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<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="utfZPAXjhugeryccubzg3ZsGYJxJAPhvLgZ4sqqFR53IlZi27ucix7YkwNfVtqPP">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="utfZPAXjhugeryccubzg3ZsGYJxJAPhvLgZ4sqqFR53IlZi27ucix7YkwNfVtqPP">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="utfZPAXjhugeryccubzg3ZsGYJxJAPhvLgZ4sqqFR53IlZi27ucix7YkwNfVtqPP">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="utfZPAXjhugeryccubzg3ZsGYJxJAPhvLgZ4sqqFR53IlZi27ucix7YkwNfVtqPP">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="utfZPAXjhugeryccubzg3ZsGYJxJAPhvLgZ4sqqFR53IlZi27ucix7YkwNfVtqPP">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="35087538">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://doi.org/10.3389/fimmu.2021.817604"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=6956&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=35087538&amp;db=pubmed&amp;nlmid=101560960"
title="See full text options at Frontiers Media SA"
data-ga-category="full_text"
data-ga-action="Frontiers Media SA"
data-ga-label="35087538"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--www.frontiersin.org-alerts-logo-PubMed.png" alt="Frontiers Media SA full text link"><span class="text">
Frontiers Media SA
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/35087538/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=35087538&amp;db=pubmed&amp;nlmid=101560960"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="35087538"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/35087538/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/35087538/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="35087538" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F35087538%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="utfZPAXjhugeryccubzg3ZsGYJxJAPhvLgZ4sqqFR53IlZi27ucix7YkwNfVtqPP"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
id="collections-action-dialog-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="collections_button"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection"><div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div></div><div class="action-panel-control-wrap existing-collections-controls"><label for="collections-action-dialog-add-to-existing" class="action-panel-label">
Choose a collection:
</label><select id="collections-action-dialog-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
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><sup class="key">1</sup> Division of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.</li>
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><sup class="key">2</sup> Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.</li>
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><sup class="key">4</sup> Division Regulation of the Immune System, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.</li>
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Abstract
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<p>
Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.
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Keywords:
</strong>
TLR; bacterial contamination; biosensor; reporter cell; toll-like receptor.
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Copyright © 2022 Radakovics, Battin, Leitner, Geiselhart, Paster, Stöckl, Hoffmann-Sommergruber and Steinberger.
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
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Figures
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data-image-width="602"
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data-pmc-id="PMC8786796"
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<img class="figure-thumb" itemprop="thumbnail"
id="article-image-0"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2d7/8786796/0ee2c2fb9392/fimmu-12-817604-g001.gif"
alt="Figure 1" />
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<p> Figure 1 </p>
</strong>
<div class="figure-caption-contents"><p> Jurkat-NF-κB::eGFP reporter cells detect the… </p></div>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 1 </p>
</strong>
<div class="figure-caption-contents"><p> Jurkat-NF-κB::eGFP reporter cells detect the TLR5 ligand flagellin with high sensitivity and specificity.… </p></div>
</div>
<figcaption id="figure-caption-0" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 1
</strong>
