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1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, 2Center for Biotechnology, University of Nebraska, Lincoln, 3Nebraska Center for Virology and School of Biological Sciences, University of Nebraska, Lincoln
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The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.
Massilamany, C., Gangaplara, A., Jia, T., Elowsky, C., Li, Q., Zhou, Y., et al. In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers. J. Vis. Exp. (90), e51679, doi:10.3791/51679 (2014).
This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler’s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the ‘Z’ serial images.
The use of major histocompatibility complex (MHC) class II tetramers for detection of antigen-specific CD4 T cells has been a challenge 1-7. To enhance the detection sensitivity of MHC class II tetramers, the research team has created next-generation tetramers, designated “dextramers” 8. Dextramers contain dextran backbones in which multiple streptavidin-fluorophore moieties are conjugated, such that several peptide-tethered, biotinylated MHC monomers can be attached to one dextran molecule9. Thus, large aggregates of MHC dextramers that contain several MHC-peptide complexes can interact with multiple T cell receptors (TCRs).
This group recently generated dextramers for various self-antigens and demonstrated that the detection sensitivity of dextramers was approximately five-fold more than tetramers 8. The experimental systems include experimental autoimmune encephalomyelitis (EAE) induced with proteolipid protein (PLP) 139-151 in SJL mice; EAE induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 in C57Bl/6 mice; and experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain-α (Myhc) 334-352 in A/J mice. This study demonstrates the use of PLP 139-151- and Myhc 334-352 dextramers to detect antigen-specific CD4 T cells in situ with specificity by developing a direct staining protocol, thereby eliminating the need to amplify the signals from using fluorophore antibodies, an approach commonly employed with the use of tetramers. In addition, a comprehensive method of evaluating tissues has also been devised to reliably enumerate the frequencies of antigen-specific CD4 T cells in situ 10. Detection of antigen-specific T cells in situ permits delineation of events caused by specific antigenic stimuli from those caused by bystander activation. Likewise, the in situ technique also provides opportunities to track the kinetics of antigen-specific T cell infiltrations in the target organs.
All animal procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the University of Nebraska-Lincoln, Lincoln, NE.
1. Induction of EAE
2. Clinical Scoring of EAE Mice and Harvesting of Cerebrums
3. In Situ Staining of Cerebral Sections with MHC Class II (IAs)/PLP 139-151 Dextramers
4. Induction of EAM and Collection of Hearts
5. In Situ Staining of Heart Sections with MHC Class II (IAk)/Myhc 334-352 Dextramers
6. Analysis of Dextramer+ (dext+) CD4+ T Cells by Laser Scanning Confocal Microscope (LSCM) (Common to Both Brain and Heart Sections)
In this report, in situ detection of antigen-specific, autoreactive CD4 T cells by direct staining with MHC class II dextramers is described. Freshly cut cerebral sections were derived from EAE mice and stained with cocktails containing PLP 139-151 (specific) or TMEV 70-86 (control) dextramers and anti-CD4. The top panels in Figure 1 shows LSCM analysis of cerebral sections costained with PLP 139-151 dextramers (red) and CD4 antibody (green), where the double-positive cells appeared yellow. Such a feature was absent in sections stained with TMEV 70-86 (control) dextramers (bottom panels). The costained (dext+ CD4+) cells showed punctate dots around the periphery. Quantitative analysis of PLP-specific T cells in the cerebral sections involved the following steps (Figure 2): 1) Sections of three pairs were made, and one section from each pair was stained individually with anti-CD4 and PLP 139-151 dextramers. Likewise, another set of three sections were stained with anti-CD4 and control (TMEV 70-86) dextramers. 2) From a given section, inflammatory foci (10 to 15) were identified based on anti-CD4 staining. 3) A set of ‘Z’ serial images (15 to 25) were taken from each focus sequentially from top to bottom with an interval of 2 µm between each. In essence, 10 to 15 similar sets of ‘Z’ serial images representing equal number of inflammatory foci were analyzed in every section. 4) In individual images, CD4+ cells (green), the cells positive for both dextramers (red) and CD4 (green) which appeared as yellow punctate cells were enumerated by marking them individually using the open source software, ImageJ, and finally, a grand total was obtained for each section by adding the number of cells counted in all the ‘Z’ serial images. Because that the cells were marked, the possibility of recounting the cells in multiple images that are overlapping was eliminated. Thus, reliable enumeration of dext+ cells relative to the total number of cells expressing CD4 can be achieved.
