Publications by authors named "Stephen M Hedrick"

71 Publications

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

J Exp Med 2021 Aug 26;218(8). Epub 2021 May 26.

Division of Biological Sciences, University of California San Diego, La Jolla, CA.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.
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http://dx.doi.org/10.1084/jem.20202512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160575PMC
August 2021

CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.

Nat Commun 2021 05 11;12(1):2625. Epub 2021 May 11.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.
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http://dx.doi.org/10.1038/s41467-021-22927-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113449PMC
May 2021

FOXO1 constrains activation and regulates senescence in CD8 T cells.

Cell Rep 2021 Jan;34(4):108674

Division of Biological Sciences, Molecular Biology Section, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA. Electronic address:

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span.
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http://dx.doi.org/10.1016/j.celrep.2020.108674DOI Listing
January 2021

Delineation of a molecularly distinct terminally differentiated memory CD8 T cell population.

Proc Natl Acad Sci U S A 2020 10 25;117(41):25667-25678. Epub 2020 Sep 25.

Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093;

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated that circulating memory cells can be further divided into effector memory (Tem) and central memory (Tcm) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was terminally fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally differentiated effector memory CD8 T cell population (terminal-Tem) was conflated within the conventional Tem population, prompting redefinition of the classical characteristics of Tem cells. Murine terminal-Tem were transcriptionally, functionally, and developmentally unique compared to Tem cells. Through mass cytometry and single-cell RNA sequencing (RNA-seq) analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-Tem population of CD8 T cells that was transcriptionally distinct from Tem and Tcm Key findings from this study show that parsing of terminal-Tem from conventionally defined Tem challenge the reported characteristics of Tem biology, including enhanced presence in lymphoid tissues, robust IL-2 production, and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-Tem and clarification of Tem biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.
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http://dx.doi.org/10.1073/pnas.2008571117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568335PMC
October 2020

Editorial overview: Lymphocyte effector subsets: blurring the frontiers.

Curr Opin Immunol 2020 04;63:iii-v

Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, TATA Institute for Genetics and Society, University of California, San Diego, La Jolla, CA 92093-0377, United States. Electronic address:

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http://dx.doi.org/10.1016/j.coi.2020.04.001DOI Listing
April 2020

A key control point in the T cell response to chronic infection and neoplasia: FOXO1.

Curr Opin Immunol 2020 04 2;63:51-60. Epub 2020 Mar 2.

Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, TATA Institute for Genetics and Society, University of California, San Diego, La Jolla, CA 92093-0377, United States. Electronic address:

T cells able to control neoplasia or chronic infections display a signature gene expression profile similar or identical to that of central memory T cells. These cells have qualities of self-renewal and a plasticity that allow them to repeatedly undergo activation (growth, proliferation, and differentiation), followed by quiescence. It is these qualities that define the ability of T cells to establish an equilibrium with chronic infectious agents, and also preserve the ability of T cells to be re-activated (by checkpoint therapy) in response to malignant cancers. Here we describe distinctions between the forms of inhibition mediated by tumors and persistent viruses, we review the properties of T cells associated with long-term immunity, and we identify the transcription factor, FOXO1, as the control point for a program of gene expression that allows CD8 T cells to undergo serial reactivation and self-renewal.
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http://dx.doi.org/10.1016/j.coi.2020.02.001DOI Listing
April 2020

The Imperative to Vaccinate.

J Pediatr 2018 10 25;201:259-263. Epub 2018 Jul 25.

UC San Diego, Molecular Biology, La Jolla, CA. Electronic address:

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http://dx.doi.org/10.1016/j.jpeds.2018.06.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7131101PMC
October 2018

Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Expression of FOXO1.

