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. 2015 Sep 17:12:169.
doi: 10.1186/s12974-015-0378-5.

Immune profile of an atypical EAE model in marmoset monkeys immunized with recombinant human myelin oligodendrocyte glycoprotein in incomplete Freund's adjuvant

S Anwar Jagessar  1   2 , Nicole Heijmans  3 , Erwin L A Blezer  4 , Jan Bauer  5 , Robert Weissert  6 , Bert A 't Hart  7   8   9
Affiliations

Immune profile of an atypical EAE model in marmoset monkeys immunized with recombinant human myelin oligodendrocyte glycoprotein in incomplete Freund's adjuvant

S Anwar Jagessar et al. J Neuroinflammation. .

Abstract

Background: Experimental autoimmune encephalomyelitis (EAE) in the common marmoset monkey (Callithrix jacchus) is a relevant preclinical model for translational research into immunopathogenic mechanisms operating in multiple sclerosis (MS). Prior studies showed a core pathogenic role of T and B cells specific for myelin oligodendrocyte glycoprotein (MOG). However, in those studies, the quality of the response against MOG epitopes was strongly biased by bacterial antigens in the complete Freund's adjuvant (CFA), in which the immunizing recombinant human (rh) MOG protein had been formulated. In response to the need of a more refined EAE model, we have tested whether disease could also be induced with rhMOG in incomplete Freund's adjuvant (IFA).

Method: Marmosets were immunized with rhMOG emulsified in IFA in the dorsal skin. Monkeys that did not develop neurological deficit were given booster immunizations at 28-day interval with the same antigen preparation. In a second experiment, three marmoset twin pairs were sensitized against MOG peptides in IFA to study a possibility for suppressive activity towards pathogenic T cells directed against the encephalitogenic epitope MOG40-48.

Results: Despite the absence of strong danger signals in the rhMOG/IFA inoculum, all monkeys developed clinically evident EAE symptoms. Moreover, in all monkeys, demyelinated lesions were present in the white matter and in two cases also in the cortical grey matter. Immune profiling at height of the disease showed a dominant T cell response against the overlapping peptides 14-36 and 24-46, but reactivity against the pathogenically most relevant peptide 34-56 was conspicuously absent. In the second experiment, there was an indication for a possible suppressive mechanism.

Conclusions: Immunization of marmoset monkeys with rhMOG in IFA elicits clinical EAE in all animals. Moreover, rhMOG contains pathogenic and regulatory epitopes, but the pathogenic hierarchy of rhMOG epitopes is strongly influenced by the adjuvant in which the protein is formulated.