This study was further extended to demonstrate the use of dextramer reagents for detecting antigen-sensitized T cells in situ in EAM induced with Myhc 334-352 in A/J mice, which is also a T cell-mediated disease 13,14. Eight paired heart sections were obtained, each 200 µm thick, from EAM mice (Figure 2). From each pair, one section (with a total of 8 sections) was stained with Myhc 334-352 dextramers and anti-CD4, whereas another set of sections (8 in total) was stained with RNase 43-56 dextramers (control) and anti-CD4. Twenty sets of ‘Z’ serial images corresponding to that many inflammatory foci representing all eight sections were acquired sequentially as above (Figure 2). Essentially, a given set of ‘Z’ serial images consisted of 10 to 15 individual represented one single inflammatory focus. Analysis of these images by LSCM showed the presence of cells positive for Myhc 334-352 dextramers (red), and CD4 (green) and expectedly, the cells bound with both dextramers and CD4 appeared as yellow punctate cells (Figure 3, top panels). The background staining for control dextramers was negligible (Figure 3, bottom panels). The total numbers of dext+ CD4+ T cells and the total number of CD4+ T cells were determined for each section and counts from all the sections were added together to derive the total number for each subset (dext+ CD4+ T cells; CD4+ T cells).
Figure 1. Detection of PLP-specific CD4 T cells by in situ staining with PLP 139-151 dextramers. 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. Top panels: two separate sections stained with PLP 139-151 dextramers/anti-CD4. Bottom panels: two separate sections stained with TMEV 70-86 dextramers/anti-CD4. Left panel: CD4, green; Middle panel: dextramers, red; Right panel: merged (arrows, dext+ CD4+ T cells). Original magnification 1,000X (adopted: Massilamany et al., 2014. Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ. PLOS ONE 9 (1): e87519). Please click here to view a larger version of this figure.
Figure 2. An approach to quantitate dext+ cells in brains and hearts. Paired sections are made from cerebrum or auricle-excised heart as shown (Panel 1). One section from each pair corresponding to the indicated organs was designated for staining with specific dextramers (cerebrum, PLP 139-151 dextramers; heart, Myhc 334-352 dextramers), and the other set of sections for control dextramers (cerebrum, TMEV 70-86; heart, RNase 43-56). After fixing and mounting, the sections were examined to locate inflammatory foci based on staining with CD4 by LSCM (original magnification, 40X) (Panel 2). Each inflammatory focus as visualized in three-dimensional (3D) view (Panel 3) was subjected to a set of ‘Z’ serial images sequentially (Panel 4). In all the images, cells positive for both specific or control dextramers and CD4, or CD4 alone were counted using ‘ImageJ’ software (adopted: Massilamany et al., 2014. Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ. PLOS ONE 9 (1): e87519). Please click here to view a larger version of this figure.
Figure 3. Detection of cardiac myosin-specific CD4 T cells by in situ staining with Myhc 334-352 dextramers. 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) was 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. Images showing the cells positive for both dextramers and CD4 or CD4 alone are shown. Top panels: two separate sections stained with Myhc 334-352 dextramers/anti-CD4. Bottom panels: two separate sections stained with RNase 43-56 dextramers /anti-CD4. Left panel: CD4, green; middle panel: dextramers, red; right panel: merged (arrows, dext+ CD4+ T cells). Original magnification 1,000 X (adopted: Massilamany et al., 2014. Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ. PLOS ONE 9 (1): e87519). Please click here to view a larger version of this figure.
Unlike MHC class I tetramers, the use of MHC class II tetramers for routine applications continues to be challenging, especially to enumerate the precursor frequencies of low-affinity autoreactive CD4 T cells, due in part to their activation dependency 6,15-17. Recently, this problem was circumvented by creating the next generation of tetramers, called “dextramers,” permitting us to detect antigen-specific CD4 T cells for a number of autoantigens, such as PLP 139-151, MOG 35-55 and Myhc 334-352 8. In this report, for the first time, the utility of MHC class II dextramers to detect and quantify self-reactive CD4 T cells in situ has been demonstrated using brain sections from EAE mice, and heart sections from EAM mice. On a reaction basis, dextramers offer the advantage of requiring a small amount of MHC/peptide complexes. In this protocol, each reaction requires only 0.75 µg of MHC/peptide monomers. In addition, staining with dextramers is a one-step reaction, since dextramers are coincubated with anti-CD4, and the whole procedure, from tissue sectioning to staining, can be finished in less than a day. In contrast, the published protocols for in situ staining with conventional tetramers commonly involve amplification procedures in which the fluorescent signals are amplified using secondary antibodies for fluorophores As a result, staining procedures may take up to three days to complete 18-20.
Furthermore, the use of dextramers in cerebral sections permits detection of antigen-specific T cells deep in the tissue sections, even up to 50 µm. It was noted that PLP 139-151 dext+ CD4+ cells were found to be located both perivascularly and also within the parenchyma. Furthermore, the intensity of signals generated from Myhc 334-352 dextramers was low in cardiac sections when compared with the PLP 139-151 dextramers in brain sections (Figure 1), and such a variation may reflect the characteristics unique to each organ. One such variable is the lipid content which is present in high amounts in the brain tissue as opposed to cardiac tissue that contains mainly striated muscle fibers that can potentially restrict easy diffusion of dextramers into the inflammatory focus. In support of this notion, Myhc 334-352 dext+ cells were detected only to a depth up to 20 µm in majority of cardiac sections. Additionally, Myhc 334-352 dext+ cells were present scattered all through the myocardium suggesting the diffused nature of inflammatory infiltrates in myocarditic lesions.