Cell Rep 2018 03;22(13):3454-3467

Molecular Biology Section, Division of Biological Sciences, UC San Diego, La Jolla, CA 92093, USA; Cellular and Molecular Medicine, UC San Diego, La Jolla, CA 92093, USA. Electronic address:

Immunity following an acutely resolved infection or the long-term equipoise of chronic viral infections often depends on the maintenance of antigen-specific CD8 T cells, yet the ongoing transcriptional requirements of these cells remain unclear. We show that active and continuous programming by FOXO1 is required for the functional maintenance of a memory population. Upon Foxo1 deletion following resolution of an infection, memory cells rapidly lost their characteristic gene expression, gradually declined in number, and were impaired in self-renewal. This was extended to chronic infections, as a loss of FOXO1 during a persistent viral infection led to a rapid decline of the TCF7 (a.k.a. TCF1)-expressing memory-like subset of CD8 T cells. We further establish FOXO1 regulation as a characteristic of human memory CD8 T cells. Overall, we show that the molecular and functional longevity of a memory T cell population is actively maintained by the transcription factor FOXO1.
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http://dx.doi.org/10.1016/j.celrep.2018.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942184PMC
March 2018

The Effects of Dendritic Cell Hypersensitivity on Persistent Viral Infection.

J Immunol 2018 02 8;200(4):1335-1346. Epub 2018 Jan 8.

Division of Biological Sciences, Molecular Biology Section, University of California, San Diego, La Jolla, CA 92093; and

Caspase-8 (CASP8) is known as an executioner of apoptosis, but more recent studies have shown that it participates in the regulation of necroptosis and innate immunity. In this study, we show that CASP8 negatively regulates retinoic acid-inducible gene I (RIG-I) signaling such that, in its absence, stimulation of the RIG-I pathway in dendritic cells (DCs) produced modestly enhanced activation of IFN regulatory factor 3 with correspondingly greater amounts of proinflammatory cytokines. In addition, mice lacking DC-specific CASP8 ( mice) develop age-dependent symptoms of autoimmune disease characterized by hyperactive DCs and T cells, spleen and liver immunopathology, and the appearance of Th1-polarized CD4 T cells. Such mice infected with chronic lymphocytic choriomeningitis virus, an RNA virus detected by RIG-I, mounted an enhanced lymphocytic choriomeningitis virus-specific immune response as measured by increased proportions of Ag-specific CD4 T cells and multicytokine-producing CD4 and CD8 T cells. These results show that CASP8 subtly modulates DC maturation, which controls the spontaneous appearance of autoimmune T cells while simultaneously attenuating the acquired immune system and its potential to control a persistent viral infection.
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http://dx.doi.org/10.4049/jimmunol.1601870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863746PMC
February 2018

Continuous activity of Foxo1 is required to prevent anergy and maintain the memory state of CD8 T cells.

J Exp Med 2018 02 27;215(2):575-594. Epub 2017 Dec 27.

Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla CA

Upon infection with an intracellular pathogen, cytotoxic CD8 T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.
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http://dx.doi.org/10.1084/jem.20170697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789410PMC
February 2018

FOXO1 opposition of CD8 T cell effector programming confers early memory properties and phenotypic diversity.

Proc Natl Acad Sci U S A 2017 10 2;114(42):E8865-E8874. Epub 2017 Oct 2.

Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0377;

The factors and steps controlling postinfection CD8 T cell terminal effector versus memory differentiation are incompletely understood. Whereas we found that naive TCF7 (alias "Tcf-1") expression is FOXO1 independent, early postinfection we report bimodal, FOXO1-dependent expression of the memory-essential transcription factor TCF7 in pathogen-specific CD8 T cells. We determined the early postinfection TCF7 population is marked by low TIM3 expression and bears memory signature hallmarks before the appearance of established memory precursor marker CD127 (IL-7R). These cells exhibit diminished TBET, GZMB, mTOR signaling, and cell cycle progression. Day 5 postinfection, TCF7 cells express higher memory-associated BCL2 and EOMES, as well as increased accumulation potential and capacity to differentiate into memory phenotype cells. TCF7 retroviral transduction opposes GZMB expression and the formation of KLRG1 phenotype cells, demonstrating an active role for TCF7 in extinguishing the effector program and forestalling terminal differentiation. Past the peak of the cellular immune response, we report a gradient of FOXO1 and TCF7 expression, which functions to oppose TBET and orchestrate a continuum of effector-to-memory phenotypes.
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http://dx.doi.org/10.1073/pnas.1618916114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651728PMC
October 2017

Understanding Immunity through the Lens of Disease Ecology.

