many retain affinity, showing that redundant components of a primary immune response establish durable protection from evolving pathogens.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross–SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection. CA extern

Profiling coronaviruses Among the coronaviruses that infect humans, four cause mild common colds, whereas three others, including the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), result in severe infections. Shrock et al. used a technology known as VirScan to probe the antibody repertoires of hundreds of coronavirus disease 2019 (COVID-19) patients and pre–COVID-19 era controls. They identified hundreds of antibody targets, including several antibody epitopes shared by the mild and severe coronaviruses and many specific to SARS-CoV-2. A machine-learning model accurately classified patients infected with SARS-CoV-2 and guided the design of an assay for rapid SARS-CoV-2 antibody detection. The study also looked at how the antibody response and viral exposure history differ in patients with diverging outcomes, which could inform the production of improved vaccine and antibody therapies. Science , this issue p. eabd4250

Critical to managing the spread of COVID-19 is the ability to diagnose infection and define the acquired immune response across the population. While genomic tests for the novel Several Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) detect the presence of viral RNA for a limited time frame, when the virus is shed in the upper respiratory tract, tests able to define exposure and infection beyond this short window of detectable viral replication are urgently needed. Following infection, antibodies are generated within days, providing a durable read-out and archive of exposure and infection. Several antibody tests have emerged to diagnose SARS-CoV-2. Here we report on a qualified quantitative ELISA assay that displays all the necessary characteristics for high-throughput sample analysis. Collectively, this test offers a quantitative opportunity to define both exposure and levels of immunity to SARS-CoV-2.

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE+ B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE+ clones in blood, stomach, duodenum and esophagus of 19 peanut allergic patients, using high-throughput DNA sequencing. IgE+ cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally-related IgE+ and non-IgE-expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE+ B-lineage cells in food allergy.

Whether Ig engagement by antigen is required to initiate somatic evolution and affinity maturation is not well defined. Silver and colleagues show that germinal center flexibility permits nonspecific B cells to achieve de novo antigen recognition and antibody affinity maturation., Two immunoglobulin (Ig) diversification mechanisms collaborate to provide protective humoral immunity. Combinatorial assembly of IgH and IgL V region exons from gene segments generates preimmune Ig repertoires, expressed as B cell receptors (BCRs). Secondary diversification occurs when Ig V regions undergo somatic hypermutation (SHM) and affinity-based selection toward antigen in activated germinal center (GC) B cells. Secondary diversification is thought to only ripen the antigen-binding affinity of Igs that already exist (i.e., cognate Igs) because of chance generation during preimmune Ig diversification. However, whether stochastic activation of noncognate B cells can generate new affinity to antigen in GCs is unclear. Using a mouse model whose knock-in BCR does not functionally engage with immunizing antigen, we found that chronic immunization induced antigen-specific serological responses with diverse SHM-mediated antibody affinity maturation pathways and divergent epitope targeting. Thus, intrinsic GC B cell flexibility allows for somatic, noncognate B cell evolution, permitting de novo antigen recognition and subsequent antibody affinity maturation without initial preimmune BCR engagement.

Antibodies are key immune effectors that confer protection against pathogenic threats. The nature and longevity of the antibody response to SARS-CoV-2 infection is not well defined. We charted longitudinal antibody responses to SARS-CoV-2 in 92 subjects after symptomatic COVID-19. Antibody responses to SARS-CoV-2 are unimodally distributed over a broad range, with symptom severity correlating directly with virus-specific antibody magnitude. Seventy-six subjects followed longitudinally to ∼100 days demonstrated marked heterogeneity in antibody duration dynamics. Virus-specific IgG decayed substantially in most individuals, whereas a distinct subset had stable or increasing antibody levels in the same timeframe despite similar initial antibody magnitudes. These individuals with increasing responses recovered rapidly from symptomatic COVID-19 disease, harbored increased somatic mutations in virus-specific memory B cell antibody genes, and had persistent higher frequencies of previously activated CD4+ T cells. These findings illuminate an efficient immune phenotype that connects rapid symptom clearance to differential antibody durability dynamics., Highlights SARS-CoV-2 antibody responses range from negligible to robust in mild COVID-19 Some individuals maintain stable or increased SARS-CoV-2 IgG, while most decline Those who sustain virus-specific IgG production tend to have shorter disease courses Virus-specific B cells from “sustainers” have more SHM early after disease resolution, Longitudinal analyses of antibody responses to SARS-CoV-2 demonstrate that individuals with sustained virus-specific IgG production have shorter disease trajectories, with a subset demonstrating increased somatic hypermutation and higher levels of activated CD4+ cells.

