Supported in part by grants from the National Institutes of Health (R01 NS37253 and N01 NS055167) and the National Multiple Sclerosis Society (RG 3288-A6).

1. Preparing CFA-emulsified Antigen
<ol>
<li>Dissolve guinea pig MBP (prepared according to the method of Swanborg1 and stored in lyophilized form at 4 &deg;C) in phosphate buffer solution (PBS) at a concentration of 2 mg/mL. If synthesized MBP peptides (MBP60-80, S H H A A R T T H Y G S L P Q K S Q R)2 are used, prepare a 2 mg/mL solution. Draw up an appropriate amount of antigen solution (0.1 mL per mouse to be immunized) into a glass syringe with luer-lok.</li>
 <li>Draw up a volume of complete Freund's adjuvant H37Ra (0.1WT%) (CFA, Difco Laboratories) equal to the antigen solution into another glass syringe equipped with luer-lok.</li>
 <li>Connect the two glass syringes with a three-way stopcock. Mix the MBP solution with the CFA by pushing the plungers back and forth. Keep the motion until an emulsion is formed.</li>
 <li>To test if an emulsion is formed, push all of the mixture into one syringe. Disconnect the empty syringe, pull the plunger back a little and discharge a drop of the residual emulsion into a beaker of clean water at room temperature. If the emulsion drop remains intact on the surface of the water, an emulsion is formed. If the emulsion drop disperses, reconnect the syringes and continue the mixing motion.</li>
 <li>Use a 1 mL plastic syringe for the immunization process to ensure delivery of an accurate dose of antigen. To transfer the antigen emulsion to the plastic syringe, pull out the plunger and insert the empty plastic syringe into the opening of three-way stopcock where the previous glass syringe was disconnected as in 1.4.</li>
 <li>Fill the plastic syringe to the rim by pushing the plunger of the glass syringe. Insert the plunger of the plastic syringe back and push back excess emulsion till reaching the 1.2 mL mark on the plastic syringe. This maneuver assures that no air bubble is trapped inside the plastic syringe.</li>
 <li>Remove the plastic syringe from the three-way stopcock. Attach a #26 gauge needle to the plastic syringe. Push the plunger to the 1 mL mark so that the emulsion now fills the void volume of the needle.</li>
</ol>
2. Immunization of Donor Mice
Note: Female C57BL/6 mice are typically injected with antigen emulsion in four sites in the flanks, following the methods originally designed by McFarlin's group3 at the National Institutes of Health. For an experienced investigator, one person can perform the whole task. For beginners, help may be solicited so that one person holds the mouse and the other person does the injection. Alternatively, the mouse can be anesthetized for the injection.
<ol>
 <li>Hold the mouse firmly by the neck skin and hold back the tail between the 4th and 5th fingers such that the thorax and abdomen are easily accessible.</li>
 <li>Place the needle of the 1mL plastic syringe at a site in the thorax slightly above the right armpit (of the mouse) and inject 0.05 mL of the antigen emulsion subcutaneously. A little bulge of the whitish CFA can be seen through the pale skin. Repeat the same for the site in the thorax slightly above the left armpit.</li>
 <li>Release the tail and take hold of the right foot (of the mouse), turn it and hold it between the 4thand 5th fingers. Locate the site just below the rib case in the right flank and inject 0.05 mL of the antigen emulsion.</li>
 <li>Release the right foot, turn the mouse the other way and hold the left foot (of the mouse) between the 4th and 5th fingers. Locate the site just below the rib case in the left flank and inject 0.05 mL of the antigen emulsion.</li>
 <li>Change to a fresh needle after injection of 10 mice, or if the needle becomes dulled.</li>
</ol>
3. Isolation of Lymph Node Cells for Tissue Culture 7 to 10 Days after Immunization
<ol>
 <li>Seven to 10 days after immunization, sacrifice the mice and remove the draining lymph nodes. The preferred method of euthanasia is by CO2 inhalation. After sacrifice, lay the mouse on its back on a dissection board with the limbs stretched out and secured to the board with pins. The mice are wetted with a spray of 70% ethanol.</li>
 <li>Cut a slit in the skin longitudinally in the middle from the lower end of the abdomen up to the chin with sterile scissors and forceps.</li>
 <li>Cut the abdomen skin from the area of the groin down to each leg. Peel back and pin down the skin, exposing the inguinal lymph nodes.</li>
 <li>Cut the skin along the forelimbs (left and right) so that the skin can be peeled back and pinned down to expose the axillary lymph nodes near the armpits.</li>
