Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ
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{"title"=>"Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ", "type"=>"journal", "authors"=>[{"first_name"=>"Chandirasegaran", "last_name"=>"Massilamany", "scopus_author_id"=>"35763905900"}, {"first_name"=>"Arunakumar", "last_name"=>"Gangaplara", "scopus_author_id"=>"36990232400"}, {"first_name"=>"Ting", "last_name"=>"Jia", "scopus_author_id"=>"56154703000"}, {"first_name"=>"Christian", "last_name"=>"Elowsky", "scopus_author_id"=>"7801360953"}, {"first_name"=>"Guobin", "last_name"=>"Kang", "scopus_author_id"=>"55391891100"}, {"first_name"=>"Jean Jack", "last_name"=>"Riethoven", "scopus_author_id"=>"7801487890"}, {"first_name"=>"Qingsheng", "last_name"=>"Li", "scopus_author_id"=>"35198421400"}, {"first_name"=>"You", "last_name"=>"Zhou", "scopus_author_id"=>"7405369258"}, {"first_name"=>"Jay", "last_name"=>"Reddy", "scopus_author_id"=>"7201745796"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"373058858", "sgr"=>"84900320048", "doi"=>"10.1371/journal.pone.0087519", "scopus"=>"2-s2.0-84900320048", "pmid"=>"24475302"}, "id"=>"f68e3e9a-9ccb-38d6-93cd-78e06d221e68", "abstract"=>"We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139-151; the sections were costained with IA(s)/PLP 139-151 (specific) or Theiler's murine encephalomyelitis virus (TMEV) 70-86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139-151 but not for TMEV 70-86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139-151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigen-specific T cells by counting the number of dextramer⁺ CD4⁺ T cells in the 'Z' serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-α (Myhc) 334-352. Heart sections prepared from immunized mice were costained with Myhc 334-352 (specific) or bovine ribonuclease 43-56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.", "link"=>"http://www.mendeley.com/research/direct-staining-major-histocompatibility-complex-class-ii-dextramers-permits-detection-antigenspecif", "reader_count"=>14, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>4, "Researcher"=>3, "Student > Ph. D. Student"=>2, "Student > Postgraduate"=>1, "Other"=>2, "Student > Bachelor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>4, "Researcher"=>3, "Student > Ph. D. Student"=>2, "Student > Postgraduate"=>1, "Other"=>2, "Student > Bachelor"=>2}, "reader_count_by_subject_area"=>{"Agricultural and Biological Sciences"=>8, "Medicine and Dentistry"=>3, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>8}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1362372"], "description"=>"*<p>Number of dext<sup>+</sup> CD4<sup>+</sup> T cells/total number of CD4<sup>+</sup> T cells.</p>", "links"=>[], "tags"=>["immunology", "Immune cells", "t cells", "autoimmunity", "Major histocompatibility complex", "Model organisms", "Animal models", "mouse", "Clinical immunology", "Autoimmune diseases", "Immunologic techniques", "myhc", "cells", "hearts", "eam", "mice", "confocal"], "article_id"=>914704, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Chandirasegaran Massilamany", "Arunakumar Gangaplara", "Ting Jia", "Christian Elowsky", "Guobin Kang", "Jean-Jack Riethoven", "Qingsheng Li", "You Zhou", "Jay Reddy"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087519.t002", "stats"=>{"downloads"=>5, "page_views"=>42, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_analysis_of_Myhc_334_352_dext_CD4_T_cells_in_the_hearts_of_EAM_mice_by_confocal_microscopy_/914704", "title"=>"Quantitative analysis of Myhc 334–352 dext<sup>+</sup> CD4<sup>+</sup> T cells in the hearts of EAM mice by confocal microscopy.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-27 04:01:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1362373"], "description"=>"*<p>Number of dext<sup>+</sup> CD4<sup>+</sup> T cells/total number of CD4<sup>+</sup> T cells</p>", "links"=>[], "tags"=>["immunology", "Immune cells", "t cells", "autoimmunity", "Major histocompatibility complex", "Model organisms", "Animal models", "mouse", "Clinical immunology", "Autoimmune diseases", "Immunologic techniques", "plp", "cells", "brains", "eae", "mice", "confocal"], "article_id"=>914705, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Chandirasegaran Massilamany", "Arunakumar Gangaplara", "Ting Jia", "Christian Elowsky", "Guobin Kang", "Jean-Jack Riethoven", "Qingsheng Li", "You Zhou", "Jay Reddy"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087519.t001", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_analysis_of_PLP_139_151_dext_CD4_T_cells_in_the_brains_of_EAE_mice_by_confocal_microscopy_/914705", "title"=>"Quantitative analysis of PLP 139–151-dext<sup>+</sup> CD4<sup>+</sup> T cells in the brains of EAE mice by confocal microscopy.