{"id":12338,"date":"2019-04-18T21:28:37","date_gmt":"2019-04-18T21:28:37","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/?p=12338"},"modified":"2019-04-18T21:35:05","modified_gmt":"2019-04-18T21:35:05","slug":"phospho-cd247-how-to-add-a-tcr-functionality-marker-to-your-t-cell-panel","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/phospho-cd247-how-to-add-a-tcr-functionality-marker-to-your-t-cell-panel\/","title":{"rendered":"Phospho-CD247: How to Add a TCR Functionality Marker to your T Cell Panel"},"content":{"rendered":"<h2><strong><span style=\"font-size: 14pt\">What type of T cell stimulation protocol should I use for my experiments?<\/span><\/strong><\/h2>\n<p><span style=\"font-size: 10pt\">Stimulation of peripheral blood mononuclear cells (PBMCs) is useful to expand and differentiate populations of immune cells.&nbsp; Often, stimulated cells will express different cell surface receptors, up or down regulate expression of intracellular cytokines, and modify proteins, as compared to resting or na&iuml;ve cells.&nbsp; Stimulants can include phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), staphylococcal enterotoxin B (SEB), antibodies, or peptide pools [2].&nbsp; The appropriate stimulant and stimulation condition are dependent on the cell type and cytokine of interest.&nbsp;<\/span><\/p>\n<p><span style=\"font-size: 10pt\">CD3 zeta phosphorylation is an indicator of T cell activation via the TCR complex, and treatment of T cells with monoclonal antibodies that engage the TCR can be used as a model for antigen-induced activation [3,4].&nbsp; The first step in this process is to treat cells with anti-CD3 antibodies to engage the TCR, and anti-CD28 antibodies to provide a co-stimulatory signal.&nbsp; This is followed by a secondary antibody that recognizes the host IgG of the anti-CD3 and -28 antibodies, to cross-link the subunits of the TCR complex for optimal signal transduction and T cell activation.<\/span><\/p>\n<p><span style=\"font-size: 14pt\"><strong>How do you know if my T cell stimulation protocol worked?<\/strong><\/span><\/p>\n<p>TCR activation involves the cytoplasmic tails of the CD3 subunits: CD3 gamma, CD3 delta, CD3 epsilon and CD3 zeta (alternatively named CD247).&nbsp; One of the early steps in the TCR cascade include CD3 zeta phosphorylation.&nbsp; It occurs immediately downstream of Lck activation, and upstream of ZAP-70, LAT, and SLP-76, and allows for direct identification of TCR engagement.&nbsp; While Lck activation is upstream of CD3 zeta phosphorylation, it is also involved in other receptor signaling pathways, such as IL-2 and CD2, making CD3 zeta a more TCR-specific method of identifying T cell activation.<\/p>\n<p>Based on known expression patterns, phospho-CD247 (Tyr142) stains stimulated CD3+ T cells following a T cell-specific stimulation with no staining observed without stimulation (figure 1A), in CD3- cells, or in cells stimulated with a B cell-specific stimulation (figure 1B).&nbsp;<\/p>\n<p>Figure 1: Intracellular staining of stimulated human peripheral blood cells.&nbsp; (A) Human peripheral blood cells unstimulated (blue histogram) or stimulated for 15 minutes with CD3 (clone OKT3), Functional Grade and CD28 (clone 28.6), Functional Grade, followed by a 5 minute incubation with F(ab&rsquo;)2-Goat anti-Mouse IgG (H+L) Secondary Antibody, Functional Grade (purple histogram) were fixed and permeabilized with the IC Fixation &amp; Permeabilization Buffer Set and protocol, followed by intracellular staining with CD3 (clone UCHT1) and Phospho-CD247 (Tyr142) (clone 3ZBR4S). Staining of Phospho-CD247 in CD3+ cells is shown on the left, and on CD3- cells on the right, with no stimulation observed in CD3- cells. Cells in the lymphocyte gate were used for analysis. (B) Human peripheral blood cells unstimulated (blue histogram) or stimulated for 5 minutes with F(ab&#8217;)2-Goat anti-Human IgG, IgM (H+L) Secondary Antibody, Functional Grade (purple histogram) were fixed and permeabilized with the IC Fixation &amp; Permeabilization Buffer Set and protocol, followed by intracellular staining with CD19 (clone HIB19) and Phospho-CD247 (Tyr142) (clone 3ZBR4S). Staining of Phospho-CD247 in CD19+ cells is shown on the left, and on CD19- cells on the right, and no stimulation is observed in either group following this B cell-specific treatment. Cells in the lymphocyte gate were used for analysis.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/www.thermofisher.com\/blog\/wp-content\/uploads\/sites\/9\/2019\/04\/phospho20image-2.png\" style=\"vertical-align: middle;border: 1px solid black;margin: 2px auto\" width=\"553\" height=\"608\" \/><\/p>\n<p><span style=\"font-size: 14pt\"><strong>What type of research can utilize phospho-CD247?<\/strong><\/span><\/p>\n<p>Those using the phospho-CD247 antibody would likely be studying T cell activation and CAR T cells.&nbsp; The zeta chain of the CD3 complex is the part responsible for initiating the signaling cascade upon engagement of the T cell receptor.