 <li>With two pairs of forceps working together, pull away the connective tissues covering and around the inguinal lymph nodes. When the connective tissues are cleared, place one pair of forceps under the lymph node and pull away from the body. Place the lymph nodes in a 35 mm Petri dish filled with sterile PBS.</li>
 <li>Pull away the connective tissues covering the two axillary lymph nodes using two pairs of forceps. Isolate the lymph nodes and place them in the Petri dish. Be very careful not to break the blood vessels crossing the area as bleeding makes identifying the lymph nodes very difficult.</li>
 <li>After all the lymph nodes are collected, move the Petri dish to a biosafety hood. Break and tease the lymph nodes apart with the two pairs of forceps, grinding one against the other. Alternatively, the lymph nodes can be broken up by using the flat end of the plunger of a 3 mL plastic syringe pressing against the lymph nodes on the bottom of the Petri dish.</li>
 <li>Pass the teased lymph node cell suspension through a nylon mesh to remove the membranes and debris.</li>
 <li>Make up the lymph node cell suspension to 50 mL using sterile PBS. Centrifuge at 1000 rpm for 10 mins. Resuspend the pellet in culture medium. Do a viable cell count and adjust the concentration to 4 x 106 cells/mL. UseRPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM MEM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 100 U/mL penicillin, 100 &mu;g/mL streptomycin, 10 mM Hepes buffer and 5 x 10-5 M 2-ME. Pre-warm the culture before resuspending the cells.</li>
 <li>Culture the cells in 24-well culture plates with 2 mL volume per well (final cell concentration 8 x 106 cells/well). Add antigen to each well at a final concentration of 100 &mu;g/mL. Place the culture plate in a 37 &deg;C incubator with 5% CO2. Incubate cells for 5 days.</li>
</ol>
4. Harvesting Cultured Lymph Node Cells 5 Days after Initiation of Cultures
<ol>
 <li>Mix the cells in each well by pipetting up and down with a Pasteur glass pipette or a small volume pipette. Harvest the cells to a 50 mL centrifuge tube.</li>
 <li>Centrifuge the cells at 1000 rpm for 10 mins. Resuspend the cell pellet into a small volume of PBS.</li>
 <li>Count cells and adjust the viable cell concentration to 2.5 x 108 cells/mL with sterile PBS. Draw the cells into a syringe fitted with a #26 gauge needle.</li>
 <li>Place a heat lamp over the cage of recipient mice to help dilate the tail vein.</li>
 <li>Using a mouse holder, extend the tail of the mouse through the slit in the holder. Pull to extend the tail. Locate the vein.</li>
 <li>Inject 0.2 mL/mouse of the cell suspension, equaling 5 x 107 cells.</li>
</ol>
5. Antigenic Challenge of Recipient Mice 20 Days Post Cell Transfer and Development of Disease Symptoms
<ol>
 <li>Challenge recipient mice not showing signs of disease with the antigen. The procedures are the same as section 2 on "Immunization of Donor Mice".</li>
 <li>Observe development of paralytic disease 7 to 10 days after antigenic challenge.
 <ol><li>Disease starts with weakness in the tail. Disease is graded as 1 when the tail becomes flaccid. Disease is graded as 2 when the mouse is paralyzed in one hind limb. Disease is graded 3 when both hind limbs are paralyzed and the mouse drags both hind limbs when crawling forward. Disease is graded 4 when the mouse loses use of both forelimbs, leaving the mouse totally immobile. Disease is graded 5 when the mouse is moribund and the body curls up. Death is scored as disease grade 64.</li></ol>
 </li>
 <li>MBP-specific EAE induced in C57BL/6 mice following this protocol is monophasic. The mice may recover from the disease but do not undergo spontaneous relapses.</li>
</ol>
6. Representative Results
Mice that received primed and in vitro-expanded donor lymph node cells did not develop EAE disease symptoms, reflecting their resistance to disease induction. However, severe disease developed after antigenic challenge (Table I), demonstrating the ability of the antigenic challenge in reversing the resistance mechanism. Two special features of the antigenic challenge noted are the doses of antigen and the kinetics of challenge to induce disease. Very low dose of antigen is needed as shown in Table III. Surprisingly, mice that received donor cells a year earlier could still develop severe disease when challenged with the antigen (Table IV). These two observations strongly suggest the involvement of "long-lived" T cells in maintaining EAE resistance. This protocol is applicable to many EAE resistant mouse strains4.
<img alt="Figure 1." src="/files/ftp_upload/3778/3778fig1.jpg" /> 
Figure 1. Flow chart of the experimental design for induction of EAE in reputed resistant mouse strains.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td colspan="3">Clinical EAE 
