Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells.

Follicular regulatory T cells (Tfr) can play opposite roles in the regulation of germinal center (GC) responses. Depending on the studies, Tfr suppress or support GC and B cell affinity maturation. However, which factors determine positive vs. negative effects of Tfr on the GC B cell is unclear. In this study, we show that GC centrocytes that express MYC up-regulate expression of CCL3 chemokine that is needed for both the positive and negative regulation of GC B cells by Tfr. B cell-intrinsic expression of CCL3 contributes to Tfr-dependent positive selection of foreign Ag-specific GC B cells. At the same time, expression of CCL3 is critical for direct Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1 receptors promote Tfr migration to CCL3 and highlights Ccr5 expression on the Tfr subset that expresses Il10. Based on our findings and previous studies, we suggest a model of chemotactically targeted checkpoint control of B cells undergoing positive selection in GCs by Tfr, where Tfr directly probe and license foreign antigen-specific B cells to complete their positive selection in GCs but, at the same time, suppress GC B cells that present self-antigens cognate to Tfr.

Germinal center B cells that acquire nuclear proteins are specifically suppressed by follicular regulatory T cells.

Follicular regulatory T cells (Tfr) restrict development of autoantibodies and autoimmunity while supporting high-affinity foreign antigen-specific humoral response. However, whether Tfr can directly repress germinal center (GC) B cells that acquire autoantigens is unclear. Moreover, TCR specificity of Tfr to self-antigens is not known. Our study suggests that nuclear proteins contain antigens specific to Tfr. Targeting of these proteins to antigen-specific B cells in mice triggers rapid accumulation of Tfr with immunosuppressive characteristics. Tfr then exert negative regulation of GC B cells with predominant inhibition of the nuclear protein-acquiring GC B cells, suggesting an important role of direct cognate Tfr-GC B cells interactions for the control of effector B cell response.

TNF-α Limits Serological Memory by Disrupting the Bone Marrow Niche.

Both infection and autoimmune disease can disrupt pre-existing Ab titers leading to diminished serological memory, yet the underlying mechanisms are not well understood. In this article, we report that TNF-α, an inflammatory cytokine, is a master regulator of the plasma cell (PC) niche in the bone marrow (BM). Acute rTNF-α treatment depletes previously existing Ab titers after vaccination by limiting PC occupancy or retention in the BM. Consistent with this phenomenon, mice lacking TNF-α signaling have elevated PC capacity in the BM and higher Ab titers. Using BM chimeric mice, we found that PC egress from the BM is regulated in a cell-extrinsic manner, by radiation-resistant cells via TNF-α receptor 1 signaling, leading to increased vascular permeability and CD138 downregulation on PCs. PC motility and egress in the BM are triggered within 6 h of recombinant TNF-α treatment. In addition to promoting egress, TNF-α signaling also prevented re-engraftment into the BM, leading to reduced PC survival. Although other inflammatory stimuli can promote PC egress, TNF-α signaling is necessary for limiting the PC capacity in the BM. Collectively, these data characterize how TNF-α-mediated inflammation attenuates the durability of serological memory and shapes the overall size and composition of the Ab-secreting cell pool in the BM.

Discovery of an agonistic Siglec-6 antibody that inhibits and reduces human mast cells.

Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease.

The Inhibitory Receptor Siglec-8 Interacts With FcεRI and Globally Inhibits Intracellular Signaling in Primary Mast Cells Upon Activation.

Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement.

Memory CD8+ T cells mediate early pathogen-specific protection via localized delivery of chemokines and IFNγ to clusters of monocytes.

While cognate antigen drives clonal expansion of memory CD8+ T (CD8+ TM) cells to achieve sterilizing immunity in immunized hosts, not much is known on how cognate antigen contributes to early protection before clonal expansion occurs. Here, using distinct models of immunization, we establish that cognate antigen recognition by CD8+ TM cells on dendritic cells initiates their rapid and coordinated production of a burst of CCL3, CCL4, and XCL1 chemokines under the transcriptional control of interferon (IFN) regulatory factor 4. Using intravital microscopy imaging, we reveal that CD8+ TM cells undergo antigen-dependent arrest in splenic red pulp clusters of CCR2+Ly6C+ monocytes to which they deliver IFNγ and chemokines. IFNγ enables chemokine-induced microbicidal activities in monocytes for protection. Thus, rapid and effective CD8+ TM cell responses require spatially and temporally coordinated events that quickly restrict microbial pathogen growth through the local delivery of activating chemokines to CCR2+Ly6C+ monocytes.

Plasma cell dynamics in the bone marrow niche.