<div class="figure-caption-contents">Jurkat-NF-κB::eGFP reporter cells detect the TLR5 ligand flagellin with high sensitivity and specificity. <b>(A)</b> eGFP expression in Jurkat JE6-1 cells stably transfected with an NF-κB::eGFP reporter gene treated with different TLR ligands and PMA/Ionomycin as a positive control for 24 h (n = 3 independent experiments performed in duplicates). <b>(B)</b> Representative histograms of eGFP expression in JE6-1 reporter cells stimulated with flagellin at the indicated concentrations (experiment shown is representative for three experiments independently performed). <b>(C)</b> Concentration-response curves upon stimulation of Jurkat-NF-κB::eGFP and THP-1-NF-κB::eGFP reporter cells with flagellin are shown (n = 3 for Jurkat and n = 2 for THP-1 independent experiments were performed in duplicates).</div>
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<img class="figure-thumb" itemprop="thumbnail"
id="article-image-1"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2d7/8786796/b253595d17d5/fimmu-12-817604-g002.gif"
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<strong class="figure-label">
<p> Figure 2 </p>
</strong>
<div class="figure-caption-contents"><p> Generation of TLR reporter cells.… </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 2 </p>
</strong>
<div class="figure-caption-contents"><p> Generation of TLR reporter cells. Scheme for the generation of JE6-1-TLR reporter cell… </p></div>
</div>
<figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
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Figure 2
</strong>
<div class="figure-caption-contents">Generation of TLR reporter cells. Scheme for the generation of JE6-1-TLR reporter cell lines (created with BioRender.com) and exemplary histograms representative of at least three independent experiments showing the NF-κB::eGFP expression (standard logarithmic scale) after 24 h incubation with the typical TLR ligands Pam3CSK4 (violet, TLR2/1), Fsl-1 (light blue, TLR2/6), LPS (pink, TLR4) and flagellin (green, TLR5), as well as medium (grey, negative control) and PMA/Ionomycin (dark violet, positive control).</div>
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<strong class="figure-label">
<p> Figure 3 </p>
</strong>
<div class="figure-caption-contents"><p> Sensitivity and specificity of TLR… </p></div>
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</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 3 </p>
</strong>
<div class="figure-caption-contents"><p> Sensitivity and specificity of TLR reporter cells. <b> (A) </b> Titration curves for typical ligands… </p></div>
</div>
<figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 3
</strong>
<div class="figure-caption-contents">Sensitivity and specificity of TLR reporter cells. <b>(A)</b> Titration curves for typical ligands of each TLR reporter cell, with a dashed line at fold induction of 1.5 representing the limit of detection; summary of representative experiments performed in duplicates (n = 2 for TLR2/1/6, TLR2/1, TLR2/6, TLR4 reporter, n = 3 for TLR5 reporter). <b>(B)</b> Reactivity of each reporter cell line to the typical ligands of TLR2/1 (Pam3CSK4, 100 ng/mL), TLR2/6 (Fsl-1, 100 ng/mL), TLR4 (LPS-B5 ultrapure, 10 ng/mL) and TLR5 (Fla-ST ultrapure, 10 ng/mL) (n = 6, each experiment was performed in duplicates).</div>
</figcaption>
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<img class="figure-thumb" itemprop="thumbnail"
id="article-image-3"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2d7/8786796/a30d87e87737/fimmu-12-817604-g004.gif"
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<div class="caption-wrap">
<strong class="figure-label">
<p> Figure 4 </p>
</strong>
<div class="figure-caption-contents"><p> Comparison of LPS-detection systems. <b> (A)… </b> </p></div>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 4 </p>
</strong>
<div class="figure-caption-contents"><p> Comparison of LPS-detection systems. <b> (A) </b> Fold induction of eGFP expression in JE6-1-NF-κB::eGFP TLR4… </p></div>
</div>
<figcaption id="figure-caption-3" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 4
</strong>
<div class="figure-caption-contents">Comparison of LPS-detection systems. <b>(A)</b> Fold induction of eGFP expression in JE6-1-NF-κB::eGFP TLR4 reporter cells compared to fold induction of SEAP activity in HEK-Blue™-hTLR4 cells after incubation with different LPS concentrations overnight. Mean and standard deviation of two experiments performed in duplicates are shown. <b>(B)</b> Fold induction of eGFP expression in JE6-1-NF-κB::eGFP TLR4 reporter cells and THP-1-NF-κB::eGFP TLR4 reporter cells after 24 h incubation with different concentrations of LPS. Mean and standard deviation of two experiments (JE6-1) or one representative experiment (THP-1) performed in duplicates are shown. <b>(C)</b> Reactivity of the typical TLR ligands LPS (LPS-B5 ultrapure, TLR4), Pam3CSK4 (TLR2/1), Fsl-1 (TLR2/6), LTA-SA (TLR2) and MPLA-SM (TLR4) in the recombinant Factor C assay EndoZyme<sup>®</sup>II. The fold change of RFU in the recombinant Factor C assay was compared with the fold induction of gMFI (eGFP) in Jurkat-NF-κB::eGFP TLR4 reporter cells after 24 h incubation with the same ligands (n = 2 independent experiments performed in duplicates or triplicates).</div>