 after adoptive transfer </td>
 <td colspan="3">Clinical EAE 
 after antigenic challenge </td>
 </tr>
 <tr>
 <td>Strain</td>
 <td>Antigen </td>
 <td>incidence</td>
 <td>Av. disease grade 
 (range)</td>
 <td>Av. day of onset 
 (range)</td>
 <td>Incidence</td>
 <td>Av. disease grade 
 (range)</td>
 <td>Av. day of onset 
 (range)</td>
 </tr>
 <tr>
 <td>SJL</td>
 <td>MBP</td>
 <td>21/21</td>
 <td>4.0 (3-5)</td>
 <td>8.0 (6-10)</td>
 <td>N.D.</td>
 <td>N.D.</td>
 <td>N.D.</td>
 </tr>
 <tr>
 <td>C57BL/6</td>
 <td>MBP</td>
 <td>0/10</td>
 <td>- -</td>
 <td>- -</td>
 <td>7/7</td>
 <td>3.85 (3-5)</td>
 <td>9.28 (9-10)</td>
 </tr>
 <tr>
 <td>Balb/c</td>
 <td>MBP</td>
 <td>0/7</td>
 <td>- -</td>
 <td>- -</td>
 <td>5/5</td>
 <td>3.2 (3-4)</td>
 <td>7.0 (7)</td>
 </tr>
 <tr>
 <td>C3H/HeJ</td>
 <td>MBP</td>
 <td>0/7</td>
 <td>- -</td>
 <td>- -</td>
 <td>4/4</td>
 <td>3.2 (3-4)</td>
 <td>8.0 (8)</td>
 </tr>
 <tr>
 <td>C57BL/6</td>
 <td>MBP60-80</td>
 <td>0/4</td>
 <td>- -</td>
 <td>- -</td>
 <td>4/4</td>
 <td>2.75 (2-4)</td>
 <td>10.2 (9-11)</td>
 </tr>
 </tbody>
</table>
Table I. Induction of MBP-mediated EAE in C57BL/6 mice. Adoptive transfer of primed and in vitro-expanded donor cells did not induce EAE in C57Bl/6 mice. Antigenic challenge post cell transfer rendered the mice susceptible to disease induction. Av., average. N.D., not done.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td colspan="3">Clinical EAE after antigenic challenge</td>
 </tr>
 <tr>
 <td>Priming Antigen </td>
 <td>Challenge antigen</td>
 <td>Incidence</td>
 <td>Av. disease grade 
 (range)</td>
 <td>Av. day of onset 
 (range)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>CFA alone</td>
 <td>0/3</td>
 <td>- -</td>
 <td>- -</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>OVA-CFA</td>
 <td>0/3</td>
 <td>- -</td>
 <td>- -</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>MBP-CFA</td>
 <td>3/3</td>
 <td>4.0 (4.0)</td>
 <td>7.7 (7-9)</td>
 </tr>
 </tbody>
</table>
Table II. Specificity of antigenic challenge. Recipient mice were challenged with the priming antigen as well as other irrelevant antigens. Only the priming antigen induced severe disease. OVA: chicken ovalbumin. Av., average.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td colspan="3">Clinical EAE after antigenic challenge</td>
 </tr>
 <tr>
 <td>Challenge antigen doses (&mu;g/mouse) </td>
 <td>Incidence</td>
 <td>Av. disease grade 
 (range)</td>
 <td>Av. day of onset 
 (range)</td>
 </tr>
 <tr>
 <td>200</td>
 <td>4/4</td>
 <td>3.75 (3-4)</td>
 <td>8.25 (7-9)</td>
 </tr>
 <tr>
 <td>100</td>
 <td>4/4</td>
 <td>3.67 (3-4)</td>
 <td>7.5 (7-8)</td>
 </tr>
 <tr>
 <td>50</td>
 <td>4/4</td>
 <td>3.25 (3-4)</td>
 <td>8.25 (7-10)</td>
 </tr>
 <tr>
 <td>25</td>
 <td>4/4</td>
 <td>3.0 (3.0)</td>
 <td>8.25 (8-9)</td>
 </tr>
 <tr>
 <td>10</td>
 <td>4/4</td>
 <td>3.0 (3.0)</td>
 <td>8.76 (8-9)</td>
 </tr>
 <tr>
 <td>5</td>
 <td>4/4</td>
 <td>2.5 (2-3)</td>
 <td>9.75 (9-10)</td>
 </tr>
 <tr>
 <td>1</td>
 <td>2/4</td>
 <td>1.0 (1.0)</td>
 <td>16 (14-18)</td>
 </tr>
 <tr>
 <td>0</td>
 <td>0/4</td>
 <td>- -</td>
 <td>- -</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table III. Challenge antigen doses required for disease induction. Various concentrations of MBP used to challenge recipient mice were tested. Av., average.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td colspan="3">Clinical EAE after antigenic challenge</td>
 </tr>
 <tr>
 <td>Challenge antigen </td>
 <td>Day of Challenge</td>
 <td>Incidence</td>
 <td>Av. Disease grade 
 (range)</td>
 <td>Av. Day of onset 
 (range)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>5</td>
 <td>3/4</td>
 <td>1.33 (1-2)</td>
 <td>9.67 (9-10)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>10</td>
 <td>4/4</td>
 <td>3.0 (3.0)</td>
 <td>8.25 (7-9)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>15</td>
 <td>4/4</td>
 <td>3.75 (3-4)</td>
 <td>7.25 (7-8)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>25</td>
 <td>4/4</td>
 <td>4.0 (4.0)</td>
 <td>8.0 (7-9)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>35</td>
 <td>4/4</td>
 <td>3.5 (3-4)</td>
 <td>7.5 (7-8)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>60</td>
 <td>4/4</td>
 <td>3.75 (3-4)</td>
 <td>9.0 (8-10)</td>
 </tr>
 <tr>
 <td colspan="5">- - - - - - - - - - - - - - - - - - - - - - - - - -</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>343</td>
 <td>3/3</td>
 <td>3.0 (3.0)</td>
 <td>10.33 (10-11)</td>
 </tr>
 <tr>
 <td>MBP-CFA</td>
 <td>445</td>
 <td>3/3</td>
 <td>3.0 (3.0)</td>
 <td>7.0 (7.0)</td>
 </tr>
 </tbody>
</table>
Table IV. Kinetics of antigenic challenge. Recipient mice were challenged at various time point post adoptive cell transfer. Av., average.

