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.
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 μg/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 μg/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.
1. Preparing CFA-emulsified Antigen
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.
3. Isolation of Lymph Node Cells for Tissue Culture 7 to 10 Days after Immunization
4. Harvesting Cultured Lymph Node Cells 5 Days after Initiation of Cultures
5. Antigenic Challenge of Recipient Mice 20 Days Post Cell Transfer and Development of Disease Symptoms
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.
Figure 1. Flow chart of the experimental design for induction of EAE in reputed resistant mouse strains.
Clinical EAE after adoptive transfer |
Clinical EAE after antigenic challenge |
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Strain | Antigen | incidence | Av. disease grade (range) |
Av. day of onset (range) |
Incidence | Av. disease grade (range) |
Av. day of onset (range) |
SJL | MBP | 21/21 | 4.0 (3-5) | 8.0 (6-10) | N.D. | N.D. | N.D. |
C57BL/6 | MBP | 0/10 | – – | – – | 7/7 | 3.85 (3-5) | 9.28 (9-10) |
Balb/c | MBP | 0/7 | – – | – – | 5/5 | 3.2 (3-4) | 7.0 (7) |
C3H/HeJ | MBP | 0/7 | – – | – – | 4/4 | 3.2 (3-4) | 8.0 (8) |
C57BL/6 | MBP60-80 | 0/4 | – – | – – | 4/4 | 2.75 (2-4) | 10.2 (9-11) |
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.
Clinical EAE after antigenic challenge | ||||
Priming Antigen | Challenge antigen | Incidence | Av. disease grade (range) |
Av. day of onset (range) |
MBP-CFA | CFA alone | 0/3 | – – | – – |
MBP-CFA | OVA-CFA | 0/3 | – – | – – |
MBP-CFA | MBP-CFA | 3/3 | 4.0 (4.0) | 7.7 (7-9) |
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.
Clinical EAE after antigenic challenge | ||||
Challenge antigen doses (μg/mouse) | Incidence | Av. disease grade (range) |
Av. day of onset (range) |
|
200 | 4/4 | 3.75 (3-4) | 8.25 (7-9) | |
100 | 4/4 | 3.67 (3-4) | 7.5 (7-8) | |
50 | 4/4 | 3.25 (3-4) | 8.25 (7-10) | |
25 | 4/4 | 3.0 (3.0) | 8.25 (8-9) | |
10 | 4/4 | 3.0 (3.0) | 8.76 (8-9) | |
5 | 4/4 | 2.5 (2-3) | 9.75 (9-10) | |
1 | 2/4 | 1.0 (1.0) | 16 (14-18) | |
0 | 0/4 | – – | – – | |
Table III. Challenge antigen doses required for disease induction. Various concentrations of MBP used to challenge recipient mice were tested. Av., average.
Clinical EAE after antigenic challenge | ||||
Challenge antigen | Day of Challenge | Incidence | Av. Disease grade (range) |
Av. Day of onset (range) |
MBP-CFA | 5 | 3/4 | 1.33 (1-2) | 9.67 (9-10) |
MBP-CFA | 10 | 4/4 | 3.0 (3.0) | 8.25 (7-9) |
MBP-CFA | 15 | 4/4 | 3.75 (3-4) | 7.25 (7-8) |
MBP-CFA | 25 | 4/4 | 4.0 (4.0) | 8.0 (7-9) |
MBP-CFA | 35 | 4/4 | 3.5 (3-4) | 7.5 (7-8) |
MBP-CFA | 60 | 4/4 | 3.75 (3-4) | 9.0 (8-10) |
– – – – – – – – – – – – – – – – – – – – – – – – – – | ||||
MBP-CFA | 343 | 3/3 | 3.0 (3.0) | 10.33 (10-11) |
MBP-CFA | 445 | 3/3 | 3.0 (3.0) | 7.0 (7.0) |
Table IV. Kinetics of antigenic challenge. Recipient mice were challenged at various time point post adoptive cell transfer. Av., average.
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 resistant to disease induction with MBP.
Much of the previous effort in elucidating the mechanisms of EAE centered on the induction mechanisms of disease, the effects of cytokine production and the recovery mediated by regulatory T cells5-7. How reputed EAE resistant mice can subvert the induction of disease, on the other hand, had not been widely investigated. This may be due to the difficulty of quantifying unresponsiveness. Arnon8 in 1981 first showed that treating B6 mice with cyclophosphamide rendered these mice susceptible to EAE induced with MBP. Others showed that treatment with anti-gamma interferon also successfully induced disease in B6 and Balb/c mice9,10. It should be noted that these studies all targeted non-antigen specific markers. On the other hand, the protocol described here focuses on a different aspect of EAE resistance. Here only specific antigen used in the challenge can overcome EAE unresponsiveness and allows induction of disease. This protocol encompasses both the EAE resistance phase with adoptive transfer of primed donor T cells and the susceptibility phase with antigenic challenge (Table I). Experiments can be designed to study the intrinsic factors that maintain EAE resistance as well as factors that reverse this unresponsiveness. Initially, it was shown that EAE resistant mice were able to mount autoimmune responses when immunized with MBP4. Later, it was shown that encephalitogenic T cells existed in these mice2. Some recent studies11,12 showed that treatment of B10.S mice with anti-CD25 antibodies prior to immunization of the mice to proteolipid protein (PLP) converted the mice from resistant to susceptible, implying the role of regulatory T cells (Tregs) in maintaining EAE resistance. However, this is not the case when C57BL/6 mice are similarly treated and immunized with MBP13. Most of the mice remain unresponsive. Thus, EAE resistance may be multi-factorial. It is postulated that, in the case of C57BL/6 mice responding to MBP, thymic selection has deleted most MBP-reactive T cells such that the frequencies of MBP-reactive T cells in the periphery are very low. This below-threshold low frequency would render the mice not able to respond to immunization with MBP. The challenge dose, through unknown mechanisms, is able to overcome the frequency issue and mount an autoimmune response. Further investigations of the possible mechanisms would include the role of IL-612 during antigenic challenge in affecting the susceptibility of encephalitogenic to inhibition by Tregs.
The authors have nothing to disclose.
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).
Name of the reagent | Company | Catalogue number | Comments |
Guinea pig spinal cords | Rockland Immuno-chemicals, Inc. Gilbertsville, PA 19525 www.rockland-inc.com |
GP-T065 | frozen |
Freund’s Adjuvant, complete H37Ra | Difco Laboratories Detroit, MI |
231131 | |
Multifit interchange-able syringe | B-D www.bd.com/us |
512133 | |
RPMI 1640 | Mediatech Inc. Manassas, VA www.cellgro.com |
10-040-CM | |
OVA | Sigma-Aldrich www.sigmaaldrich.com |
A5503 | |
Mice | Jackson Laboratory Bar Harbor, Maine |
B6 mice: 0000664 |