Symbiotic relationships help shape immune fitness. Chen et al. demonstrate that microbial symbionts influence host immunity by enriching frequencies of antibacterial specificities within the naive B cell receptor repertoire and that this may have consequences for mucosal and systemic immunity., The ability of immunoglobulin (Ig) to recognize pathogens is critical for optimal immune fitness. Early events that shape preimmune Ig repertoires, expressed on IgM+ IgD+ B cells as B cell receptors (BCRs), are poorly defined. Here, we studied germ-free mice and conventionalized littermates to explore the hypothesis that symbiotic microbes help shape the preimmune Ig repertoire. Ig-binding assays showed that exposure to conventional microbial symbionts enriched frequencies of antibacterial IgM+ IgD+ B cells in intestine and spleen. This enrichment affected follicular B cells, involving a diverse set of Ig-variable region gene segments, and was T cell–independent. Functionally, enrichment of microbe reactivity primed basal levels of small intestinal T cell–independent, symbiont-reactive IgA and enhanced systemic IgG responses to bacterial immunization. These results demonstrate that microbial symbionts influence host immunity by enriching frequencies of antibacterial specificities within preimmune B cell repertoires and that this may have consequences for mucosal and systemic immunity.

Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighe/e mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighe/e mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgμ > mIgγ1 > mIge, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighe/e mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells—which essentially do not exist under normal conditions—to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

For decades, intravenous immunoglobulin (IVIg) has provided safe and effective therapy for immunodeficient patients. This proof-of-principle study describes a novel approach to generate personalized IVIg for chronic, antibiotic-resistant infection in real time.

Primary B-cell development is thought to be restricted to the bone marrow; here it is shown to occur also in intestinal tissues of postnatal mice, that it peaks at the time of weaning and is increased upon colonization of germ-free mice, and is thus influenced by commensal microbes. Primary B-cell development is thought to be restricted to the bone marrow, but here Frederick Alt and colleagues present the surprising finding that it also occurs in the gut, where it is stimulated by the gut microbes. The authors describe a population of early B-lineage cells developing within the intestinal mucosa — specifically in the lamina propria — of postnatal mice. B-cell production peaks at the time of weaning and is increased upon colonization of germ-free mice. The repertoire of these B cells differs from that of cells derived from the bone marrow, and may be shaped by commensal microbes. The RAG1/RAG2 endonuclease (RAG) initiates the V(D)J recombination reaction that assembles immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B-cell antigen-binding receptor (BCR). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3,4,5. This ‘receptor editing’ process, which can also lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen encounter at the Rag-expressing immature B-cell stage helps shape pre-immune BCR repertoires. As the major site of postnatal B-cell development, the bone marrow is the principal location of primary immunoglobulin repertoire diversification in mice. Here we report that early B-cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP immunoglobulin repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B-lineage cells that harbour intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing bone marrow counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B-cell development occurs in the intestinal mucosa, where it is regulated by extracellular signals from commensal microbes that influence gut immunoglobulin repertoires.

To be added., Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cμ (IgM) constant regions (CH) exons with downstream CH exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cμ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cμ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cμ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cμ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.

SUMMARY Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/ degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity.

The loss of a classical non-homologous end-joining (NHEJ) repair factor, XLF, shows strong effects in non-lymphoid cells, but in lymphoid cells its absence surprisingly has only modest effects on V(D)J recombination. Frederick Alt and colleagues show that in lymphoid cells, two other repair factors — ATM kinase and histone protein H2AX — have functional redundancy with XLF. Thus, mice that are deficient in both ATM and XLF have compromised conventional NHEJ, although alternative end-joining is retained. The results hint that the redundant function in end-joining that XLF has with both ATM and H2AX may be related to a role for ATM in chromatin accessibility. Although loss of XLF, a classical non-homologous DNA end-joining (NHEJ) repair factor, shows strong effects in non-lymphoid cells, in lymphoid cells its absence has only modest effects on V(D)J recombination. This study now shows that in lymphoid cells, two other repair factors — ATM kinase and histone protein H2AX — have functional redundancy with XLF. Thus, mice deficient in both ATM and XLF have compromised conventional NHEJ, although alternative end-joining is retained. The results hint that the redundant function in end-joining that XLF has with both ATM and H2AX may have to do with an ATM role in chromatin accessibility. Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ3,4,5,6; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes7,8,9. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.

