Antibody
Clone
Provider*
CD1d
LY38
1
CD3
SP34-2
1
CD4
L200
1
CD5
UCHT2
3
CD8
LT8
4
CD9
ML13
1
CD11b
D12
1
CD11c
S-HCL-3
1
CD16
3G8
1
CD20
H299
5
CD21
HB5
3
CD23
ML233
1
CD25
2A3
1
CD27
MT271
1
CD39
eBioA1
3
CD40
B-B20
6
CD45RA
5H9
1
CD56
Ncam 16-2
1
CD80
L307.4
1
CD83
HB15a
5
CD86
IT2.2
1
CD95
DX2
1
CD127
eBioRDR5
3
CD355
BAB281
5
CD278
isa3
3
CD279
J105
3
CCR4
205410
7
CCR7
150503
7
Foxp3
PCH101
3
HLA–DR
L243
1
Another important immunological marker in an EAE experiment is the humoral activity. Therefore, the antibody profile is usually determined by ELISA (enzyme linked immuno-sorbent assay), a biochemical assay to determine whether a certain antigen is present in a sample. The method that we use to determine marmoset antibody levels is highly similar to other protocols, although some small modifications were made to test antibody responses against as much antigens as possible. Plasma obtained from repeated blood draws during the study gives us the opportunity to monitor longitudinal antibody responses.
Since several methods have already been mentioned to profile the marmoset immune response ex vivo, we would also like to draw attention to a more peptide-specific cellular experiment, such as a T cell cytotoxicity assay. For such assays T cell lines are required which are specific for a single antigen and that can be cultured for a longer period of time and to greater numbers. After the first round of antigen exposure and outgrowth of T cells, the lines need to be restimulated with antigen presenting cells (APC) in the presence of peptide to facilitate proper stimulation of the T cells. In this protocol autologous B cell lines immortalized with EBV are used as APC. Depending on the immunization protocol T cell lines can be generated against several MOG peptides, for example against MOG34-56.
Immortalized marmoset B cell lines are generated by infecting PBMC with EBV supernatant of the B95-8 strain. The B95-8 strain is derived from humans infected with EBV, who suffered from infectious mononucleosis [9]. This has been kept for several decades in cotton-top tamarins cells, also a New World monkey, and thereafter in marmoset cells. Infection with EBV immortalizes B cells, so they can be kept in culture for extended periods of time without the need for any other stimulation. These B cells can then be used as antigen presenting cells in various immunological assays, such as cytotoxicity assays, or to restimulate peptide-specific T cell lines.
To determine the cytotoxicity of peptide-specific T cell lines acquired from marmosets with EAE, a classic cytotoxicity assay is used, where immortalized B cells labeled with radioactive 51-chromium and MOG peptide serve as target cells for increasing amounts of T cells. Each ratio of T cells is tested in quadruplicate to correct for variation in the measurements. Next to each incubation with peptide-labeled B cell sample with T cells, also B cells incubated without T cells have to be tested to determine the spontaneous release of 51-chromium and in the presence of 2 % Triton X-100 to determine the maximum release of 51-chromium. These measurements are then used to determine the percentage of B cells killed by the antigen-specific T cell lines.
2 Materials
Prepare all mixtures in a sterile laminar flow hood at room temperature, and use only sterile equipment and disposables. All the steps involving physical handling of the marmosets, e.g., sedation and immunization, should be executed by personnel trained according to local animal ethic guidelines. All personnel working with radioactive material should have proper training and employ the necessary safety procedures when working with 3[H]-thymidine or 51-Chromium. Contact the radiation safety officer for more information if unsure how to proceed.
2.1 EAE Induction Components
1.
10 ml glass vials.
2.
Small stir bar (±1 cm).
3.
Magnetic stirrer.
4.
Buffered phosphate saline (PBS).
5.
MOG34-56 peptide.
6.
rhMOG (purified from E. coli).
7.
IFA or CFA.
8.
1 ml syringe.
9.
26 G × ½″ needle.
2.2 Isolation of Mononuclear Cells from Peripheral Blood and Lymphoid Tissue Components
1.
Ethylenediaminetetraacetic (EDTA) or heparin coated blood collection tubes.
