Eosinophils are required for the maintenance of plasma cells in the bone marrow.

Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils localized together with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported the survival of plasma cells by secreting the proliferation-inducing ligand APRIL and interleukin-6 (IL-6). In eosinophil-deficient mice, plasma cell numbers were much lower in the bone marrow both at steady state and after immunization. Reconstitution experiments showed that eosinophils were crucial for the retention of plasma cells in the bone marrow. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. Our findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils.

Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis.

Although in normal lamina propria (LP) large numbers of eosinophils are present, little is known about their role in mucosal immunity at steady state. Here we show that eosinophils are needed to maintain immune homeostasis in gut-associated tissues. By using eosinophil-deficient ΔdblGATA-1 and PHIL mice or an eosinophil-specific depletion model, we found a reduction in immunoglobulin A(+) (IgA(+)) plasma cell numbers and in secreted IgA. Eosinophil-deficient mice also showed defects in the intestinal mucous shield and alterations in microbiota composition in the gut lumen. In addition, TGF-ß-dependent events including class switching to IgA in Peyer's patches (PP), the formation of CD103(+) T cells including Foxp3(+) regulatory (Treg), and also CD103(+) dendritic cells were disturbed. In vitro cultures showed that eosinophils produce factors that promote T-independent IgA class switching. Our findings show that eosinophils are important players for immune homeostasis in gut-associated tissues and add to data suggesting that eosinophils can promote tissue integrity.

Physiology and pathology of eosinophils: Recent developments: Summary of the Focus Workshop Organized by DGAKI.

Over the last century, eosinophils have been regarded ambiguously either as 'friends' or 'foes'. Recent developments have greatly enhanced our understanding of the role and function of eosinophils in health and disease. Pathogenic eosinophilic inflammation can lead to severe diseases in various organs, such as the gastrointestinal tract, airways, heart and skin. In a 2-day focus workshop of the German Society for Allergology and Clinical Immunology (DGAKI), the state of the art was discussed and practical recommendations for diagnosis and treatment of eosinophilic diseases, with a particular focus on new biologics, such as anti-interleukin 5 and anti-interleukin 5R, were derived.

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line.

Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.

A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry.

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

The establishment of the plasma cell survival niche in the bone marrow.

Antibodies continuously secreted by plasma cells play a central role in humoral immune protection of the organism. These plasma cells are generated during the germinal center reaction, and it is likely that they here acquire the potential to develop into long-lived cells. To achieve longevity, these cells require factors provided by the microenvironment. Indeed, only a few of the plasmablasts arising during an immune response will differentiate into mature plasma cells, which may survive for decades in specialized survival niches in the bone marrow. Here, we discuss how the survival niche in the bone marrow is established and what is known about the cell-cell interactions needed to support the long-term survival of plasma cells. A particular emphasis is put on the role of eosinophils, which have been shown to be key providers of plasma cell survival factors. The data suggest that the reticulum of bone marrow stromal cells supports a dynamic survival niche, in which long-lived plasma cells are provided with essential factors by a continuously turning over population of eosinophils and other cells.

Prevalence and gene characteristics of antibodies with cofactor-induced HIV-1 specificity.

The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses.

A cryptic polyreactive antibody recognizes distinct clades of HIV-1 glycoprotein 120 by an identical binding mechanism.

Polyreactive antibodies play an important role for neutralization of human immunodeficiency virus (HIV). In addition to intrinsic polyreactive antibodies, the immune system of healthy individuals contains antibodies with cryptic polyreactivity. These antibodies acquire promiscuous antigen binding potential post-translationally, after exposure to various redox-active substances such as reactive oxygen species, iron ions, and heme. Here, we characterized the interaction of a prototypic human antibody that acquires binding potential to glycoprotein (gp) 120 after exposure to heme. The kinetic and thermodynamic analyses of interaction of the polyreactive antibody with distinct clades of gp120 demonstrated that the antigen-binding promiscuity of the antibody compensates for the molecular heterogeneity of the target antigen. Thus, the polyreactive antibody recognized divergent gp120 clades with similar values of the binding kinetics and quantitatively identical changes in the activation thermodynamic parameters. Moreover, this antibody utilized the same type of noncovalent forces for formation of complexes with gp120. In contrast, HIV-1-neutralizing antibodies isolated from HIV-1-infected individuals, F425 B4a1 and b12, demonstrated different binding behavior upon interaction with distinct variants of gp120. This study contributes to a better understanding of the physiological role and binding mechanism of antibodies with cryptic polyreactivity. Moreover, this study might be of relevance for understanding the basic aspects of HIV-1 interaction with human antibodies.

IgA response in preterm neonates shows little evidence of antigen-driven selection.