The primary immunoglobulin repertoire develops via opposing forces of expanding diversification balanced by contracting selection mechanisms. The resulting shape is essential for host health and immune fitness. While the molecular mechanisms of Ig diversification have largely been defined, selection forces shaping emerging Ig repertoires are poorly understood. During lifetime, human and mouse early B cell development occurs at distinct locations — beginning in fetal liver before transferring to bone marrow and spleen by the end of gestation. During an early life window of time, the murine gut lamina propria harbors developing immature B cells in proximity to intestinal contents such as commensal microbes and dietary antigens. Location and timing of early B cell development may thus endow neighboring antigens with primary repertoire-shaping capabilities.

Immunoglobulin (Ig) E is the most tightly regulated of all Ig heavy chain (IgH) isotypes and plays a key role in atopic disease. The gene encoding for IgH in mature B cells consists of a variable region exon—assembled from component gene segments via V(D)J recombination during early B cell development—upstream of a set of IgH constant region CH exons. Upon activation by antigen in peripheral lymphoid organs, B cells can undergo IgH class switch recombination (CSR), a process in which the initially expressed IgH μ constant region exons (Cμ) are deleted and replaced by one of several sets of downstream CH exons (e.g., Cγ, Ce, and Cα). Activation of the IL-4 receptor on B cells, together with other signals, can lead to the replacement of Cμ with Ce resulting in CSR to IgE through a series of molecular events involving irreversible remodeling of the IgH locus. Here, we discuss the molecular mechanisms of CSR and the unique features surrounding the generation of IgE-producing B cells.

Animals and many of their chronic microbial inhabitants form relationships of symbiotic mutualism, which occurs when coexisting life-forms derive mutual benefit from stable associations. While microorganisms receive a secure habitat and constant food source from vertebrate hosts, they are required for optimal immune system development and occupy niches otherwise abused by pathogens. Microbes have also been shown to provide vertebrate hosts with metabolic capabilities that enhance energy and nutrient uptake from the diet. The immune system plays a central role in the establishment and maintenance of host-microbe homeostasis, and B lineage cells play a key role in this regulation. Here, I reviewed the structure and function of the microbiota and the known mechanisms of how nonpathogenic microbes influence B cell biology and immunoglobulin repertoire development early in life. I also discuss what is known about how B lineage cells contribute to the process of shaping the composition of commensal/mutualistic microbe membership.

Inflammatory events in the central nervous system (CNS) contribute to the disease process in a variety of neuroinflammatory diseases such as multiple sclerosis (MS), Alzheimer's Disease (AD), and cerebral ischemia, and activated macrophages/microglia are central to this response. Immunological activation of these cells leads to the production of a wide array of cytokines, chemokines, matrix metalloproteinases and neurotoxins, and ultimately to glial/neuronal injury and death. The CD40 molecule has an important role in promoting inflammatory responses by macrophages/microglia, since interaction with its cognate ligand, CD154, leads to secretion of cytokines and neurotoxins. Aberrant CD40 expression by macrophages/microglia, induced by cytokines such as IFN-gamma and TNF-alpha, contributes to neuroimmunologic cascades in the CNS. Strategies to suppress CD40 expression may attenuate inflammation and neuronal damage within the CNS, which will ultimately be of benefit in neuroinflammatory diseases. The mediators that regulate expression of CD40 in macrophages/microglia (both induction and inhibition) function at the level of gene transcription. In this review, we present an overview of the molecular basis of CD40 expression in macrophages/microglia. The signal transduction pathways and transcription factors employed by IFN-gamma and TNF-alpha to induce CD40 expression are described, as are the cis-elements in the CD40 promoter that are critical for CD40 transcription. Information is provided on the mechanism(s) underlying suppression of CD40 in macrophages/microglia by immunomodulatory agents such as IL-4, TGF-beta, neuropeptides, neurotrophins, and statins. A comprehensive assessment of CD40 production and function in macrophages/microglia will establish the foundation for future therapeutic manipulation of this critical immunoregulatory protein.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

