Class-Switch Recombination Occurs Infrequently in Germinal Centers.

Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.

Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Regulation of germinal center B-cell differentiation.

Germinal centers (GC) are the main sites where antigen-activated B-cell clones expand and undergo immunoglobulin gene hypermutation and selection. Iterations of this process will lead to affinity maturation, replicating Darwinian evolution on the cellular level. GC B-cell selection can lead to four different outcomes: further expansion and evolution, apoptosis (non-selection), or output from the GC with differentiation into memory B cells or plasma cells. T-helper cells in GC have been shown to have a central role in regulating B-cell selection by sensing the density of major histocompatibility complex (MHC):peptide antigen complexes. Antigen is provided on follicular dendritic cells in the form of immune complex. Antibody on these immune complexes regulates antigen accessibility by shielding antigen from B-cell receptor access. Replacement of antibody on immune complexes by antibody generated from GC-derived plasma cell output will gradually reduce the availability of antigen. This antibody feedback can lead to a situation where a slow rise in selection stringency caused by a changing environment leads to directional evolution toward higher affinity antibody.

Toll-like receptor 4 signaling by follicular dendritic cells is pivotal for germinal center onset and affinity maturation.

Germinal centers (GCs) are specialized microenvironments where antigen-activated B cells undergo proliferation, immunoglobulin (Ig) class switch recombination, somatic hypermutation (SHM), and affinity maturation. Within GCs, follicular dendritic cells (FDCs) are key players in driving these events via direct interaction with GC B cells. Here, we provide in vivo evidence that FDCs express and upregulate Toll-like-receptor (TLR) 4 in situ during germinal center reactions, confirm that their maturation is driven by TLR4, and associate the role of FDC-expressed TLR4 with quantitative and qualitative affects of GC biology. In iterative cycles of predictions by in silico modeling subsequently verified by in vivo experiments, we demonstrated that TLR4 signaling modulates FDC activation, strongly impacting SHM and generation of Ig class-switched high-affinity plasma and memory B cells. Thus, our data place TLR4 in the heart of adaptive humoral immunity, providing further insight into mechanisms driving GCs arising in both health and disease.

IgG1 Is Required for Optimal Protection after Immunization with the Purified Porin OmpD from Salmonella Typhimurium.

In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Salmonella Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as S. Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified S. Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to S. Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as S. Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs.

The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22-blocking agents in B-cell-mediated autoimmune conditions.

Selective Expression of Flt3 within the Mouse Hematopoietic Stem Cell Compartment.

The fms-like tyrosine kinase 3 (Flt3) is a cell surface receptor that is expressed by various hematopoietic progenitor cells (HPC) and Flt3-activating mutations are commonly present in acute myeloid and lymphoid leukemias. These findings underscore the importance of Flt3 to steady-state and malignant hematopoiesis. In this study, the expression of Flt3 protein and Flt3 mRNA by single cells within the hematopoietic stem cell (HSC) and HPC bone marrow compartments of C57/BL6 mice was investigated using flow cytometry and the quantitative reverse transcription polymerase chain reaction. Flt3 was heterogeneously expressed by almost all of the populations studied, including long-term reconstituting HSC and short-term reconstituting HSC. The erythropoietin receptor (EpoR) and macrophage colony-stimulating factor receptor (M-CSFR) were also found to be heterogeneously expressed within the multipotent cell compartments. Co-expression of the mRNAs encoding Flt3 and EpoR rarely occurred within these compartments. Expression of both Flt3 and M-CSFR protein at the surface of single cells was more commonly observed. These results emphasize the heterogeneous nature of HSC and HPC and the new sub-populations identified are important to understanding the origin and heterogeneity of the acute myeloid leukemias.

Versatility of stem and progenitor cells and the instructive actions of cytokines on hematopoiesis.