&nbsp; It is phosphorylated on Tyr142 upon TCR activation, and functions to recruit ZAP70, which further propagates the signal. Staining for phospho-CD247 would demonstrate TCR receptor activation and functionality. In particular, this antibody would be useful for the CAR T workflow, as this would be a way to determine that the engineered TCR is functional and can receive and propagate a signal.<\/p>\n<p><span style=\"font-size: 14pt\"><strong>If incorporating CD247 into a flow cytometry panel, what other markers would be recommended for use and why?<\/strong><\/span><\/p>\n<p>As far as other markers that would be associated with it, some of the classic T cell markers, such as CD3, CD8, and CD28 would be appropriate.&nbsp; We also have antibodies against other phosphorylated proteins involved in T cell signaling, such as phospho-ZAP70, phospho-LCK, and phospho-SLP76.<\/p>\n<p><span style=\"font-size: 14pt\"><strong>What can you tell me about the relative expression of CD247 and picking a fluorophore conjugate?<\/strong><\/span><\/p>\n<p>CD3 is highly expressed and can be stained with both brighter fluorophores such as PE, APC, and PerCP-eFluor 710 conjugates, but also dimmer ones too including FITC or eFluor 450.&nbsp;<\/p>\n<p><span style=\"font-size: 14pt\"><strong>Where can I find information about the key antibodies to T cell markers that are available?<\/strong><\/span><\/p>\n<p>The first resource we recommend is the Thermo Fisher Scientific website. All listed antibodies are presented with a description of the antigen they recognize, and the flow plot data on primary human or mouse cells.&nbsp; Application notes are also available with detail protocols and guidance with gating.&nbsp; For building a T cell panel we recommend reading our application notes including a <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/flow-cytometry\/flow-cytometry-learning-center\/flow-cytometry-resource-library\/flow-cytometry-application-notes\/validated-ten-color-tcell-panel-attune-nxt-flow-cytometer.html?icid=BID_FCREG_Phospho_Natalie1\">10-color T cell panel<\/a>.&nbsp; Then, try building your own immunophenotyping panel with a comprehensive <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/cell-analysis\/flow-cytometry\/antibodies-for-flow-cytometry\/flow-cytometry-panel-builder.html?icid=BID_FCREG_Phospho_Natalie2\">panel builder<\/a>.<\/p>\n<p><strong>Key T cell References:<\/strong><\/p>\n<ol>\n<li>Lin J, Weiss A. (2001) T cell receptor signaling. J Cell Sci. 114: 243-4.<\/li>\n<li>Kay, J.E. (1991). Mechanisms of T lymphocyte activation. Immunology Letters. 29, 51 &ndash; 54.<\/li>\n<li>Trickett A, Kwan YL. (2003) T cell stimulation and expansion using anti-CD3\/CD28 beads. J Immunol Methods. 275(1-2):251-5.<\/li>\n<li>Risue&ntilde;o RM, Schamel WW, Alarc&oacute;n B. (2002) T cell receptor engagement triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked conformation.&nbsp;<em>PLoS One<\/em>. 3(3):e1747.<\/li>\n<\/ol>\n<p>For Research Use Only. Not for Use in Diagnostic Procedures.<\/p>\n<p>_______________________________________________________________________________________________________________________________________________________________________________________<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/www.thermofisher.com\/blog\/wp-content\/uploads\/sites\/9\/2019\/04\/phospho20natalie-2.png\" width=\"182\" height=\"182\" style=\"float: left;margin-left: 5px;margin-right: 5px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p><span><span style=\"font-size: 12pt\"><strong>Natalie Oxford<\/strong><\/span>, <strong>antibody development scientist at Thermo Fisher Scientific, is responsible for the development of anti-phospho CD247 by flow cytometry.&nbsp; She has over 11 years of R&amp;D experience and is providing her guidance in adding antibodies like anti-phospho CD247 to complex immunophenotyping flow cytometry applications.<\/strong><\/span><\/p>\n<p><!-- BEGIN KAPOST ANALYTICS CODE --><\/p>\n<p><!--\nvar _kaq = _kaq || [];\n_kaq.push([2, \"5ca784e269d63f0076000020\", \"5674227b963491538f00011d\"]);\n(function(){\nvar ka = document.createElement(\"script\"); ka.async=true; ka.id=\"ka_tracker\";\nka.src = (document.location.protocol == \"https:\" ? \"https\" : \"http\") + \":\/\/analytics.kapost.com\/ka.js\";\nvar s = document.getElementsByTagName(\"script\")[0]; s.parentNode.insertBefore(ka, s);\n})();\n\/\/--><\/p>\n<p><!-- END KAPOST ANALYTICS CODE --><\/p>\n","protected":false},"excerpt":{"rendered":"<p>What type of T cell stimulation protocol should I use for my experiments? Stimulation of peripheral blood mononuclear cells (PBMCs) is useful to expand and differentiate populations of immune cells.&nbsp; Often, stimulated cells will express different cell surface receptors, up or down regulate expression of intracellular cytokines, and modify proteins, as compared to resting or<\/p>\n","protected":false},"author":120,"featured_media":12344,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[172],"tags":[],"division":[],"class_list":{"0":"post-12338","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-general","8":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- 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