Publications by authors named "Irina Grigorova"

23 Publications

  • Page 1 of 1

B and Th cell response to Ag in vivo: Implications for vaccine development and diseases.

Immunol Rev 2020 07;296(1):5-8

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA.

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http://dx.doi.org/10.1111/imr.12899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405089PMC
July 2020

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

Immunol Rev 2020 07 29;296(1):9-23. Epub 2020 May 29.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

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.
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http://dx.doi.org/10.1111/imr.12865DOI Listing
July 2020

Macropinocytosis drives T cell growth by sustaining the activation of mTORC1.

Nat Commun 2020 01 10;11(1):180. Epub 2020 Jan 10.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, 48109, USA.

Macropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.
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http://dx.doi.org/10.1038/s41467-019-13997-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954116PMC
January 2020

Self-Antigens Displayed on Liposomal Nanoparticles above a Threshold of Epitope Density Elicit Class-Switched Autoreactive Antibodies Independent of T Cell Help.

J Immunol 2020 01 13;204(2):335-347. Epub 2019 Dec 13.

Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109;

Epitope density has a profound impact on B cell responses to particulate Ags, the molecular mechanisms of which remain to be explored. To dissect the role of epitope density in this process, we have synthesized a series of liposomal particles, similar to the size of viruses, that display a model self-antigen peptide at defined surface densities. Immunization of C57BL/6J mice using these particles elicited both IgM and class-switched IgG1, IgG2b, and IgG3 autoreactive Abs that depended on the epitope density. In C57BL/6 gene knockout mice lacking either functional TCRs or MHC class II molecules on B cells, the liposomal particles also elicited IgM, IgG1, IgG2b, and IgG3 responses that were comparable in magnitudes to wild-type mice, suggesting that this B cell response was independent of cognate T cell help. Notably, the titer of the IgG in wild-type animals could be increased by more than 200-fold upon replacement of liposomes with bacteriophage Qβ virus-like particles that displayed the same self-antigen peptide at comparable epitope densities. This enhancement was lost almost completely in gene knockout mice lacking either TCRs or MHC class II molecules on B cells. In conclusion, epitope density above a threshold on particulate Ags can serve as a stand-alone signal to trigger secretion of autoreactive and class-switched IgG in vivo in the absence of cognate T cell help or any adjuvants. The extraordinary immunogenicity of Qβ viral-like particles relies, in large part, on their ability to effectively recruit T cell help after B cell activation.
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http://dx.doi.org/10.4049/jimmunol.1801677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946842PMC
January 2020

B Cell Receptor Crosslinking Augments Germinal Center B Cell Selection when T Cell Help Is Limiting.

Cell Rep 2018 11;25(6):1395-1403.e4

Department of Microbiology and Immunology, Michigan Medicine University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Antigen-dependent engagement of germinal center (GC) B cell receptors (BCRs) promotes antigen internalization and presentation for follicular helper T cells. However, whether BCR signaling is critical or synergistic with T cell help for GC B cell selection or differentiation is unclear. Here, by adapting an experimental approach that enables independent delivery of BCR-crosslinking antigen or T cell help to GC B cells in vivo, we showed that T cell help was sufficient to induce GC B cell expansion and plasmablast formation. However, although BCR crosslinking could not by itself promote GC B cell selection or differentiation, it could synergize with T cell help to enhance the GC and plasmablast responses when T cell help was limiting. These findings indicate that GC B cells can integrate variable inputs from T cell help and BCR signaling in vivo for an optimal process of selection and differentiation, critical for potent long-term humoral immunity.
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http://dx.doi.org/10.1016/j.celrep.2018.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289055PMC
November 2018

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

Front Immunol 2018 13;9:2044. Epub 2018 Sep 13.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, United States.

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 . 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 .
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http://dx.doi.org/10.3389/fimmu.2018.02044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146081PMC
September 2019

TLR7-Mediated Lupus Nephritis Is Independent of Type I IFN Signaling.

J Immunol 2018 07 8;201(2):393-405. Epub 2018 Jun 8.

Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109;

Systemic lupus erythematosus is an autoimmune disease characterized by increased type I IFNs, autoantibodies, and inflammatory-mediated multiorgan damage. TLR7 activation is an important contributor to systemic lupus erythematosus pathogenesis, but the mechanisms by which type I IFNs participate in TLR7-driven pathologic conditions remain uncertain. In this study, we examined the requirement for type I IFNs in TLR7-stimulated lupus nephritis. Lupus-prone NZM2328, INZM (which lack a functional type I IFN receptor), and NZM2328 IL-1β mice were treated at 10 wk of age on the right ear with R848 (TLR7 agonist) or control (DMSO). Autoantibody production and proteinuria were assessed throughout treatment. Multiorgan inflammation was assessed at the time of decline in health. Renal infiltrates and mRNA expression were also examined after 14 d of treatment. Both NZM2328 and INZM mice exhibited a decline in survival after 3-4 wk of R848 but not vehicle treatment. Development of splenomegaly and liver inflammation were dependent on type I IFN. Interestingly, autoantibody production, early renal infiltration of dendritic cells, upregulation of IL-1β, and lupus nephritis occurred independent of type I IFN signaling. Development of TLR7-driven lupus nephritis was not abolished by the deletion of IL-1β. Thus, although IFN-α is sufficient to induce nephritis acceleration, our data emphasize a critical role for IFN-independent signaling in TLR7-mediated lupus nephritis. Further, despite upregulation of IL-1β after TLR7 stimulation, deletion of IL-1β is not sufficient to reduce lupus nephritis development in this model.
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http://dx.doi.org/10.4049/jimmunol.1701588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039244PMC
July 2018

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

PLoS One 2017 29;12(8):e0183877. Epub 2017 Aug 29.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183877PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574538PMC
October 2017

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

J Immunol 2017 08 7;199(4):1301-1307. Epub 2017 Jul 7.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620

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.
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http://dx.doi.org/10.4049/jimmunol.1700267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548600PMC
August 2017

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

Nat Commun 2017 04 21;8:15072. Epub 2017 Apr 21.

Department of Microbiology and Immunology, University of Michigan Medical School, 1150W. Medical Center Drive, Ann Arbor, Michigan 48109, USA.

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.
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http://dx.doi.org/10.1038/ncomms15072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413946PMC
April 2017

Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress.

Nature 2013 Jan 23;493(7434):684-8. Epub 2012 Dec 23.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.

The splenic marginal zone is a unique microenvironment where resident immune cells are exposed to the open blood circulation. Even though it has an important role in responses against blood-borne antigens, lymphocyte migration in the marginal zone has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a two-photon microscopy procedure to study marginal zone and follicular B-cell movement in the live mouse spleen. We show that marginal zone B cells are highly motile and exhibit long membrane extensions. Marginal zone B cells shuttle between the marginal zone and follicles with at least one-fifth of the cells exchanging between compartments per hour, a behaviour that explains their ability to deliver antigens rapidly from the open blood circulation to the secluded follicles. Follicular B cells also transit from follicles to the marginal zone, but unlike marginal zone B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. Follicular B-cell egress via the marginal zone is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that marginal zone B cells migrate continually between marginal zone and follicles and establishes the marginal zone as a site of S1PR1-dependent B-cell exit from follicles. The results also show how adhesive differences of similar cells critically influence their behaviour in the same microenvironment.
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http://dx.doi.org/10.1038/nature11738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561487PMC
January 2013

Lymph node cortical sinus organization and relationship to lymphocyte egress dynamics and antigen exposure.

Proc Natl Acad Sci U S A 2010 Nov 8;107(47):20447-52. Epub 2010 Nov 8.

The Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USA.

Recent studies have identified cortical sinuses as sites of sphingosine-1-phosphate receptor-1 (S1P(1))-dependent T- and B-cell egress from the lymph node (LN) parenchyma. However, the distribution of cortical sinuses in the entire LN and the extent of lymph flow within them has been unclear. Using 3D reconstruction and intravital two-photon microscopy we describe the branched organization of the cortical sinus network within the inguinal LN and show that lymphocyte flow begins within blunt-ended sinuses. Many cortical sinuses are situated adjacent to high endothelial venules, and some lymphocytes access these sinuses within minutes of entering a LN. However, upon entry to inflamed LNs, lymphocytes rapidly up-regulate CD69 and are prevented from accessing cortical sinuses. Using the LN reconstruction data and knowledge of lymphocyte migration and cortical sinus entry dynamics, we developed a mathematical model of T-cell egress from LNs. The model suggests that random walk encounters with lymphatic sinuses are the major factor contributing to LN transit times. A slight discrepancy between predictions of the model and the measured transit times may be explained by lymphocytes undergoing a few rounds of migration between the parenchyma and sinuses before departing from the LN. Because large soluble antigens gain rapid access to cortical sinuses, such parenchyma-sinus shuttling may facilitate antibody responses.
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http://dx.doi.org/10.1073/pnas.1009968107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996652PMC
November 2010

Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning.

J Exp Med 2010 Jan 21;207(1):17-27. Epub 2009 Dec 21.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.

Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Galphai-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell-cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.
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http://dx.doi.org/10.1084/jem.20091619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812554PMC
January 2010

Visualizing B cell capture of cognate antigen from follicular dendritic cells.