Using intravital imaging, we report that bone marrow (BM) plasma cells (PCs) are motile. BM PCs exhibit a unique migration pattern, characterized by intermittent periods of high motility and longer stretches of confined migration or arrest. BM PCs accumulate into clusters, which have reduced cell motility. APRIL promotes cluster formation and overall PC motility in the BM. Although CXCL12 and its receptor, CXCR4, promote PC motility in the BM, VLA4 activity promotes arrest. However, blocking either pathway promotes PC egress from the BM. Under steady-state conditions, BM PCs recirculate to other bones and spleen. In older mice, overall PC motility and recirculation increase, and this is correlated with increased CXCR4 expression, which depends on PC age or maturation rather than mouse age. Altogether, these results suggest that changes in PC motility and CXCR4 expression are linked with survival of long-lived PCs in the BM.

Signals 1, 2 and B cell fate or: Where, when and for how long?

Diverse B cell responses are important for generating antibody-mediated protection against highly variable pathogens. While some antigens can trigger T-independent B cell proliferation and short-term antibody production, development of long-term humoral immunity requires T-dependent B cell responses. The "two-signal" model of B cell activation has long been invoked to explain alternate B cell recruitment into immune response to foreign antigens vs. induction of tolerance to self-antigens. However, a number of other factors appear to influence the fate of mature B cells responding to antigen in vivo. In this review, we will discuss how various spatiotemporal scenarios of antigen access into secondary lymphoid organs, antigen valency and cellular environment of antigen acquisition by B cells, duration of B cell access to antigen and the timing of T cell help may affect follicular B cell fate, including death, survival, anergy, and recruitment into T-dependent responses. We will also highlight unresolved questions related to B cell activation and tolerance in vivo that may have important implications for vaccine development and autoimmunity.

CCL3 Promotes Germinal Center B Cells Sampling by Follicular Regulatory T Cells in Murine Lymph Nodes.

Previous studies and our findings suggest upregulated expression of proinflammatory chemokines CCL3/4 in germinal center (GC) centrocytes. However, the role of CCL3/4 for centrocyte interactions with follicular T cells and regulation of humoral immunity is poorly understood. We found that CCL3 promotes chemotaxis of Tfr cells ex vivo. Two-photon imaging revealed that B cells-intrinsic production of CCL3 promotes their probing by follicular regulatory T cells (Tfr) within GCs of murine lymph nodes. Overall this study suggests that CCL3 facilitates direct interactions of foreign antigen-specific GC B cells and their negative regulation with Tfr cells in vivo.

Transiently antigen primed B cells can generate multiple subsets of memory cells.

Memory B cells are long-lived cells that generate a more vigorous response upon recognition of antigen (Ag) and T cell help than naïve B cells and ensure maintenance of durable humoral immunity. Functionally distinct subsets of murine memory B cells have been identified based on isotype switching of BCRs and surface expression of the co-stimulatory molecule CD80 and co-inhibitory molecule PD-L2. Memory B cells in a subpopulation with low surface expression of CD80 and PD-L2 are predominantly non-isotype switched and can be efficiently recruited into germinal centers (GCs) in secondary responses. In contrast, a CD80 and PD-L2 positive subset arises predominantly from GCs and can quickly differentiate into antibody-secreting plasma cells (PCs). Here we demonstrate that single transient acquisition of Ag by B cells may be sufficient for their long-term participation in GC responses and for development of various memory B cell subsets including CD80 and PD-L2 positive effector-like memory cells that rapidly differentiate into class-switched PCs during recall responses.

Antigen Acquisition Enables Newly Arriving B Cells To Enter Ongoing Immunization-Induced Germinal Centers.

Modern vaccines must be designed to generate long-lasting, high-affinity, and broadly neutralizing Ab responses against pathogens. The diversity of B cell clones recruited into germinal center (GC) responses is likely to be important for the Ag-neutralization potential of the Ab-secreting cells and memory cells generated upon immunization. However, the factors that influence the diversity of B cell clones recruited into GCs are unclear. As recirculating naive Ag-specific B cells arrive in Ag-draining secondary lymphoid organs, they may join the ongoing GC response. However, the factors that limit their entry are not well understood, and it is not known how that depends on the stage of the ongoing follicular T cell and GC B cell response. In this article, we show that, in mice, naive B cells have a limited window of time during which they can undergo Ag-driven activation and join ongoing immunization-induced GC responses. However, preloading naive B cells with even a threshold-activating amount of Ag is sufficient to rescue their entry into the GC response during its initiation, peak, and contraction. Based on these results, we suggest that productive acquisition of Ag may be one of the main factors limiting entry of new B cell clones into ongoing immunization-triggered GC responses.

Transiently antigen-primed B cells return to naive-like state in absence of T-cell help.