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Figures

Fig. 1
Fig. 1
rhMOG in IFA induces clinical EAE in marmoset monkeys. Six unrelated healthy marmosets were immunized with rhMOG in IFA (post-sensitization day 0). Monkeys not developing clinically evident EAE within 28 days were given one or two booster immunizations with the same antigen-adjuvant emulsion (arrowheads). Clinical scores and body weights are shown for all marmosets. Clinical scores are depicted on the left y-axis (solid line) and the percentage body weight loss compared with day 0 on the right y-axis (dotted line)
Fig. 2
Fig. 2
MRI images. MRI scans were made of formalin fixed brains. A representative slide of T2-weighted (T2W) and white matter attenuated inversion recovery (WAIR) images are depicted. In addition, a 3D reconstruction was made of all T2W images. The left two columns display typical slices through the brains on which lesions in the white (red arrowheads) and grey (blue arrowheads) matter lesions can be discerned. The 3D volume shows the total outlined volume of white (red) and grey matter (blue) lesions for each animal. Note that grey matter lesions were only observed in M08093 and M09083
Fig. 3
Fig. 3
Plasma antibody levels. Plasma samples were collected longitudinally (a) and at necropsy (b) and tested for the presence of IgM (left column) and IgG (right column) antibodies binding ELISA-plate bound rhMOG or MOG peptides 14–36, 24–46, 34–56 and 54–76. Results are quantitatively expressed as fold increase relative to pre-immune plasma of the same monkeys. Fold increase ≥2.0 is considered positive. PSD post-sensitization day
Fig. 4
Fig. 4
Plasma IgG binding to native MOG. Necropsy plasma samples were tested for IgG binding with healthy marmoset myelin particles. a Gating strategy: myelin particles were plotted at a logarithmic FSC and SCC scale. Middle-sized particles were selected to determine the mean fluorescence intensity (MFI) in the FITC channel. b Marmoset myelin particles were incubated with 2000× diluted necropsy plasma samples, and IgG binding was detected. IgG binding to myelin was expressed as mean fluorescence activity (MFI). c Plasma samples of monkey M09083 were pre-incubated in two dilutions (1:500 and 1:5000) for 1 h at 37 °C with a dose titration of rhMOG to block anti-MOG IgG molecules. The pre-incubated plasma samples were subsequently tested for residual IgG reactivity with marmoset myelin particles. All data were corrected with background staining with the secondary only
Fig. 5
Fig. 5
MNC proliferation against MOG antigens. a MNC were isolated at 2-week interval from venous blood and tested for proliferation against a panel of 23-mer overlapping MOG peptides. The positive proliferation data from individual monkeys, i.e. against MOG14-36, 24–46 and 34–56, are shown. b At necropsy, MNC were prepared from blood (PBMC), spleen, axillary (ALN), inguinal (ILN), lumbar (LLN) and cervical (CLN) lymph nodes and were tested for their proliferation against MOG peptides and rhMOG. Data of the six monkeys are presented as mean ± SEM. Data are expressed as stimulation index (SI) relative to unstimulated cell cultures. SI values ≥2.0 (dotted line) are considered positive. PSD post-sensitization day
Fig. 6
Fig. 6
Cytokine levels in culture supernatants. MNC from the blood, spleen or ALN were cultured ex vivo with rhMOG under conditions as explained in Figure 5. After 48-h culture, supernatants were collected. Levels of IL-17A, TNF-α and IFN-γ were measured with ELISA. Data is expressed as pg/ml
Fig. 7
Fig. 7
Biomarker mRNA expression levels in blood and lymphoid organs. mRNA was extracted from PBMC, spleen or ALN that were collected at necropsy. Specificity and levels of mRNA was determined with qPCR. Expression of mRNA for IL-17A, TNF-α, CD3, IL-10, IL-7 and IFN-γ was tested in all three compartments. The other markers (IL-6, CD19, CD28, IL-1β, CCR7, Il-2, TGF-β, CCR4, CCR6 and CD14) were only tested in the spleen and ALN. Data is expressed as relative to the household gene ABL
Fig. 8
Fig. 8
Clinical response to immunization with linked or unlinked epitopes in IFA. One sibling of each twin was immunized on days 0 and 28 with peptide MOG20-50 (left column); the other sibling was immunized on the same days with a mixture of MOG14-36 and MOG34-56 (right column). All monkeys not displaying EAE symptoms were immunized on days 56 and 84 with the encephalitogenic peptide MOG34-56. All immunizations were with peptides emulsified in IFA. Clinical scores are depicted on the left y-axis (solid line) and the percentage body weight loss compared with day 0 on the right y-axis (dotted line)
Fig. 9
Fig. 9
Plasma antibody levels in the linked or non-linked epitope group. Immune plasma samples were collected at day 0 and every 14 days thereafter. All plasmas were tested for the presence of IgM and IgG binding to the indicated antigens coated on ELISA plates. Results of day 70 were excluded because of technical failure. Data is expressed as fold increase relative to pre-immune plasma
Fig. 10
Fig. 10
Linked suppression paradigm for the absent activation of MOG34-56-reactive T cells. We postulate that the marmoset’s immune repertoire contains pre-sensitized autoreactive T cells against two dominant MOG epitopes, i.e. the CD4 cells/Th1 epitope 24–36 (blue triangle) and the CTL epitope 40–48 (red circle), which are juxta-positioned in an evolutionary conserved region in the extracellular domain, residues 20–50. In monkeys immunized with the MOG20-50 peptide, both epitopes are processed and presented simultaneously on the APC surface. Conceptually, this implies that reactivation of CTL cells by the 40–48 epitope (red repertoire) may be blunted by Tr1 cells re-activated at the same APC by the MOG24-36 epitope (blue repertoire)
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