J Exp Med 2009 Jul 8;206(7):1485-93. Epub 2009 Jun 8.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.

The prominent display of opsonized antigen by follicular dendritic cells (FDCs) has long favored the view that they serve as antigen-presenting cells for B cells. Surprisingly, however, although B cell capture of antigen from macrophages and dendritic cells has been visualized, acquisition from FDCs has not been directly observed. Using two-photon microscopy, we visualized B cell capture of cognate antigen from FDCs. B cell CXCR5 expression was required, and encounter with FDC-associated antigen could be detected for >1 wk after immunization. B cell-FDC contact times were often brief but occasionally persisted for >30 min, and B cells sometimes acquired antigen together with FDC surface proteins. These observations establish that FDCs can serve as sites of B cell antigen capture, with their prolonged display time ensuring that even rare B cells have the chance of antigen encounter, and they suggest possible information transfer from antigen-presenting cell to B cell.
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http://dx.doi.org/10.1084/jem.20090209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715076PMC
July 2009

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

Nat Immunol 2009 Jan 7;10(1):58-65. Epub 2008 Dec 7.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA.

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.
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http://dx.doi.org/10.1038/ni.1682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710451PMC
January 2009

The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse strain and in a patient with severe combined immunodeficiency.

Nat Immunol 2008 Nov 5;9(11):1307-15. Epub 2008 Oct 5.

Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California 94143, USA.

Mice carrying the recessive locus for peripheral T cell deficiency (Ptcd) have a block in thymic egress, but the mechanism responsible is undefined. Here we found that Ptcd T cells had an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus showed a point substitution of lysine for glutamic acid at position 26 in the actin regulator coronin 1A that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. The discovery of another coronin 1A mutant during an N-ethyl-N-nitrosourea-mutagenesis screen for T cell-lymphopenic mice prompted us to evaluate a T cell-deficient, B cell-sufficient and natural killer cell-sufficient patient with severe combined immunodeficiency, whom we found had mutations in both CORO1A alleles. Our findings establish a function for coronin 1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation and demonstrate that actin regulation is a biological process defective in human and mouse severe combined immunodeficiency.
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http://dx.doi.org/10.1038/ni.1662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672406PMC
November 2008

Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces.

Nat Immunol 2007 Oct 26;8(10):1076-85. Epub 2007 Aug 26.

Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.
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http://dx.doi.org/10.1038/ni1499DOI Listing
October 2007

Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells.

Nat Immunol 2007 Sep 29;8(9):992-1000. Epub 2007 Jul 29.

Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.

The mechanism of B cell-antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells. Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune complexes by a complement receptor-dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore, cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an important site of B cell encounter with immune complexes and show that intrafollicular B cell migration facilitates the transport of immune complexes as well as encounters with cognate antigen.
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http://dx.doi.org/10.1038/ni1494DOI Listing
September 2007

Design principles of the proteolytic cascade governing the sigmaE-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction.

Genes Dev 2007 Jan;21(1):124-36

Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94158, USA.

Proteolytic cascades often transduce signals between cellular compartments, but the features of these cascades that permit efficient conversion of a biological signal into a transcriptional output are not well elucidated. sigma(E) mediates an envelope stress response in Escherichia coli, and its activity is controlled by regulated degradation of RseA, a membrane-spanning anti-sigma factor. Examination of the individual steps in this protease cascade reveals that the initial, signal-sensing cleavage step is rate-limiting; that multiple ATP-dependent proteases degrade the cytoplasmic fragment of RseA and that dissociation of sigma(E) from RseA is so slow that most free sigma(E) must be generated by the active degradation of RseA. As a consequence, the degradation rate of RseA is set by the amount of inducing signal, and insulated from the "load" on and activity of the cytoplasmic proteases. Additionally, changes in RseA degradation rate are rapidly reflected in altered sigma(E) activity. These design features are attractive as general components of signal transduction pathways governed by unstable negative regulators.
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http://dx.doi.org/10.1101/gad.1496707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1759897PMC
January 2007

Module-based analysis of robustness tradeoffs in the heat shock response system.

PLoS Comput Biol 2006 Jul 13;2(7):e59. Epub 2006 Apr 13.

Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan.