2.
Culture media: RPMI supplemented with 10 % fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μM Glutamax, 50 μM MEM Non-Essential amino acid solution, and 20 μM β-mercaptoethanol.
3.
Cryotubes 2 ml.
4.
Lymphoprep (AXIS-SHIELD, Oslo, Norway).
5.
Leucosep tubes 12 ml (Greiner bio-one, Frickenhausen, Germany).
6.
5 ml syringe.
7.
70 μm cell strainer.
2.3 T Cell Proliferation by Thymidine Incorporation Components
2.
Culture media: RPMI supplemented with 10 % fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μM Glutamax, 50 μM MEM Non-Essential amino acid solution, and 20 μM β-mercaptoethanol.
3.
MOG peptide.
4.
Concanavalin A (ConA).
5.
Ovalbumin (OVA) or any other irrelevant protein or peptide.
6.
96-well plates U-bottom.
7.
3[H]-thymidine, 50× diluted in culture media.
8.
UniFilter-96 GF/C plate (Perkin Elmer, Waltman, USA).
9.
Cell harvester for 96-well plates.
10.
MicroScint-E (Perkin Elmer, Waltman, USA).
11.
TopSeal-A 96 classic, seal for 96-well plate (Perkin Elmer, Waltman, USA).
12.
Beta-counter.
2.4 MNC Phenotyping by Flow Cytometer Components
1.
96-well plates V-bottom.
2.
1 %BSA/PBS.
4.
1 % paraformaldehyde or BD Cytofix Fixation buffer.
5.
Flow cytometer.
2.5 Antibody Measurement by ELISA Components
1.
96-well ELISA plates F-shape.
2.
Bovine serum albumin (BSA).
3.
Tween-20.
4.
3 M NaOH.
5.
Antigen, for example MOG peptides.
6.
Plasma samples.
7.
Secondary antibodies: goat-anti-monkey IgM—alkaline phosphatase (AP) (Rockland Immunochemicals, PA, USA), rabbit-anti-human IgG—AP (Abcam, Cambridge, UK).
8.
Substrate SIGMA Fast™ p-Nitrophenyl phosphate tablets.
9.
Wash buffer: PBS containing 0.05 % Tween-20.
10.
Blocking buffer: PBS containing 2 % BSA.
11.
Dilution buffer: PBS containing 1 % BSA.
12.
ELISA plate reader.
2.6 Generation of Peptide-Specific T Cell Lines Components
3.
MOG peptides.
4.
24-well cell culture plate.
5.
Interleukin 2.
6.
Gamma or X-ray radiation cell irradiator.
7.
Culture medium: RPMI supplemented with 10 % fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μM Glutamax, 50 μM MEM Non-Essential amino acid solution, and 20 μM β-mercaptoethanol.
8.
IL2 starter medium: culture medium supplemented with 40 U IL2/ml.
9.
T cell medium: culture medium supplemented with 20 U IL2/ml.
2.7 Generating B Cell Lines Components
1.
Blood derived lymphocytes.
2.
EBV supernatant strain B95-8.
3.
Culture media: RPMI supplemented with 10 % fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 μM Glutamax.
4.
Phytohemagglutinin (PHA-M).
5.
24-well cell culture plate.
2.8 Assaying Cytotoxic T Cells Components
3.
MOG peptides.
4.
Culture medium: RPMI supplemented with 10 % fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μM Glutamax, 50 μM MEM Non-Essential amino acid solution, and 20 μM β-mercaptoethanol.
5.
Chromium-51.
6.
96-well U-bottom plates with lid (Perkin Elmer, Waltham, USA).
7.
Triton-X100, 2 % solution in culture medium.
8.
Gamma radiation counter.
3 Methods
3.1 EAE Induction
1.
Thaw rhMOG or MOG peptide and once thawed, keep it on ice.
2.
Dilute the MOG antigen to a final concentration of 1 mg/ml in PBS.
3.
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Each animal has to be immunized with 100 μg MOG antigen in a final volume of 400 μl. The immunization emulsion consists of a 1:1 mixture of antigen in PBS and adjuvant (CFA or IFA) (see Note 2 ).
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