Generally, two major factors can negatively influence the detection of antigen-specific T cells in situ by LSCM. First, insufficient signals emitted by fluorophores can lead to the need to amplify the signals using fluorophore antibodies. Second, such an indirect staining can undermine the specificity, as the background staining for control reagents also can increase proportionately 18,19. These limitations were circumvented by direct staining with the dextramers. Although, detection of CD4 T cells has been successfully shown with fresh sections, the use of dextramer reagents in frozen tissues requires additional studies, which can be a challenge as the retention of tissue-integrity may be difficult because of the tendency for tissues to get disintegrated during various staining steps 18,19 .
No conflicts of interests were declared.
This work was supported by the American Heart Association and the Children’s Cardiomyopathy Foundation (SDG2462390204001), and the National Institutes of Health (HL114669). CM is a recipient of a postdoctoral research fellowship grant awarded by the Myocarditis Foundation, NJ.
|CFA||Sigma Aldrich, St Louis, MO||5881||Store at 4 °C|
|MTB H37Rv extract||Difco Laboratories, Detroit, MI||231141||Store at 4 °C|
|PT||List Biologicals Laboratories, Campbell, CA||181||Store at 4 °C|
|1x PBS||Corning, Manassas, VA||21-040-CV||Store at 4 °C|
|Female A/J mice||Jackson Laboratories, Bar Harbor, ME||646|
|Female SJL/J mice||Jackson Laboratories, Bar Harbor, ME||686|
|Leur-lok sterile 1 ml syrringe||BD, Franklin Lakes, NJ||309628|
|Leur-lok sterile 3 ml syrringe||BD, Franklin Lakes, NJ||309657|
|Sterile needle, 18 G||BD, Franklin Lakes, NJ||305195|
|Sterile needle, 27 1/2 G||BD, Franklin Lakes, NJ||305109|
|3-way stopcock||Smiths Medical ASD, Inc. Dublin, OH||MX5311L|
|Kerlix gauze bandage rolls||Covidien, Mansfield, MA||6720|
|Kimwipes||Kimberly-Clark Professional, Roswell, GA||34155|
|Agarose, Low Melting Point, Analytical Grade||Promega corporation, Madison, WI||V2111|
|Immunopure normal goat serum||Thermo Scientific, Waltham, MA||31873||Store at -20 °C|
|Anti-mouse CD4 conjugated with PE dye||eBioscience, San Diego, CA||12004185||Store at 4 °C|
|Paraformaldehyde||Electron Microscopy Sciences, Hatfield, PA||19202||Store at 4 °C|
|Faramount Aqueous Mounting Medium||Dako, Carpinteria, CA||10073021|
|Conical tube (15 ml)||BD Biosciences, San Jose, CA||352099|
|Conical tube (50 ml)||BD Biosciences, San Jose, CA||352070|
|24-well flat bottomed tissue culture plates||BD Biosciences, San Jose, CA||356775|
|Water color #2 brushes||Charles Leonard, Inc. Hauppauge, NY||73502|
|Plain Microscope slides, size: 3" x 1" x 1.2 mm||Fisher Scientific, Pittsburgh, PA||1255010|
|Premium Cover glass, 22 x 22-1||Fisher Scientific, Pittsburgh, PA||12548B|
|Disposable deep base mold histology cassettes||Laboratory prodcust, Rochester, NY||M-475-10|
|Loctite Super glue||Henkel Corporation, Westlake, OH||1471879|
|Razor blades||Gillette SuperSilver|
|Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)||Neopeptide, Cambridge, MA||Store at 4 °C|
|PLP 139-151 (HSLGKWLGHPDKF)||Neopeptide, Cambridge, MA||Store at 4 °C|
|MHC class II/IAs PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye||Store at 4 °C|
|MHC class II/IAk Myhc 334-352 or or RNase 40-56 dextramers conjugated with APC dye||Store at 4 °C|
|Dextran conjugated SA-APC||Immundex, Copenhagen, Demark||Store at 4 °C|
|Sterile surgical scissors and forceps||INOX tool Corporation|
|Micro oven||GE Healthcare, Pittsuburgh, PA|
|Leica VT 1200 Vibratome||Leica Microsystems, Inc. Buffalo Grove, IL|
|Olympus BX 60 Laser scanning confocal microscope||Olympus America, Inc. Center Valley, PA|
|Nikon A1-Eclipse 90i confocal microscope||Nikon Inc. Melville, NY|
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