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-27 04:01:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1362364"], "description"=>"<p>For dextramer staining, two cell sources were used: (a) PLP 139-151-sensitized LNCs obtained from immunized mice; and (b) PLP 139-151-specific T cell hybridoma clone (B8). Viable cells harvested from LNC cultures on day 6 poststimulation, and also hybridoma cells were stained with PLP 139–151 dextramers/anti-CD4 or TMEV 70–86 (control) dextramers/anti-CD4. After washing, cells were fixed with 0.5% paraformaldehyde and examined by LSCM. <u>Top panels (panels, a and b)</u>: cells stained with PLP 139–151 dextramers and anti-CD4. <u>Bottom panels (panels, a and b)</u>: cells stained with TMEV 70–86 dextramers (control) and anti-CD4. <u>Left panels</u>: CD4, green; <u>Middle panels</u>: dextramers, red; <u>Right panels</u>: merged. Dext<sup>+</sup> CD4<sup>+</sup> T cells are shown with arrows for LNCs, whereas in PLP 139-151-specific hybridoma cells, the dext<sup>+</sup> CD4<sup>+</sup> cells are shown as such throughout the field. Original magnification 600×; bar  = 20 µm.</p>", "links"=>[], "tags"=>["immunology", "Immune cells", "t cells", "autoimmunity", "Major histocompatibility complex", "Model organisms", "Animal models", "mouse", "Clinical immunology", "Autoimmune diseases", "Immunologic techniques", "specificity", "plp", "dextramers", "confocal", "microscopy"], "article_id"=>914698, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Chandirasegaran Massilamany", "Arunakumar Gangaplara", "Ting Jia", "Christian Elowsky", "Guobin Kang", "Jean-Jack Riethoven", "Qingsheng Li", "You Zhou", "Jay Reddy"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087519.g001", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Evaluation_of_the_specificity_of_PLP_139_151_dextramers_by_confocal_microscopy_in_vitro_/914698", "title"=>"Evaluation of the specificity of PLP 139–151 dextramers by confocal microscopy <i>in vitro</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-27 04:01:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1362365"], "description"=>"<p>EAE was induced in SJL mice by immunizing the animals with PLP 139–151 in CFA. At termination, cerebrums collected from EAE mice were embedded in 4% agarose, and the sections were made using vibratome. After staining with cocktails containing either PLP 139–151 dextramers/anti-CD4 or TMEV 70–86 dextramers (control)/anti-CD4 and fixing with 4% PBS-buffered paraformaldehyde, sections were washed and mounted for examination by LSCM. <u>Top panels</u>: sections stained with PLP 139–151 dextramers and anti-CD4. <u>Bottom panels</u>: sections stained with TMEV 70–86 dextramers and anti-CD4. <u>Left panel</u>: CD4, green; <u>Middle panel</u>: dextramers, red; <u>Right panel</u>: merged (circles, dext<sup>+</sup> CD4<sup>+</sup> T cells; insets represent enlarged views of dext<sup>+</sup> CD4<sup>+</sup> T cells). Original magnification 1000×; bar  = 20 µm.</p>", "links"=>[], "tags"=>["immunology", "Immune cells", "t cells", "autoimmunity", "Major histocompatibility complex", "Model organisms", "Animal models", "mouse", "Clinical immunology", "Autoimmune diseases", "Immunologic techniques", "plp-specific", "cells", "staining", "plp"], "article_id"=>914699, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Chandirasegaran Massilamany", "Arunakumar Gangaplara", "Ting Jia", "Christian Elowsky", "Guobin Kang", "Jean-Jack Riethoven", "Qingsheng Li", "You Zhou", "Jay Reddy"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087519.g002", "stats"=>{"downloads"=>11, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_of_PLP_specific_T_cells_by_in_situ_staining_with_PLP_139_151_dextramers_/914699", "title"=>"Detection of PLP-specific T cells by <i>in situ</i> staining with PLP 139–151 dextramers.