The SWHEL model for studying B cell responses in tolerance and immunity

Classical immunoglobulin transgenic (Ig-Tg) mouse models such as the MD4 anti-hen egg lysozyme (-HEL) Ig-Tg line have been used extensively to study B cell responses in tolerance and immunity. This thesis describes a new generation of gene-targeted mice (designated SWHEL mice) whereby the VH10 Ig variable gene encoding the HyHEL-10 specificity of the original anti-HEL Ig-Tg mouse was targeted to the Ig heavy chain locus. B cells in the SWHEL mouse are therefore capable of undergoing class switch recombination (CSR) and somatic hypermutation (SHM), representing a major advance on the original MD4 mouse model. SWHEL mice were found to not only contain a large population of HEL-specific (HEL+) B cells but also a significant population of non-HEL-binding (HEL-) B cells generated by VH gene replacement. HEL+ SWHEL B cells were found to belong to the B2 lineage and displayed high levels of surface IgM. Nevertheless, they matured normally and colonised the primary B cell follicle and marginal zone (MZ) of the spleen. The SWHEL model thus provided an opportunity to re-examine some of the original observations made in the MD4 system and also to extend these observations, particularly with regard to the regulation of CSR by self-reactive B cells. As expected, analysis of SWHEL B cells exposed to high avidity membrane-bound HEL revealed that they underwent clonal deletion in the bone marrow (BM). More interestingly, analysis of HEL+ B cells exposed to low avidity soluble HEL revealed that they were able to emigrate from the BM to the spleen as anergic B cells. However, unlike anergic MD4 B cells, anergic SWHEL B cells were reduced in frequency, displayed an immature B cell phenotype, were excluded from the follicle and had a reduced lifespan. Direct measurement of B cell antigen receptor (BCR) occupancy by HEL and the frequency of HEL- competitor B cells was combined with mixed BM irradiation chimeras to demonstrate unequivocally that the difference in phenotype and fate of HEL+ B cells in the two systems was due solely to competition from HEL- B cells. In addition, the SWHEL model of B cell self-tolerance was used to show that while self-reactive B cells were hypo-responsive to BCR stimulation, BCR-independent signals delivered via anti-CD40 plus IL-4 or lipopolysaccharide could trigger them to undergo CSR and secretion of potentially pathogenic isotype-switched autoantibodies. Finally, the SWHEL model was used to study the responses of adoptively transferred follicular (Fo) and MZ B cells to in vivo activation with HEL conjugated to sheep red blood cells (HEL-SRBC). These studies revealed that both HEL+ MZ and Fo B cells were capable of mounting a robust T cell-dependent IgG1 antibody response to HEL-SRBC. However, HEL+ MZ B cells did not efficiently localise to the T cell-B cell border following antigen engagement and preferentially migrated to the bridging channels and red pulp. In contrast, HEL+ Fo B cells rapidly localised to the T cell-B cell border and subsequently colonised numerous germinal centres. As a result, the rate and pattern of SHM by HEL+ Fo and MZ B cells was shown to be distinct, with preferential targeting of mutations to the second complementarity-determining region in the former and to the second framework region in the latter. Together these data indicate illustrate the value of the SWHEL model and its potential to greatly advance the current understanding of B cell responses in tolerance and immunity

Clearing the complexity: immune complexes and their treatment in lupus nephritis

Systemic lupus erythematosus (SLE) is a classic antibody-mediated systemic autoimmune disease characterised by the development of autoantibodies to ubiquitous self-antigens (such as antinuclear antibodies and antidouble-stranded DNA antibodies) and widespread deposition of immune complexes in affected tissues. Deposition of immune complexes in the kidney results in glomerular damage and occurs in all forms of lupus nephritis. The development of nephritis carries a poor prognosis and high risk of developing end-stage renal failure despite recent therapeutic advances. Here we review the role of DNA-anti-DNA immune complexes in the pathogenesis of lupus nephritis and possible new treatment strategies aimed at their control

Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation.

Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity

Mobile microscopy on the move

In this paper, we demonstrate the application of low cost light weight imaging device that amplifies the imaging resolution of a smartphone camera by three orders of magnitude from millimeters to sub-micrometers. We attached the lens onto a commercial smartphone camera and imaged micrometer graticules, pathological biological tissue slides and skin which validate the imaging quality of lenses

Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells.

The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph

Challenges and opportunities for non-antibody scaffold drugs

The first candidates from the promising class of small non-antibody protein scaffolds are now moving into clinical development and practice. Challenges remain, and scaffolds will need to be further tailored toward applications where they provide real advantages over established therapeutics to succeed in a rapidly evolving drug development landscape

Gene expression predicts dormant metastatic breast cancer cell phenotype

BACKGROUND: Breast cancer can recur months to decades after an initial diagnosis and treatment. The mechanisms that control tumor cell dormancy remain poorly understood, making it difficult to predict which patients will recur and thus benefit from more rigorous screening and treatments. Unfortunately, the extreme rarity of dormant DTCs has been a major obstacle to their study. METHODS: To overcome this challenge, we developed an efficient system to isolate and study rare dormant breast cancer cells from metastatic organs including bones, which represent a major site of metastasis. After isolation of cells from the long bones, we used single cell RNA-sequencing (scRNA-seq) to profile proliferative and dormant PyMT-Bo1 breast cancer cells. We also compared this signature to dormant versus proliferative tumor cells isolated from the lungs. Finally, we compared our dormant signature to human datasets. RESULTS: We identified a group of genes including Cfh, Gas6, Mme and Ogn that were highly expressed in dormant breast cancer cells present in the bone and lung. Expression of these genes had no impact on dormancy in murine models, but their expression correlated with disease-free survival in primary human breast cancer tumors, suggesting that these genes have predictive value in determining which patients are likely to recur. CONCLUSIONS: Dormant breast cancer cells exhibit a distinct gene expression signature regardless of metastatic site. Genes enriched in dormant breast cancer cells correlate with recurrence-free survival in breast cancer patients

High affinity germinal center B cells are actively selected into the plasma cell compartment

A hallmark of T cell–dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this “quality control” over plasma cell differentiation is likely critical for establishing effective humoral immunity

B Cell Receptor–independent Stimuli Trigger Immunoglobulin (Ig) Class Switch Recombination and Production of IgG Autoantibodies by Anergic Self-Reactive B Cells

In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are required to reinforce B cell self-tolerance. We have used gene targeting to produce mice in which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SWHEL mice). When crossed with soluble HEL transgenic (Tg) mice, self-reactive SWHEL B cells became anergic. However, in contrast to anergic B cells from the original nonswitching anti-HEL × soluble HEL double Tg model, self-reactive SWHEL B cells also displayed an immature phenotype, reduced lifespan, and exclusion from the splenic follicle. These differences were not related to their ability to Ig class switch, but instead to competition with non-HEL–binding B cells generated by VH gene replacement in SWHEL mice. When activated in vitro with B cell receptor (BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or lipopolysaccharide (LPS), anergic SWHEL double Tg B cells proliferated and produced IgG anti-HEL antibodies as efficiently as naive HEL-binding B cells from SWHEL Ig Tg mice. These results demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive B cells. Instead, production of IgG autoantibodies is prevented by separate controls that reduce the likelihood of anergic B cells encountering BCR-independent stimuli. That bacteria-derived LPS could circumvent these controls may explain the well-known association between autoantibody-mediated diseases and episodes of systemic infection

MicroRNA-155 controls affinity-based selection by protecting c-MYC+ B cells from apoptosis.

The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes.R. Nakagawa was supported by a Marie Curie Incoming Fellowship from the European Union’s Seventh Framework Programme for research, technological development, and demonstration. E. Vigorito was supported by the Medical Research Council grants G1001781 and G0700287 and by the Biotechnology and Biological Sciences Research Council. M. Meyer-Hermann was supported by the German Federal Ministry of Education and Research within the Measures for the Establishment of Systems Medicine, project SYSIMIT (BMBF eMed project SYSIMIT, FKZ: 01ZX1308B) and by the Human Frontier Science Program (RGP0033/2015)