Induced pluripotent stem cells (iPSCs) can be formed from somatic cells by a defined set of genetic factors; however, aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster often hinders their developmental potency and ability to contribute to high-grade chimerism in mice. Here, we describe an approach that allows splenic B cells activated to undergo Ig heavy-chain ( IgH ) class-switch recombination (CSR) to be reprogrammed into iPSCs that contribute to high-grade chimerism in mice. Treatment of naïve splenic B cells in culture with anti-CD40 plus IL-4 induces IgH CSR from IgM to IgG1 and IgE. CSR leads to irreversible IgH locus deletions wherein the IgM-producing Cμ exons are permanently excised from the B-cell genome. We find that anti-CD40 plus IL-4–activated B cells produce iPSCs that are uniformly hypermethylated in the imprinted Dlk1-Dio3 gene cluster and fail to produce chimerism in mice. However, treatment of activated B cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine before and at early stages of reprogramming attenuates hypermethylation of the Dlk1-Dio3 locus in resultant iPSCs and enables them to form high-grade chimerism in mice. These conditions allowed us to produce chimeric mice in which all mature B cells were derived entirely from IgG1-expressing B-cell–derived iPSCs. We conclude that culture conditions of activated B cells before and at early stages of reprogramming influence the developmental potency of resultant iPSCs.

Since the discovery of the STAT proteins, there have been a great many reports on the involvement of STAT signals with other signaling pathways. These interactions can be classified into at least four general groups. First, STAT-mediated signals may be the means of the transcriptional upregulation of genes that impact on other pathways. Second, STATs interact with other transcriptional regulators at the promoter level. These interactions occur as STATs bind regions of DNA that are adjacent to or overlap with binding sites for other transcription factors. Moreover, STATs contain binding sites for a number of different co-activators such as the CREB binding protein (CBP) that enables the indirect association with many other transcriptional regulators such as those involved in TGF-β signaling. Third, STAT function can be manipulated by post-translational modification induced by other signaling pathways. An example of such is serine phosphorylation that can be induced by kinases that are activated by other signaling pathways (i.e. TNF-α and IL-1β). And fourth, STATs have been shown to interact with components from other signaling pathways that are not linked to DNA binding or transcription, such as the TNF Receptor 1 (TNFR1) the TNF-Associated Death Domain protein (TRADD). Demonstration of these interactions has provided clues to the mechanisms involved in how cells can integrate multiple signals and respond in a specific manner to a given set of signals from the environment. This chapter will review the interactions of various STAT molecules with signaling components from the TNF, IL-1β, TGF-β and hormone receptor signaling pathways. Specifically, the focus will be on STAT interactions with NF-кB, TNFR1, TRADD, SMADs and glucocorticoid receptor. Since this is not a comprehensive outline of all of the interaction partners of the STATs, emphasis will be placed on the general modes of interaction and the relation to functional modification.

Microglia and macrophages are important antigen-presenting cells (APCs) in the central nervous system (CNS). By virtue of their ability to express class II MHC antigens and costimulatory molecules such as CD40 and B7, microglia/macrophages promote Th1 cell activation and subsequent immune/inflammatory responses within the CNS. We have previously demonstrated that IFN-γ is the most potent inducer of CD40 expression on microglia. Our more recent studies have focused on the molecular basis of IFN-γ induced CD40 expression, and mechanisms by which this gene can be inhibited. The suppressive effects of IL-4 on CD40 expression will be discussed, as will the involvement of SH2 containing proteins called SOCS (for Suppressors Of Cytokine Signalling). Expression of CD40 by activated microglia/macrophages may contribute to the complex neuroimmunologic cascades that result in inflammation, demyelination and neuronal dysfunction. As such, understanding the mechanisms of inhibition of this molecule will be beneficial in diseases such as multiple sclerosis, HIV-1 associated dementia and Alzheimer's disease.

Nature Immunology 5, 199–207 (2004). On page 204, column 2, line 3, the number should read “(1,700%).” Nature Immunology regrets the error.

Nature Immunology 5, 199–207 (2004). Figure 3b should have a line separating the top and bottom halves.