Publications by authors named "Genita Metzler"

10 Publications

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Activated PIK3CD drives innate B cell expansion yet limits B cell-intrinsic immune responses.

J Exp Med 2018 10 7;215(10):2485-2496. Epub 2018 Sep 7.

Seattle Children's Research Institute, Seattle, WA

Activated PI3K-delta syndrome (APDS) is an immunodeficiency caused by gain-of-function mutations in PIK3CD. This disease exhibits complex immune phenotypes including increased IgM, recurrent infection, and impaired vaccine responses. To better understand the impact of B cells in this disease, we generated an inducible model of the common APDS mutation (h-E1021K; referred to as aPIK3CD) and intercrossed these mice with B cell-specific Cre models. Mb1-aPIK3CD mice exhibited bone marrow B lymphopenia and, conversely, expansion of the peripheral innate B1a and MZ B cell compartments. aPIK3CD B cells manifest increased pS6 and increased survival at several stages, without alterations in cycling, and baseline increases in plasma cells, natural IgM, and IgG3. Finally, Mb1-aPIK3CD mice exhibited blunted T cell-independent immune responses, and both AID- and CD21-aPIK3CD mice displayed reduced class-switched antibodies following T cell-dependent immunization. Thus, aPIK3CD alters B cell development and function and is counter-productive during immune responses, providing insight into B cell-intrinsic contributions to the APDS phenotype.
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http://dx.doi.org/10.1084/jem.20180617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170176PMC
October 2018

αv Integrins regulate germinal center B cell responses through noncanonical autophagy.

J Clin Invest 2018 08 20;128(9):4163-4178. Epub 2018 Aug 20.

Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA.

Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through TLRs on B cells promotes many aspects of GC B cell responses, including affinity maturation, class switching, and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here, we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and noncanonical autophagy. Using B cell-specific αv-KO mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic hypermutation, class switching, and generation of long-lived plasma cells after immunization with virus-like particles (VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells that can be targeted to enhance antibody responses to vaccination.
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http://dx.doi.org/10.1172/JCI99597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6118577PMC
August 2018

The Autoimmune Risk Variant C1858T Alters B Cell Tolerance at Discrete Checkpoints and Differentially Shapes the Naive Repertoire.

J Immunol 2017 10 11;199(7):2249-2260. Epub 2017 Aug 11.

Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101;

A common genetic variant in the gene encoding the protein tyrosine phosphatase nonreceptor type 22 ( C1858T) has been linked to a wide range of autoimmune disorders. Although a B cell-intrinsic role in promoting disease has been reported, the mechanism(s) through which this variant functions to alter the preimmune B cell repertoire remains unknown. Using a series of polyclonal and transgenic self-reactive models harboring the analogous mutation in murine , we show evidence for enhanced BCR, B cell-activating factor receptor, and CD40 coreceptor programs, leading to broadly enhanced positive selection of B cells at two discrete checkpoints in the bone marrow and spleen. We further identified a bias for selection of B cells into the follicular mature versus marginal zone B cell compartment. Using a biomarker to track a self-reactive H chain in peripheral blood, we found evidence of similarly enhanced positive selection in human carriers of the C1858T variant. Our combined data support a model whereby the risk variant augments the BCR and coreceptor programs throughout B cell development, promoting enrichment of self-reactive specificities into the follicular mature compartment and thereby likely increasing the risk for seeding of autoimmune B cell responses.
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http://dx.doi.org/10.4049/jimmunol.1700601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791366PMC
October 2017

Altered B cell signalling in autoimmunity.

Nat Rev Immunol 2017 Jul 10;17(7):421-436. Epub 2017 Apr 10.

Seattle Children's Research Institute, 1900 9th Avenue, Seattle, Washington 98101, USA.

Recent work has provided new insights into how altered B cell-intrinsic signals - through the B cell receptor (BCR) and key co-receptors - function together to promote the pathogenesis of autoimmunity. These combined signals affect B cells at two distinct stages: first, in the selection of the naive repertoire; and second, during extrafollicular or germinal centre activation responses. Thus, dysregulated signalling can lead to both an altered naive BCR repertoire and the generation of autoantibody-producing B cells. Strikingly, high-affinity autoantibodies predate and predict disease in several autoimmune disorders, including type 1 diabetes and systemic lupus erythematosus. This Review summarizes how, rather than being a downstream consequence of autoreactive T cell activation, dysregulated B cell signalling can function as a primary driver of many human autoimmune diseases.
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http://dx.doi.org/10.1038/nri.2017.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5523822PMC
July 2017

Cutting Edge: BAFF Promotes Autoantibody Production via TACI-Dependent Activation of Transitional B Cells.

J Immunol 2016 05 28;196(9):3525-31. Epub 2016 Mar 28.

Seattle Children's Research Institute, Seattle, WA 98101; Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98195

Mice overexpressing B cell activating factor of the TNF family (BAFF) develop systemic autoimmunity characterized by class-switched anti-nuclear Abs. Transmembrane activator and CAML interactor (TACI) signals are critical for BAFF-mediated autoimmunity, but the B cell developmental subsets undergoing TACI-dependent activation in settings of excess BAFF remain unclear. We report that, although surface TACI expression is usually limited to mature B cells, excess BAFF promotes the expansion of TACI-expressing transitional B cells. TACI(+) transitional cells from BAFF-transgenic mice are characterized by an activated, cycling phenotype, and the TACI(+) cell subset is specifically enriched for autoreactivity, expresses activation-induced cytidine deaminase and T-bet, and exhibits evidence of somatic hypermutation. Consistent with a potential contribution to BAFF-mediated humoral autoimmunity, TACI(+) transitional B cells from BAFF-transgenic mice spontaneously produce class-switched autoantibodies ex vivo. These combined findings highlight a novel mechanism through which BAFF promotes humoral autoimmunity via direct, TACI-dependent activation of transitional B cells.
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http://dx.doi.org/10.4049/jimmunol.1600017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868625PMC
May 2016

BCR and co-receptor crosstalk facilitate the positive selection of self-reactive transitional B cells.