The perspective that naive B-cell recognition of antigen in the absence of T-cell help causes cell death or anergy is supported by in vivo studies of B cells that are continuously exposed to self-antigens. However, intravital imaging suggests that early B-cell recognition of large foreign antigens may be transient. Whether B cells are tolerized or can be recruited into humoural immune responses following such encounters is not clear. Here we show that in the presence of T-cell help, single transient antigen acquisition is sufficient to recruit B cells into the germinal centre and induce memory and plasma cell responses. In the absence of T-cell help, transiently antigen-primed B cells do not undergo apoptosis in vivo; they return to quiescence and are recruited efficiently into humoural responses upon reacquisition of antigen and T-cell help.

Genome-wide siRNA screen of genes regulating the LPS-induced NF-κB and TNF-α responses in mouse macrophages.

The mammalian innate immune system senses many bacterial stimuli through the toll-like receptor (TLR) family. Activation of the TLR4 receptor by bacterial lipopolysaccharide (LPS) is the most widely studied TLR pathway due to its central role in host responses to gram-negative bacterial infection and its contribution to endotoxemia and sepsis. Here we describe a genome-wide siRNA screen to identify genes regulating the mouse macrophage TNF-α and NF-κB responses to LPS. We include a secondary validation screen conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits. We also provide microarray data from the same LPS-treated macrophage cells to facilitate downstream data analysis. These data provide a resource for analyzing gene function in the predominant pathway driving inflammatory signaling and cytokine expression in mouse macrophages.

Proteome and Secretome Analysis Reveals Differential Post-transcriptional Regulation of Toll-like Receptor Responses.

The innate immune system is the organism's first line of defense against pathogens. Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogen-associated molecules. The prototypic PRRs, the membrane-bound receptors of the Toll-like receptor (TLR) family, recognize pathogen-associated molecular patterns (PAMPs) and initiate an innate immune response through signaling pathways that depend on the adaptor molecules MyD88 and TRIF. Deciphering the differences in the complex signaling events that lead to pathogen recognition and initiation of the correct response remains challenging. Here we report the discovery of temporal changes in the protein signaling components involved in innate immunity. Using an integrated strategy combining unbiased proteomics, transcriptomics and macrophage stimulations with three different PAMPs, we identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level.

Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages.

Macrophages play a critical role in the innate immune response to pathogen infection, but few tools exist for systematic dissection of these responses using modern genome-wide perturbation methods. To develop an assay platform for high-throughput analysis of macrophage activation by pathogenic stimuli, we generated reporter systems in human and mouse macrophages with dynamic readouts for NF-κB and/or TNF-α responses. These reporter cells show responsiveness to a broad range of TLR ligands and to gram-negative bacterial infection. There are significant challenges to the use of RNAi in innate immune cells, including efficient small RNA delivery and non-specific immune responses to dsRNA. To permit the interrogation of the macrophage pathogen response pathways with RNAi, we employed the stably expressed reporter genes to develop efficient siRNA delivery protocols for maximal target gene silencing with minimal activation of the innate macrophage response to nucleic acids. We demonstrate the utility of these macrophage cell systems for siRNA screening of pathogen responses by targeting components of the human and mouse TLR pathways, and observe species-specific perturbation of signaling and cytokine responses. Our approach to reporter cell development and siRNA delivery optimization provides an experimental paradigm with significant potential for developing genetic screening platforms in mammalian cells.

Prevalence and detailed mapping of the gonococcal genetic island in Neisseria meningitidis.

The 57-kb gonococcal genetic island (GGI) encodes a type IV secretion system (T4SS) that is found in most strains of N. gonorrhoeae. This T4SS functions to secrete single-stranded DNA that is active in natural transformation. The GGI has also been found in some strains of N. meningitidis. We screened 126 isolates of N. meningitidis and found the GGI in 17.5% of strains, with the prevalence varying widely among serogroups. The GGI is found in a significant number of serogroup C, W-135, and X strains but was not found in strains of serogroup A, B, or Y. Through detailed PCR mapping and DNA sequencing, we identified five distinct GGI types in meningococci. DNA sequencing and a genetic assay revealed that the GGI was likely integrated into the meningococcal chromosome by the site-specific recombinase XerCD and that the GGI can be excised and lost from the genome. Functional studies showed that in contrast with the gonococcal T4SS, the meningococcal T4SS does not secrete DNA, nor does it confer Ton-independent intracellular survival. Deletion of T4SS genes did not affect association with or invasion of host cells. These results demonstrate that the GGI is found in a significant proportion of meningococcal strains and that while some strains carry multiple insertions and deletions in the GGI, other strains carry intact T4SS genes and may produce functional secretion systems.