Biological systems have evolved complex regulatory mechanisms, even in situations where much simpler designs seem to be sufficient for generating nominal functionality. Using module-based analysis coupled with rigorous mathematical comparisons, we propose that in analogy to control engineering architectures, the complexity of cellular systems and the presence of hierarchical modular structures can be attributed to the necessity of achieving robustness. We employ the Escherichia coli heat shock response system, a strongly conserved cellular mechanism, as an example to explore the design principles of such modular architectures. In the heat shock response system, the sigma-factor sigma32 is a central regulator that integrates multiple feedforward and feedback modules. Each of these modules provides a different type of robustness with its inherent tradeoffs in terms of transient response and efficiency. We demonstrate how the overall architecture of the system balances such tradeoffs. An extensive mathematical exploration nevertheless points to the existence of an array of alternative strategies for the existing heat shock response that could exhibit similar behavior. We therefore deduce that the evolutionary constraints facing the system might have steered its architecture toward one of many robustly functional solutions.
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http://dx.doi.org/10.1371/journal.pcbi.0020059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1523291PMC
July 2006

Insights into transcriptional regulation and sigma competition from an equilibrium model of RNA polymerase binding to DNA.

Proc Natl Acad Sci U S A 2006 Apr 27;103(14):5332-7. Epub 2006 Mar 27.

Graduate Group in Biophysics and Department of Microbiology, University of California-San Francisco, 600 16th Street, Genentech Hall, Box 2200, San Francisco, CA 94143, USA.

To explore scenarios that permit transcription regulation by activator recruitment of RNA polymerase and sigma competition in vivo, we used an equilibrium model of RNA polymerase binding to DNA constrained by the values of total RNA polymerase (E) and sigma(70) per cell measured in this work. Our numbers of E and sigma(70) per cell, which are consistent with most of the primary data in the literature, suggest that in vivo (i) only a minor fraction of RNA polymerase (<20%) is involved in elongation and (ii) sigma(70) is in excess of total E. Modeling the partitioning of RNA polymerase between promoters, nonspecific DNA binding sites, and the cytoplasm suggested that even weak promoters will be saturated with Esigma(70) in vivo unless nonspecific DNA binding by Esigma(70) is rather significant. In addition, the model predicted that sigmas compete for binding to E only when their total number exceeds the total amount of RNA polymerase (excluding that involved in elongation) and that weak promoters will be preferentially subjected to sigma competition.
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http://dx.doi.org/10.1073/pnas.0600828103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459355PMC
April 2006

Fine-tuning of the Escherichia coli sigmaE envelope stress response relies on multiple mechanisms to inhibit signal-independent proteolysis of the transmembrane anti-sigma factor, RseA.

Genes Dev 2004 Nov;18(21):2686-97

Graduate Group in Biophysics, University of California, San Francisco, San Francisco, California 94143, USA.

Proteolytic cascades are widely implicated in signaling between cellular compartments. In Escherichia coli, accumulation of unassembled outer membrane porins (OMPs) in the envelope leads to expression of sigma(E)-dependent genes in the cytoplasmic cellular compartment. A proteolytic cascade conveys the OMP signal by regulated proteolysis of RseA, a membrane-spanning anti-sigma factor whose cytoplasmic domain inhibits sigma(E)-dependent transcription. Upon activation by OMP C termini, the membrane localized DegS protease cleaves RseA in its periplasmic domain, the membrane-embedded protease RseP (YaeL) cleaves RseA near the inner membrane, and the released cytoplasmic RseA fragment is further degraded. Initiation of RseA degradation by activated DegS makes the system sensitive to a wide range of OMP concentrations and unresponsive to variations in the levels of DegS and RseP proteases. These features rely on the inability of RseP to cleave intact RseA. In the present report, we demonstrate that RseB, which binds to the periplasmic face of RseA, and DegS each independently inhibits RseP cleavage of intact RseA. Thus, the function of RseB, widely conserved among bacteria using the sigma(E) pathway, and the second role of DegS (in addition to RseA proteolysis initiation) is to improve the performance characteristics of this signal transduction system.
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http://dx.doi.org/10.1101/gad.1238604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC525548PMC
November 2004

Regulation of the alternative sigma factor sigma(E) during initiation, adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli.

J Bacteriol 2003 Apr;185(8):2512-9

Department of Stomatology, University of California, San Francisco 94143-0512, USA.

The alternative sigma factor sigma(E) is activated in response to stress in the extracytoplasmic compartment of Escherichia coli. Here we show that sigma(E) activity increases upon initiation of the stress response by a shift to an elevated temperature (43 degrees C) and remains at that level for the duration of the stress. When the stress is removed by a temperature downshift, sigma(E) activity is strongly repressed and then slowly returns to levels seen in unstressed cells. We provide evidence that information about the state of the cell envelope is communicated to sigma(E) primarily through the regulated proteolysis of the inner membrane anti-sigma factor RseA, as the degradation rate of RseA is correlated with the changes in sigma(E) activity throughout the stress response. However, the relationship between sigma(E) activity and the rate of degradation of RseA is complex, indicating that other factors may cooperate with RseA and serve to fine-tune the response.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC152616PMC
http://dx.doi.org/10.1128/jb.185.8.2512-2519.2003DOI Listing
April 2003