After birth, contact to environmental Ags induces the production of IgA, which represents a first line of defense for the neonate. We sought to characterize the maturation of the repertoire of IgA H chain transcripts in circulating blood B cells during human ontogeny. We found that IgA H chain transcripts were present in cord blood as early as 27 wk of gestation and that the restrictions of the primary Ab repertoire (IgM) persisted in the IgA repertoire. Thus, B cells harboring more "mature" V(H) regions were not preferred for class switch to IgA. Preterm and term neonates expressed a unique IgA repertoire, which was characterized by short CDR-H3 regions, preference of the J(H) proximal D(H)7-27 gene segment, and very few somatic mutations. During the first postnatal months, these restrictions were slowly released. Preterm birth did not measurably accelerate the maturation of the IgA repertoire. At a postconceptional age of 60 wk, somatic mutation frequency of IgA H chain transcripts reached 25% of the adult values but still showed little evidence of Ag-driven selection. These results indicate that similar to IgG, the IgA repertoire expands in a controlled manner after birth. Thus, the IgA repertoire of the newborn has distinctive characteristics that differ from the adult IgA repertoire. These observations might explain the lower affinity and specificity of neonatal IgA Abs, which could contribute to a higher susceptibility to infections and altered responses to vaccinations, but might also prevent the development of autoimmune and allergic diseases.

Immunization induces activation of bone marrow eosinophils required for plasma cell survival.

Eosinophils not only have multiple functions as effector cells of the innate immune system but also as modulators of immune responses. As producers of cytokines required for plasma cell survival, they are essential for the long-term maintenance of plasma cells in the BM. Here we show that the activation of eosinophils both in vitro and in vivo enhances the expression of the plasma cell survival factors APRIL, IL-6, IL-4, IL-10 and TNF-α. The in vivo activation of eosinophils was independent of the type of adjuvant used for primary immunization. Although eosinophils were activated by adjuvant itself, a stable activation and a constant increase in BM eosinophils were observed only in the presence of antigen. Thus, the numbers and the quality of eosinophils were dependent on priming the adaptive immune system. With secondary immunization and re-activation of antigen-dependent memory cells, the ability of eosinophils to promote plasma cell survival was further increased. These findings suggest that in T-cell-dependent immune responses eosinophils are conditioned to support the long-term survival of plasma cells in the BM, and furthermore imply that through accelerated numbers of eosinophils, stable plasma cell survival niches are established and the long-term survival of plasma cells is ensured.

Manifestation of spontaneous and early autoimmune gastritis in CCR7-deficient mice.

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

V-region gene analysis of locally defined synovial B and plasma cells reveals selected B cell expansion and accumulation of plasma cell clones in rheumatoid arthritis.

OBJECTIVE: To elucidate the development of synovial tissue-specific B cell immune responses, the clonality of individual naive B cells, memory B cells, and plasma cells and their organization and histologic localization in the inflamed tissue were investigated in patients with rheumatoid arthritis (RA). METHODS: B and plasma cells were isolated by laser capture microdissection (LCM) from the synovial tissue of patients with RA. In addition, single naive B cells, memory B cells, and plasma cells were sorted from synovial tissue cell suspensions. RNA was extracted from the cells, and Ig VH genes were amplified, cloned, and sequenced. RESULTS: Both LCM and single cell sorting analyses showed that naive and memory B cells infiltrated the RA synovial tissue. Comparison of the V-gene repertoire of B and plasma cells suggested that synovial plasma cells were generated, by and large, from locally activated B cells, indicating that a selected population of memory B cells differentiates into large plasma cell clones that then accumulate in the inflamed tissue. Clonally related plasma cells were isolated from separate and distinct localized areas of the tissue, suggesting that the newly generated plasma cells have a high migratory capacity. CONCLUSION: These results support the idea of a continuous activation of selected B cell clones, and hence a massive accumulation of plasma cells, in RA synovial tissue. As B cells and their secreted antibodies are an important factor in controlling inflammatory processes, patients with RA displaying intensive synovial tissue lymphocytic infiltrations might benefit from B cell depletion therapy. Early treatment will prevent accumulation of pathogenic plasma cells.

In silico subtraction approach reveals a close lineage relationship between follicular dendritic cells and BP3(hi) stromal cells isolated from SCID mice.

Organization of the stromal compartments in secondary lymphoid tissue is a prerequisite for an efficient immune reaction. In particular, follicular dendritic cells (FDC) are pivotal for the activation and differentiation of B cells. To investigate the development of FDC, FDC together with tightly associated B cells (FDC networks) were micro-dissected from frozen tissue sections and follicular B cells were sorted by FACS. Using an in silico subtraction approach, gene expression of FDC was determined and compared with that of follicular stromal cells micro-dissected from the spleen of SCID mice. Nearly 90% of the FDC genes were expressed in follicular stromal cells of the SCID mouse, providing further evidence that FDC develop from the residual network of reticular cells. Thus, it suggests that rather minor modifications in the gene expression profile are sufficient for differentiation into mature FDC. The analysis of different immune-deficient mouse strains shows that a complex pattern of gene regulation controls the development of residual stromal cells into mature FDC. The in silico subtraction approach provides a molecular framework within which to determine the diverse roles of FDC in support of B cells and to investigate the differentiation of FDC from their mesenchymal precursor cells.