</figcaption>
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id="article-image-4"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2d7/8786796/93067ba489e9/fimmu-12-817604-g005.gif"
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<strong class="figure-label">
<p> Figure 5 </p>
</strong>
<div class="figure-caption-contents"><p> TLR2 stimulatory capacity of different… </p></div>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 5 </p>
</strong>
<div class="figure-caption-contents"><p> TLR2 stimulatory capacity of different cell wall components. <b> (A) </b> Activation of indicated reporter… </p></div>
</div>
<figcaption id="figure-caption-4" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 5
</strong>
<div class="figure-caption-contents">TLR2 stimulatory capacity of different cell wall components. <b>(A)</b> Activation of indicated reporter cells by the microbial components peptidoglycan (PGN), isolated from the gram-positive <i>B. subtilis</i> (PGN-BS) or <i>S. aureus</i> (PGN-SA) or the gram-negative <i>E. coli</i> K12 (PGN-EK), purified lipoteichoic acid from <i>S. aureus</i> (LTA-SA) and the synthetic ligands Diprovocim-1 (TLR2/1), Pam3CSK4 (TLR2/1) and Fsl-1 (TLR2/6) (n = 8, each experiment performed in duplicates). Exemplary histograms of eGFP expression are shown for TLR2/1/6, TLR2/1 and TLR2/6 reporter cells. <b>(B)</b> TLR2 surface expression on different TLR reporter cell lines was compared by staining with an AF647-coupled anti-TLR2 antibody.</div>
</figcaption>
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2d7/8786796/a153179b72b6/fimmu-12-817604-g006.gif"
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<strong class="figure-label">
<p> Figure 6 </p>
</strong>
<div class="figure-caption-contents"><p> Detection of bacterial contamination. <b> (A)… </b> </p></div>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 6 </p>
</strong>
<div class="figure-caption-contents"><p> Detection of bacterial contamination. <b> (A) </b> Flagellin (Fla) preparations of different purity (std. –… </p></div>
</div>
<figcaption id="figure-caption-5" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
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Figure 6
</strong>
<div class="figure-caption-contents">Detection of bacterial contamination. <b>(A)</b> Flagellin (Fla) preparations of different purity (std. standard, 10% purity; u.p. ultrapure, &gt; 95% purity; rec. recombinant) from <i>S. typhimurium</i> (-ST) or <i>B. subtilis</i> (-BS) at 10 ng/mL were compared regarding their capacity to activate TLR5 as well as TLR4 reporter cells. n = 7 independent experiments performed in duplicates and exemplary histograms. ns = p &gt; 0.1; ** = p &lt; 0.01; **** = p &lt; 0.0001. <b>(B)</b> Activation of different TLR reporter cells by <i>E. coli</i> LPS extracts (10 ng/mL) of different purity (std. standard, 10% purity; u.p. ultrapure, &gt; 95% purity) from <i>E. coli</i> 055:B5 (-B5), <i>E. coli</i> K12 (-EK) or <i>E. coli</i> 0127:B8 (-B8), as well as MPLA-SM (100 ng/mL), an LPS component of <i>S. minnesota</i>. n = 6 independent experiments performed in duplicates and exemplary histograms. <b>(C)</b> Exemplary histograms of eGFP expression in Jurkat-TLR2/1/6, -TLR2/1 and -TLR2/6 reporter cells and THP-1-NF-κB::eGFP reporter cells after overnight incubation with medium or cell culture supernatant containing mycoplasma (1:1 diluted). <b>(D)</b> Exemplary histograms of eGFP expression in different TLR reporter cells after overnight incubation with protein preparations of the C4d protein, either expressed in <i>E. coli</i> BL21 or ClearColi™ (10 μg/mL) or PMA(100 ng/mL)/Ionomycin(100 nM) as positive control.</div>
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<p> Figure 7 </p>
</strong>
<div class="figure-caption-contents"><p> Detection of TLR Ligands in… </p></div>
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<p> Figure 7 </p>
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<div class="figure-caption-contents"><p> Detection of TLR Ligands in allergen extracts. Exemplary histograms with gMFI of eGFP… </p></div>
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Figure 7
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<div class="figure-caption-contents">Detection of TLR Ligands in allergen extracts. Exemplary histograms with gMFI of eGFP expression in TLR reporter cells after overnight incubation with allergenic extracts (15 μg/mL) are shown. HDM(1) - HDM(4): house dust mite extracts from different sources.</div>
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