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No financial disclosures.

Studies of EAE in mice often utilize the neuroantigens, myelin basic protein (MBP), proteolipid protein (PLP) or myelin oligodendrocyte protein (MOG). Earlier studies mostly used MBP. PLP stimulates strong and consistent responses from SJL mice. More recently, MOG is the common neuroantigen used because B6 mice are susceptible to disease induction with this antigen. Many of the gene targeted mouse strains are on the B6 genetic background. Interestingly, B6 mice are susceptible to EAE induction with MOG but are resis...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Guinea pig spinal cords</td>
 <td>Rockland Immuno-chemicals, Inc. 
 Gilbertsville, PA 19525 
 <a href="http://www.rockland-inc.com" target="_blank">www.rockland-inc.com</a></td>
 <td>GP-T065</td>
 <td> frozen</td>
 </tr>
 <tr>
 <td>Freund's Adjuvant, complete H37Ra </td>
 <td>Difco Laboratories 
 Detroit, MI</td>
 <td> 231131 </td>
 <td></td>
 </tr>
 <tr>
 <td>Multifit interchange-able syringe </td>
 <td> B-D 
 <a href="http://www.bd.com/us" target="_blank">www.bd.com/us</a></td>
 <td>512133</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Mediatech Inc. 
 Manassas, VA 
 <a href="http://www.cellgro.com" target="_blank">www.cellgro.com</a></td>
 <td>10-040-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>OVA</td>
 <td>Sigma-Aldrich 
 <a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a></td>
 <td>A5503</td>
 <td></td>
 </tr>
 <tr>
 <td>Mice</td>
 <td>Jackson Laboratory 
 Bar Harbor, Maine</td>
 <td>B6 mice: 0000664</td>
 <td></td>
 </tr>
</tbody>
 </table>

overcoming unresponsiveness in experimental autoimmune encephalomyelitis (eae) resistant mouse strains by adoptive transfer and antigenic challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Watch this Scientific Journal Video about Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge at JoVE.com

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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Research

JoVE Journal

Immunology and Infection

20.2K ビュー.  St. John-Providence Health System. 特定のマウス系統では、ミエリン塩基性タンパク質で実験的自己免疫性脳脊髄炎（EAE）の誘導に抵抗することができます。ここで説明するには、無反応を逆にし、いくつかの典型的なEAE耐性マウスの汚れに麻痺性疾患を引き起こす単純な予防接種プロトコルです。

ビデオ: Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Immunogenicity Enhancement by &lt;em&gt;Mycobacterium paratuberculosis&lt;/em&gt; in Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Author Spotlight: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...