Trends Immunol 2017 12 4;38(12):888-903. Epub 2017 Sep 4.

Departments of Molecular Biology and Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Electronic address:

As we describe the immune system in ever more exquisite detail, we might find that no matter how successful, this approach will not be sufficient to understand the spread of infectious agents, their susceptibility to vaccine therapy, and human disease resistance. Compared with the strict reductionism practiced as a means of characterizing most biological processes, I propose that the progression and outcome of disease-causing host-parasite interactions will be more clearly understood through a focus on disease ecology.
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http://dx.doi.org/10.1016/j.it.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711578PMC
December 2017

A rheostat tuning thymic selection.

Nat Immunol 2017 06;18(7):713-714

Departments of Molecular Biology and Cellular and Molecular Medicine of the University of California, San Diego, La Jolla, California, USA.

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http://dx.doi.org/10.1038/ni.3778DOI Listing
June 2017

Foxo3 Transcription Factor Drives Pathogenic T Helper 1 Differentiation by Inducing the Expression of Eomes.

Immunity 2016 10 11;45(4):774-787. Epub 2016 Oct 11.

UMR Inserm, U1043, Toulouse 31300, France; UMR CNRS, U5282, Toulouse 31300, France; Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse 31300, France. Electronic address:

The transcription factor Foxo3 plays a crucial role in myeloid cell function but its role in lymphoid cells remains poorly defined. Here, we have shown that Foxo3 expression was increased after T cell receptor engagement and played a specific role in the polarization of CD4 T cells toward pathogenic T helper 1 (Th1) cells producing interferon-γ (IFN-γ) and granulocyte monocyte colony stimulating factor (GM-CSF). Consequently, Foxo3-deficient mice exhibited reduced susceptibility to experimental autoimmune encephalomyelitis. At the molecular level, we identified Eomes as a direct target gene for Foxo3 in CD4 T cells and we have shown that lentiviral-based overexpression of Eomes in Foxo3-deficient CD4 T cells restored both IFN-γ and GM-CSF production. Thus, the Foxo3-Eomes pathway is central to achieve the complete specialized gene program required for pathogenic Th1 cell differentiation and development of neuroinflammation.
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http://dx.doi.org/10.1016/j.immuni.2016.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141513PMC
October 2016

Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells.

Elife 2016 07 4;5. Epub 2016 Jul 4.

Department of Cellular and Molecular Medicine, University of California, San Diego, United States.

Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function.
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http://dx.doi.org/10.7554/eLife.10134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931909PMC
July 2016

Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs.

Neuron 2016 05 21;90(3):535-50. Epub 2016 Apr 21.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.
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http://dx.doi.org/10.1016/j.neuron.2016.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860075PMC
May 2016

Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway.

Stem Cell Reports 2015 Nov 30;5(5):866-880. Epub 2015 Oct 30.

Broad CIRM Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, 1425 San Pablo St., Los Angeles, CA 90033, USA. Electronic address:

The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.
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http://dx.doi.org/10.1016/j.stemcr.2015.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649379PMC
November 2015

Loss of Murine FOXO3 in Cells of the Myeloid Lineage Enhances Myelopoiesis but Protects from K/BxN-Serum Transfer-Induced Arthritis.

PLoS One 2015 13;10(5):e0126728. Epub 2015 May 13.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.

