Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ.
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.
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Chandirasegaran
C
Massilamany
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Arunakumar
A
Gangaplara
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Ting
T
Jia
Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Christian
C
Elowsky
Nebraska Center for Virology and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Guobin
G
Kang
Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Jean-Jack
JJ
Riethoven
Nebraska Center for Virology and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Qingsheng
Q
Li
Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
You
Y
Zhou
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.
Jay
J
Reddy
D000818:Animals; D001921:Brain / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D058948:Cell Tracking / Q000379:methods*; D005260:Female; D005453:Fluorescence; D005802:Genes, MHC Class II / Q000235:genetics; D006649:Histocompatibility Antigens / Q000235:genetics / Q000378:metabolism*; D008222:Lymphokines / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D009205:Myocarditis / Q000276:immunology; D009206:Myocardium / Q000378:metabolism*; D055503:Protein Multimerization / Q000235:genetics*; D013194:Staining and Labeling / Q000379:methods; D018709:Statistics, Nonparametric
D006649:Histocompatibility Antigens; D008222:Lymphokines; C057264:antigen-specific helper factors