For many years, developing hematopoietic cells have been strictly compartmentalized into a rare population of multi-potent self-renewing hematopoietic stem cells (HSC), multi-potent hematopoietic progenitor cells (MPP) that are undergoing commitment to particular lineage fates, and recognizable precursor cells that mature towards functional blood and immune cells. A single route to each end-cell type is prescribed in the "classical" model for the architecture of hematopoiesis. Recent findings have led to the viewpoint that HSCs and MPPs are more versatile than previously thought. Underlying this are multiple routes to a particular fate and cells having clandestine fate options even when they have progressed some way along a pathway. The primary role of cytokines during hematopoiesis has long been seen to be regulation of the survival and proliferation of developing hematopoietic cells. Some cytokines now clearly have instructive actions on cell-fate decisions. All this leads to a new way of viewing hematopoiesis whereby versatile HSC and MPP are directed towards lineage outcomes via cytokine regulated cell-fate decisions. This means greater flexibility to the shaping of hematopoiesis.

Robo4 vaccines induce antibodies that retard tumor growth.

Tumor endothelial specific expression of Robo4 in adults identifies this plasma membrane protein as an anti-cancer target for immunotherapeutic approaches, such as vaccination. In this report, we describe how vaccination against Robo4 inhibits angiogenesis and tumor growth. To break tolerance to the auto-antigen Robo4, mice were immunised with the extracellular domain of mouse Robo4, fused to the Fc domain of human immunoglobulin within an adjuvant. Vaccinated mice show a strong antibody response to Robo4, with no objectively detectable adverse effects on health. Robo4 vaccinated mice showed impaired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism. Finally, we show that an adjuvant free soluble Robo4-carrier conjugate can retard tumor growth in carrier primed mice. These results point to appropriate Robo4 conjugates as potential anti-angiogenic vaccines for cancer patients.

Cognate interactions: extrafollicular IL-4 drives germinal-center reactions, a new role for an old cytokine.

Over the past 25 years it has become clear that B and T lymphocytes go through a range of interactions and migratory events when B cells differentiate to become high-affinity, antibody-secreting cells. This B-cell differentiation is associated with multiple sequential cognate interactions. In this issue of the European Journal of Immunology, Turqueti-Neves et al. [Eur. J. Immunol. 2014. 44: 2130-2138] show that IL-4, a cytokine well known as a regulator of Ig class switch recombination, has another as-yet-unappreciated role. The authors show that IL-4 produced by T-helper cells outside germinal centers has a major effect on the early stages of germinal-center B-cell differentiation. This Commentary will summarize their findings and relate them to what we know on the sequence of cognate interactions and migratory events B cells undergo during T-dependent immune responses.

CD8 T cells induce T-bet-dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines.

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell-dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

The capsular polysaccharide Vi from Salmonella typhi is a B1b antigen.

Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid Vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.

Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TMBIM4 safeguards IgG1+ GC B cell-positive selection.

The transition from immunoglobulin M (IgM) to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential maintenance of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different Ig isotypes within GCs is dependent on specific transcriptional mechanisms. Here, we explored IgG1+ GC B cell transcription factor dependency using a CRISPR-Cas9 screen and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor (IP3R)-mediated calcium (Ca2+) mobilization downstream of B cell receptor (BCR) signaling in IgG1+ B cells. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique Ig isotype-specific dependency on a hitherto unidentified mechanism in GC-positive selection.

Noxa mediates p18INK4c cell-cycle control of homeostasis in B cells and plasma cell precursors.