Curr Opin Immunol 2015 Dec;37:46-53

Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, United States; Department of Immunology, University of Washington School of Medicine, Seattle, WA, United States; Seattle Children's Research Institute, Seattle, WA, United States. Electronic address:

The establishment of a diverse B cell repertoire requires fine-tuning of antigen receptor selection during development in order to permit sufficient diversity while reducing the potential for autoimmunity. In this review, we highlight recent studies demonstrating the central role of the B cell antigen receptor (BCR), in coordination with other key pro-survival signals mediated by CD40, BAFF-R, TACI and/or TLRs, in regulating both negative and positive selection of autoreactive B cells. In particular, we show how altered antigen or co-stimulatory signaling can facilitate positive selection of transitional B cells with self-reactive BCRs, ultimately leading to their entry into the mature, naive B cell compartment. We propose a model wherein altered receptor signals (due to inherited genetic changes) leads: first, to enhanced positive selection of autoreactive cells into the naïve B cell repertoire; subsequently, to an increased probability of pathogenic germinal center responses in individuals with a broad range of autoimmune disorders.
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http://dx.doi.org/10.1016/j.coi.2015.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679457PMC
December 2015

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help.

PLoS One 2012 12;7(1):e30229. Epub 2012 Jan 12.

Department of Medical Oncology, Dana-Farber Cancer Institute, Massachusetts, United States of America.

Background: Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model.

Methods/principal Findings: We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells.

Conclusions: We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257268PMC
July 2012

Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.

Sci Transl Med 2011 Apr;3(80):80ra34

Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA.

Although advanced-stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of antitumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred antitumor CD8(+) T lymphocytes (CTLs) have had limited life spans unless the host has been manipulated. To generate CTLs that have an intrinsic capacity to persist in vivo, we developed a human artificial antigen-presenting cell system that can educate antitumor CTLs to acquire both a central memory and an effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. We examined whether antitumor CTLs generated using this system could function and persist in patients. We showed that MART1-specific CTLs, educated and expanded using our artificial antigen-presenting cell system, could survive for prolonged periods in advanced-stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTLs trafficked to the tumor, mediated biological and clinical responses, and established antitumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.
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http://dx.doi.org/10.1126/scitranslmed.3002207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861895PMC
April 2011

A panel of human cell-based artificial APC enables the expansion of long-lived antigen-specific CD4+ T cells restricted by prevalent HLA-DR alleles.

Int Immunol 2010 Nov 8;22(11):863-73. Epub 2010 Nov 8.

Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.

Many preclinical experiments have attested to the critical role of CD4(+) T cell help in CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. Recent clinical trials have demonstrated that reinfusion of CD4(+) T cells can induce responses in infectious diseases and cancer. However, few standardized and versatile systems exist to expand antigen-specific CD4(+) T(h) for clinical use. K562 is a human erythroleukemic cell line, which lacks expression of HLA class I and class II, invariant chain and HLA-DM but expresses adhesion molecules such as intercellular adhesion molecule-1 and leukocyte function-associated antigen-3. With this unique immunologic phenotype, K562 has been tested in clinical trials of cancer immunotherapy. Previously, we created a K562-based artificial antigen-presenting cell (aAPC) that generates ex vivo long-lived HLA-A2-restricted CD8(+) CTL with a central/effector memory phenotype armed with potent effector function. We successfully generated a clinical version of this aAPC and conducted a clinical trial where large numbers of anti-tumor CTL are reinfused to cancer patients. In this article, we shifted focus to CD4(+) T cells and developed a panel of novel K562-derived aAPC, where each expresses a different single HLA-DR allele, invariant chain, HLA-DM, CD80, CD83 and CD64; takes up soluble protein by endocytosis and processes and presents CD4(+) T-cell peptides. Using this aAPC, we were able to determine novel DR-restricted CD4(+) T-cell epitopes and expand long-lived CD4(+) T-cells specific for multiple antigens without growing bystander Foxp3(+) regulatory T cells. Our results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD8(+) CTL and CD4(+) T(h).
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http://dx.doi.org/10.1093/intimm/dxq440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994545PMC
November 2010

Diversity and complexity in DNA recognition by transcription factors.

Science 2009 Jun 14;324(5935):1720-3. Epub 2009 May 14.

Banting and Best Department of Medical Research, University of Toronto, Toronto, ON M5S 3E1, Canada.

Sequence preferences of DNA binding proteins are a primary mechanism by which cells interpret the genome. Despite the central importance of these proteins in physiology, development, and evolution, comprehensive DNA binding specificities have been determined experimentally for only a few proteins. Here, we used microarrays containing all 10-base pair sequences to examine the binding specificities of 104 distinct mouse DNA binding proteins representing 22 structural classes. Our results reveal a complex landscape of binding, with virtually every protein analyzed possessing unique preferences. Roughly half of the proteins each recognized multiple distinctly different sequence motifs, challenging our molecular understanding of how proteins interact with their DNA binding sites. This complexity in DNA recognition may be important in gene regulation and in the evolution of transcriptional regulatory networks.
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http://dx.doi.org/10.1126/science.1162327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905877PMC
June 2009