Class switching and consecutive loss of dsDNA-reactive B1a B cells from the peritoneal cavity during murine lupus development.

B1a B cells have been implicated in the pathogenesis of systemic lupus erythematosus; however, their precise contribution to the disease remains unclear. Here we analysed isotype expression, organ accumulation and autoreactivity of B1a cells in the NZB/W F1 murine model for systemic lupus erythematosus using flow cytometry, ELISPOT and adoptive cell transfer. In the course of lupus, the B1a compartment is expanded and B1a cells class switch to IgG. Class-switched B1a B cells were excluded from the peritoneal cavity but accumulated in the spleen and target organs such as kidneys and thymus. Autoreactive B1a B cells preferentially class switch, which leads to a subsequent loss of autoreactive B1a cells from the peritoneal cavity. We propose a model whereby autoreactive B1a B cells become activated early in the course of lupus and class switch to IgG. These autoreactive B1a B cells accumulate in the spleen and affected organs where they presumably secrete autoantibodies and contribute to the local and systemic pathogenesis.

The heavy chain variable segment gene repertoire in chronic Chagas' heart disease.

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.

Oxidation-Specific Epitopes (OSEs) Dominate the B Cell Response in Murine Polymicrobial Sepsis.

In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis.

Homology-directed recombination in IgH variable region genes from human neonates, infants and adults: implications for junctional diversity.

Homology-directed recombination, i.e. the preferential joining of gene segments at short sequence homologies, is found in 80% of IgH variable region genes from neonatal mice and causes a marked uniformity of their VH-DH- and DH-JH-junctions, which are predominated by one to three junctional sequences. To analyze the impact of homology-directed recombination on IgH gene diversity in humans, IgH rearrangements from fetuses and neonates (gestational age 20-42 weeks), infants (age 1-27 months) and adults were cloned and sequenced. As a marker of homology-directed recombination the VH-DH- and DH-JH-junctions were searched for nucleotides that could have been encoded by each of the two adjacent gene segments. Such overlapping sequences were rare (<3%) in VH-DH-junctions from newborns, infants, and adults. In contrast, overlapping sequences were found in 30% of the DH-JH-junctions from preterm neonates. Their frequency decreased with age to 19% in term neonates, 12% in infants and 4% in adults (p<0.001). Our analysis of the five most common DH-JH-combinations in neonates demonstrated that overlapping nucleotides reduced diversity: only 48% of junctions with overlapping nucleotides were different compared to 99% of junctions with N-insertions between DH and JH (p<0.001). However, homology-directed recombination had a much smaller effect on overall junctional diversity in human neonates than in neonatal mice because it rarely occurred in VH-DH-junctions and affected only 19% (term neonates) to 30% (preterm neonates) of the DH-JH-junctions. Therefore, unlike in mice, homology-directed recombination does not cause junctional uniformity in human neonates.

Eosinophils can more than kill.

In this issue of JEM, Arnold et al. (https://doi.org/10.1084/jem.20172049) demonstrate that eosinophils suppress mucosal inflammation by directly interacting with pro-inflammatory Th1 cells. This emphasizes the dual role of eosinophils, which can act both as effector cells that control an infection and as immunomodulatory cells that promote immune homeostasis.

Single cell analysis of synovial tissue B-cells.

Mononuclear cells often form highly organized lymphoid structures in the chronically inflamed synovial tissue of patients with rheumatoid arthritis (RA) within which B-cells are activated and may differentiate into effector plasma cells. The analysis of those activated B-cells and the determination of their specificity is of great importance for the understanding of the pathogenesis of RA. Here, we describe a technique that combines histological analysis of synovial tissue with a molecular analysis of the V-gene repertoire at the level of the single B-cell. Immunohistochemical staining of tissue sections allows us to identify the activated B-cells. Those cells are then isolated using a micromanipulator and the rearranged immunoglobulin (Ig) genes amplified, cloned and sequenced. The combination of the V(D)J gene segments and the pattern of somatic mutations in the V-region genes, allows us to identify clonal relationships between the isolated B cells. Once Ig genes for a heavy and a light chain have been isolated from individual B-cells, they can be used to generate recombinant antibodies. These antibodies can be used to determine the antigens which support the activation of B-cells in the inflamed synovial tissue