Inhibition of Cdk4/Cdk6 by p18(INK4c) (p18) is pivotal for generation of noncycling immunoglobulin (Ig)-secreting plasma cells (PCs). In the absence of p18, CD138(+) plasmacytoid cells continue to cycle and turnover rapidly, suggesting that p18 controls PC homeostasis. We now show that p18 selectively acts in a rare population of rapidly cycling CD138(hi)/B220(hi) intermediate PCs (iPCs). While retaining certain B-cell signatures, iPCs are poised to differentiate to end-stage PCs although the majority undergo apoptosis. p18 is dispensable for the development of the PC transcriptional circuitry, and Blimp-1 and Bcl-6 are expressed fully and mutually exclusively in individual iPCs. However, a minor proportion of iPCs express both, and they are preferentially protected by p18 or Bcl-xL overexpression, consistent with expansion of the iPC pool by Bcl-xL overexpression, or loss of proapoptotic Bim or Noxa. Expression of Noxa is induced during B-cell activation, peaks in iPCs, and selectively repressed by p18. It is required to promote apoptosis of cycling B cells, especially in the absence of p18. These findings define the first physiologic function for Noxa and suggest that by repressing Noxa, induction of G1 arrest by p18 bypasses a homeostatic cell-cycle checkpoint in iPCs for PC differentiation.

IFN-{gamma} produced by CD8 T cells induces T-bet-dependent and -independent class switching in B cells in responses to alum-precipitated protein vaccine.

Alum-precipitated protein (alum protein) vaccines elicit long-lasting neutralizing antibody responses that prevent bacterial exotoxins and viruses from entering cells. Typically, these vaccines induce CD4 T cells to become T helper 2 (Th2) cells that induce Ig class switching to IgG1. We now report that CD8 T cells also respond to alum proteins, proliferating extensively and producing IFN-γ, a key Th1 cytokine. These findings led us to question whether adoptive transfer of antigen-specific CD8 T cells alters the characteristic CD4 Th2 response to alum proteins and the switching pattern in responding B cells. To this end, WT mice given transgenic ovalbumin (OVA)-specific CD4 (OTII) or CD8 (OTI) T cells, or both, were immunized with alum-precipitated OVA. Cotransfer of antigen-specific CD8 T cells skewed switching patterns in responding B cells from IgG1 to IgG2a and IgG2b. Blocking with anti-IFN-γ antibody largely inhibited this altered B-cell switching pattern. The transcription factor T-bet is required in B cells for IFN-γ-dependent switching to IgG2a. By contrast, we show that this transcription factor is dispensable in B cells both for IFN-γ-induced switching to IgG2b and for inhibition of switching to IgG1. Thus, T-bet dependence identifies distinct transcriptional pathways in B cells that regulate IFN-γ-induced switching to different IgG isotypes.

IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation.

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced.

Vi polysaccharide and conjugated vaccines afford similar early, IgM or IgG-independent control of infection but boosting with conjugated Vi vaccines sustains the efficacy of immune responses.

Introduction: Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Methods and results: Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Discussion: Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S. Typhi.

Early B blasts acquire a capacity for Ig class switch recombination that is lost as they become plasmablasts.

Rapid production of neutralizing antibody can be critical for limiting the spread of infection. Such early antibody results when B-cell blasts mature directly to plasmablasts without forming germinal centers. These extrafollicular responses can involve Ig class switch recombination (CSR), producing antibody that can readily disseminate through infected tissues. The present study identifies the differentiation stage where CSR occurs in an extrafollicular response induced by 4-hydroxy-3-nitrophenyl acetyl (NP) conjugated to Ficoll (NP-Ficoll). To do this, we took advantage of the antigen dose dependency of CSR in this response. Thus, while both 30 and 1 µg NP-Ficoll induce plasmablasts, only the higher antigen dose induces CSR. Activation-induce cytidine deaminase (AID) is critical for CSR and in keeping with this a proportion of NP-specific B-cell blasts induced by 30 µg NP-Ficoll express AID. None of the B blasts responding to the non-CSR-inducing 1 µg dose of NP-Ficoll express AID. We confirmed that CSR occurs in B blasts by demonstrating the presence of rearranged heavy-chain transcripts in B blasts in the 30 µg response. CSR in this extrafollicular response is confined to B blasts, because NP-specific plasmablasts, identified by expressing CD138 and Blimp-1, no longer express AID and cannot undergo CSR.