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-27 04:01:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1362370"], "description"=>"<p>Three paired cerebral sections, each 200 µm thick, were made from brains obtained from EAEmice using vibratome by cutting the sections from the dorsal to the ventral surface with 1.5 mm intervals between pairs. One section from each pair was stained with either PLP 139–151 or TMEV 70–86 (control) dextramers together with anti-CD4. After fixing and mounting, the sections were examined by LSCM (60X), and each inflammatory focus was subjected to a set of ‘Z’ serial images (15 to 25) taken sequentially. In total, 10 to 15 sets of such ‘Z’ serial images representing as many inflammatory foci were acquired from each section. In all the images, cells positive for both PLP 139–151 dextramers and CD4 or CD4 alone were counted using ‘ImageJ’ software. Finally, the cells counted in all the ‘Z’ serial images obtained from all the inflammatory foci were added to obtain the total number per section.</p>", "links"=>[], "tags"=>["immunology", "Immune cells", "t cells", "autoimmunity", "Major histocompatibility complex", "Model organisms", "Animal models", "mouse", "Clinical immunology", "Autoimmune diseases", "Immunologic techniques", "plp-specific", "cd4", "cells", "staining", "plp", "dextramers"], "article_id"=>914702, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Chandirasegaran Massilamany", "Arunakumar Gangaplara", "Ting Jia", "Christian Elowsky", "Guobin Kang", "Jean-Jack Riethoven", "Qingsheng Li", "You Zhou", "Jay Reddy"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087519.g003", "stats"=>{"downloads"=>1, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_analysis_of_PLP_specific_CD4_T_cells_detected_by_in_situ_staining_with_PLP_139_151_dextramers_in_EAEmice_/914702", "title"=>"Quantitative analysis of PLP-specific CD4 T cells detected by <i>in situ</i> staining with PLP 139–151 dextramers in EAEmice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-27 04:01:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1362371"], "description"=>"<p>EAM was induced in A/J mice by immunizing the animals with Myhc 334–352 in CFA; at termination on day 21, hearts were collected after perfusion, and the tissue blocks were prepared using 4% PBS-buffered agarose. Eight paired sections of 200 µm thickness each were made using vibratome. One section from each pair (a total of eight sections) were stained with mixtures containing either Myhc 334–352 dextramers/anti-CD4 or RNase 43–56 (control) dextramers/anti-CD4. After fixing with 4% PBS-buffered paraformaldehyde, the sections were washed and mounted for examination by LSCM. A collage of images showing the cells positive for both dextramers and CD4 or CD4 alone is shown. <u>Top panels</u>: sections stained with Myhc 334–352 dextramers and anti-CD4. <u>Bottom panels</u>: sections stained with RNase 43–56 dextramers and anti-CD4. <u>Left panel</u>: CD4, green; <u>middle panel</u>: dextramers, red; <u>right panel</u>: merged. (arrows, dext<sup>+</sup> CD4<sup>+</sup> T cells). Original magnification 1000×; bar  = 20 µm.</p>", "links"=>[], "tags"=>["immunology", "Immune cells", "t cells", "autoimmunity", "Major histocompatibility complex", "Model organisms", "Animal models", "mouse", "Clinical immunology", "Autoimmune diseases", "Immunologic techniques", "cardiac", "myosin-specific", "cd4", "cells"], "article_id"=>914703, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Chandirasegaran Massilamany", "Arunakumar Gangaplara", "Ting Jia", "Christian Elowsky", "Guobin Kang", "Jean-Jack Riethoven", "Qingsheng Li", "You Zhou", "Jay Reddy"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087519.g004", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Detection_of_cardiac_myosin_specific_CD4_T_cells_by_in_situ_staining_/914703", "title"=>"Detection of cardiac myosin-specific CD4 T cells by <i>in situ</i> staining.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-27 04:01:47"}

PMC Usage Stats | Further Information

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Relative Metric

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