原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的
Summary
通过直接染色与主要组织相容性复合物Ⅱ类dextramers检测自身反应性CD4 + T细胞在大脑和心脏的协议已经本报告中进行了描述。为综合分析，一种可靠的方法来枚举抗原特异性CD4 + T细胞在原位的频率也被设计出来。
Abstract
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.
Introduction
使用的主要组织相容性复合体（MHC）Ⅱ类四聚体检测抗原特异性CD4 + T细胞一直是个挑战1-7。为了增强MHC II类四聚体的检测灵敏度，研究小组已经创造下一代四聚体，命名为“dextramers”8。 Dextramers含有葡聚糖主链，其中多个链亲和素-荧光基团缀合，使得一些肽-拴系的，生物素化的MHC单体可以连接到一个葡聚糖分子9。因此，包含数个MHC-肽复合物的MHC dextramers的大的聚集体可以与多个T细胞受体（TCR）进行交互。
这组最近产生dextramers各种自身抗原，并证明dextramers的检测灵敏度约为5倍以上的四聚体8。实验系统包括实验性自身免疫性脑脊髓炎（EAE）诱导蛋白脂质蛋白（PLP）在SJL小鼠139-151; EAE诱导的髓磷脂少突胶质细胞糖蛋白（MOG）在C57BL / 6小鼠35〜55;和实验性自身免疫性心肌炎（EAM）与诱导心肌肌球蛋白重链α（MYHC）中的A / J小鼠334-352段。这项研究表明，使用PLP 139-151的-和MYHC 334-352 dextramers来检测抗原特异性CD4 + T细胞在原位特异性通过开发一个直接染色方案，从而省去了使用荧光抗体放大信号，一接近常用配合使用四聚体。此外，评估组织的综合方法也被设计成可靠地列举抗原特异性CD4 + T细胞在原位 10的频率。检测抗原特异性T细胞在原位的允许由从所造成的旁观者激活特定抗原刺激事件的描述。同样地， 在原位技术也providES机会来跟踪在靶器官的抗原特异性T细胞浸润的动力学。
Protocol
Representative Results
Discussion
与MHC I类四聚体，采用MHCⅡ类四聚体的常规应用仍然是具有挑战性的，尤其是枚举低亲和力的自身反应性CD4 + T细胞，部分原因是由于其激活依赖6,15-17的前体频率。最近，这个问题是由创建下一代四聚体，称为规避“dextramers，”允许我们检测抗原特异性CD4 + T细胞为一些自身抗原，如PLP 139-151，MOG 35-55和MYHC 334 – 352 8。在此报告中，用于第一时间的推移，MHC II类dextramers的实用程序来?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国心脏协会和儿童心肌病基金会（SDG2462390204001）和美国国立卫生研究院（HL114669）的支持。 CM是荣获由心肌炎基金会，新泽西州的博士后研究奖学金补助金的获得者。
Materials
| CFA | Sigma Aldrich, St Louis, MO | 5881 | Store at 4οC |
| MTB H37Rv extract | Difco Laboratories, Detroit, MI | 231141 | Store at 4οC |
| PT | List Biologicals Laboratories, Campbell, CA | 181 | Store at 4οC |
| 1x PBS | Corning, Manassas, VA | 21-040-CV | Store at 4οC |
| 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 οC |
| Anti-mouse CD4 conjugated with PE dye | eBioscience, San Diego, CA | 12004185 | Store at 4οC |
| Paraformaldehyde | Electron Microscopy Sciences, Hatfield, PA | 19202 | Store at 4οC |
| Faramount Aqueous Mounting Medium | Dako, Carpinteria, CA | 10073021 | |
| Conical tube (15ml) | 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 4οC | |
| PLP 139-151 (HSLGKWLGHPDKF) | Neopeptide, Cambridge, MA | Store at 4οC | |
| MHC class II/IAs PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye | Store at 4οC | ||
| MHC class II/IAk Myhc 334-352 or or RNase 40-56 dextramers conjugated with APC dye | Store at 4οC | ||
| Dextran conjugated SA-APC | Immundex, Copenhagen, Demark | Store at 4οC | |
| 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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