FOXO transcription factors have a highly conserved role in regulating transcription of genes involved in differentiation, cell cycle arrest, apoptosis and DNA repair. Loss of FOXO3 in mice has previously been shown to result in a myeloproliferative disease. In agreement with this, we found that an independent Foxo3 null mouse strain, Foxo3Kca, exhibits an increase in neutrophils in the spleen, bone marrow and blood. This coincides with an expansion of myeloid progenitor cells including pre-granulocyte-macrophage progenitors (Pre-GMs) and granulocyte-macrophage progenitors (GMPs). Surprisingly, despite neutrophilia, the severity of passive serum transfer arthritis was markedly attenuated in Foxo3Kca mice. These defects appear to be at least partially intrinsic to the myeloid lineage, as deleting Foxo3 specifically from myeloid cells using LysMCre also leads to an elevated number of neutrophils and protection from K/BxN-serum transfer-induced arthritis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430473PMC
February 2016

ICOS coreceptor signaling inactivates the transcription factor FOXO1 to promote Tfh cell differentiation.

Immunity 2015 Feb;42(2):239-251

Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0377, USA; Molecular Biology Section, Division of Biological Sciences, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0377, USA. Electronic address:

T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.
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http://dx.doi.org/10.1016/j.immuni.2015.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334393PMC
February 2015

Noncanonical mode of ERK action controls alternative αβ and γδ T cell lineage fates.

Immunity 2014 Dec 28;41(6):934-46. Epub 2014 Nov 28.

Blood Cell Development and Cancer Keystone, Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA. Electronic address:

Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.
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http://dx.doi.org/10.1016/j.immuni.2014.10.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4273651PMC
December 2014

Single-cell mass cytometry of TCR signaling: amplification of small initial differences results in low ERK activation in NOD mice.

Proc Natl Acad Sci U S A 2014 Nov 31;111(46):16466-71. Epub 2014 Oct 31.

Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;

Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates.
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http://dx.doi.org/10.1073/pnas.1419337111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246343PMC
November 2014

Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88-mediated dendritic cell activation.

J Immunol 2014 Jun 7;192(12):5548-60. Epub 2014 May 7.

Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death-independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8-deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type-specific manner and acts uniquely in DCs to maintain tolerance.
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http://dx.doi.org/10.4049/jimmunol.1400122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074511PMC
June 2014

Transcription factor binding site analysis identifies FOXO transcription factors as regulators of the cutaneous wound healing process.

PLoS One 2014 19;9(2):e89274. Epub 2014 Feb 19.

Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America ; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089274PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929751PMC
October 2014

Differentiation of CD8 memory T cells depends on Foxo1.

J Exp Med 2013 Jun 27;210(6):1189-200. Epub 2013 May 27.

Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA.

The forkhead O transcription factors (FOXO) integrate a range of extracellular signals, including growth factor signaling, inflammation, oxidative stress, and nutrient availability, to substantially alter the program of gene expression and modulate cell survival, cell cycle progression, and many yet to be unraveled cell type-specific responses. Naive antigen-specific CD8(+) T cells undergo a rapid expansion and arming of effector function within days of pathogen exposure. In addition, by the peak of expansion, they form precursors to memory T cells capable of self-renewal and indefinite survival. Using lymphocytic choriomeningitis virus Armstrong to probe the response to infection, we found that Foxo1(-/-) CD8(+) T cells expand normally with no defects in effector differentiation, but continue to exhibit characteristics of effector T cells long after antigen clearance. The KLRG1(lo) CD8(+) T cells that are normally enriched for memory-precursor cells retain Granzyme B and CD69 expression, and fail to up-regulate TCF7, EOMES, and other memory signature genes. As a correlate, Foxo1(-/-) CD8(+) T cells were virtually unable to expand upon secondary infection. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing the post-effector memory program that is essential to forming long-lived memory cells capable of immune reactivation.
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http://dx.doi.org/10.1084/jem.20130392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674697PMC
June 2013

Antigen-loaded pH-sensitive hydrogel microparticles are taken up by dendritic cells with no requirement for targeting antibodies.

Integr Biol (Camb) 2013 Jan;5(1):195-203

Division of Biological Sciences and the Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

Particle-based delivery of encapsulated antigens has great potential for improving vaccine constructs. In this study, we show that antigen-loaded, pH-sensitive hydrogel microparticles are taken up and presented by bone marrow-derived dendritic cells (BMDCs) in vitro and are taken up by dendritic cells (DCs) and monocytes in vivo. This uptake is irrespective of targeting antibodies. BMDCs in vitro and DCs in vivo also display upregulation of activation markers CD80 and CD86 when treated with microparticles, again with no difference in conjugated antibodies, even the agonistic CD40 antibody. We further show that these particles induce enhanced expansion of cytokine-producing CD8 T cells in response to challenge with ovalbumin-expressing vesicular stomatitis virus, in both an accelerated vaccination strategy using pre-loaded BMDCs and a traditional mouse immunization setting.
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http://dx.doi.org/10.1039/c2ib20109gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535018PMC
January 2013

FOXO transcription factors throughout T cell biology.

Nat Rev Immunol 2012 Sep;12(9):649-61

University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0377, USA.

The outcome of an infection with any given pathogen varies according to the dosage and route of infection, but, in addition, the physiological state of the host can determine the efficacy of clearance, the severity of infection and the extent of immunopathology. Here we propose that the forkhead box O (FOXO) transcription factor family--which is central to the integration of growth factor signalling, oxidative stress and inflammation--provides connections between physical well-being and the form and magnitude of an immune response. We present a case that FOXO transcription factors guide T cell differentiation and function in a context-driven manner, and might provide a link between metabolism and immunity.
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http://dx.doi.org/10.1038/nri3278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875397PMC
September 2012

Multivalent porous silicon nanoparticles enhance the immune activation potency of agonistic CD40 antibody.

Adv Mater 2012 Aug 12;24(29):3981-7. Epub 2012 Jun 12.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, 92093-0358, USA.

One of the fundamental paradigms in the use of nanoparticles to treat disease is to evade or suppress the immune system in order to minimize systemic side effects and deliver sufficient nanoparticle quantities to the intended tissues. However, the immune system is the body's most important and effective defense against diseases. It protects the host by identifying and eliminating foreign pathogens as well as self-malignancies. Here we report a nanoparticle engineered to work with the immune system, enhancing the intended activation of antigen presenting cells (APCs). We show that luminescent porous silicon nanoparticles (LPSiNPs), each containing multiple copies of an agonistic antibody (FGK45) to the APC receptor CD40, greatly enhance activation of B cells. The cellular response to the nanoparticle-based stimulators is equivalent to a 30-40 fold larger concentration of free FGK45. The intrinsic near-infrared photoluminescence of LPSiNPs is used to monitor degradation and track the nanoparticles inside APCs.
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http://dx.doi.org/10.1002/adma.201200776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517000PMC
August 2012

Polar opposites: Erk direction of CD4 T cell subsets.

J Immunol 2012 Jul 6;189(2):721-31. Epub 2012 Jun 6.

University of California, San Diego, La Jolla, CA 92093-0377, USA.

Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-β, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.
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http://dx.doi.org/10.4049/jimmunol.1103015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392534PMC
July 2012

Positive selection in the thymus: an enigma wrapped in a mystery.

J Immunol 2012 Mar;188(5):2043-5

Department of Cellular and Molecular Medicine and Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

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http://dx.doi.org/10.4049/jimmunol.1200077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387976PMC
March 2012

Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis.

Nature 2011 Sep 14;477(7364):335-9. Epub 2011 Sep 14.

Department of Medicine 1, Friedrich-Alexander-University, D-91054 Erlangen, Germany.

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.
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http://dx.doi.org/10.1038/nature10400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373730PMC
September 2011