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. 2012 Sep;7(3):557-70.
doi: 10.1007/s11481-012-9384-x. Epub 2012 Jun 30.

Antibodies against human BLyS and APRIL attenuate EAE development in marmoset monkeys

S Anwar Jagessar  1 , Nicole Heijmans, Luke Oh, Jan Bauer, Erwin L A Blezer, Jon D Laman, Thi-Sau Migone, Matt N Devalaraja, Bert A 't Hart
Affiliations

Antibodies against human BLyS and APRIL attenuate EAE development in marmoset monkeys

S Anwar Jagessar et al. J Neuroimmune Pharmacol. 2012 Sep.

Erratum in

  • J Neuroimmune Pharmacol. 2013 Mar;8(1):370. Oh, Luke [added]

Abstract

B lymphocyte stimulator (BLyS, also indicated as BAFF (B-cell activating factor) and CD257), and A Proliferation Inducing Ligand (APRIL, CD256) are two members of the TNF superfamily with a central role in B cell survival. Antibodies against these factors have potential therapeutic relevance in autoimmune inflammatory disorders with a proven pathogenic contribution of B cells, such as multiple sclerosis (MS). In the current study we performed a multi-parameter efficacy comparison of monoclonal antibodies against human anti-BLyS and anti-APRIL in a common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE). A MS-like disease was induced by immunization with recombinant human myelin/oligodendrocyte glycoprotein (rhMOG) in complete Freund's adjuvant. The results show that the anti-BLyS and anti-APRIL antibody cause significant depletion of circulating CD20+ B cells, but a small subset of CD20 + CD40(high) B cells was not depleted. Induction of CD20+ B cell depletion from lymph nodes was only observed in the anti-BLyS treated monkeys. Both antibodies had a significant inhibitory effect on disease development, but all monkeys developed clinically evident EAE. Anti-BLyS treated monkeys were sacrificed with the same clinical signs as saline-treated monkeys, but nevertheless displayed significantly reduced spinal cord demyelination. This effect was not observed in the anti-APRIL treated monkeys. The two antibodies had a different effect on T cell subset activation and the profiles of ex vivo released cytokines. In conclusion, treatment with anti-BLyS and anti-APRIL delays the development of neurological disease in a relevant preclinical model of MS. The two mAbs achieve this effect via different mechanisms.

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Figures

Fig. 1
Fig. 1
B cell depletion by anti-BLyS and anti-APRIL antibodies. a. CD20+ and CD40+ expression by PBMC was analysed by flow cytometry. Based on CD40 expression two populations could be defined, namely CD20 + CD40high (13 %, dark shaded) and CD20 + CD40 + low (7 %, light shaded). Depicted data are from monkey M02061 as a representative example. b. CD20+ (left panel) and CD20 + CD40+ (right panel) expression was measured in PBMC collected at the indicated time points. The CD20 + CD40+ includes the CD20 + CD40 + high and CD20 + CD40 + low population The grey shaded area indicates the period of treatment, which was started at post sensitization day (psd) 21. Percentages on the y-axes are cell numbers expressed relative to the total analysed cell number (mean ± SEM). CD20+ B cells are reduced by the anti-BLyS and anti-APRIL treatment compared to untreated monkeys, but this is not the case for CD20 + CD40+ mature B cells. c. MNC were prepared from blood, spleen, ALN, ILN and BM at necropsy to measure the presence of CD20+ B cells (left panel) and CD20 + CD40+ mature B cells (right panel). Given percentages on y-axes indicates the fraction of stained cells from the total measured cells (mean ± SEM). In the anti-BLyS treated group a significant decrease of CD20+ B cells was detected in all tested organs except BM. Anti-APRIL treatment had only in blood a significant effect on CD20+ B cells. For the CD20 + CD40+ cells no differences were observed between the control and antibody treated groups. d. At necropsy also the expression levels of CD19 mRNA transcripts were measured in PBMC, spleen and ALN, showing a reduction in both antibody-treated group, although the differences with anti-APRIL were only significant for spleen and ALN. Data was normalized to the household gene ABL (mean ± SEM). *p < 0.05 Mann Whitney U test, treated group vs. control group
Fig. 2
Fig. 2
Anti-BLyS treatment reduced antibody production. Plasma samples collected at two weeks interval and necropsy were tested with ELISA for the presence of IgM (a) and IgG (b) antibodies against the B-cell epitopes MOG24-46 and MOG54-76, and against intact rhMOG. In the anti-BLyS treated group, reduction of IgM and IgG levels were observed during the entire study, but in the anti-APRIL treated groups IgM as well as IgG remained high. Mann Whitney U test has been performed but no significantly differences were observed
Fig. 3
Fig. 3
Delay of EAE course by anti-BLyS and anti-APRIL treatment. a. Clinical scores are depicted of controls (left panel) and the two antibody treated groups, with anti-BlyS (middle panel) or anti-APRIL (right panel). The solid lines represent clinical scores (left y-axes) and the dotted line body weight loss relative to the immunization day, defined as post sensitization day (psd) 0. Grey shaded boxes indicate the treatment period. All animals in the experiment developed clinically evident EAE (clinical score 2.0) and most of them were sacrificed with an EAE score of 3.0. However, monkey Mi016, M0169 and M05049 had to be sacrificed at an earlier time point due to the serious body weight loss. b. Survival curves depict the disease free survival time (time interval to development of EAE score 2.0; left panel) and overall survival (time interval to clinical end point; right panel). Disease free survival times were significantly prolonged in the anti-BLyS and anti-APRIL treated monkeys. The total survival was significantly prolonged by the anti-BLyS treatment, but the delay in anti-APRIL treated monkeys was not significant (p = 0,0646). *p < 0.05; **p < 0.001 Log-rank test, treated group vs. control group
Fig. 4
Fig. 4
Reduced spinal cord demyelination in anti-BLyS treated monkeys. Formalin-fixed tissue samples were stained to analyse the intensity of inflammation and demyelination. For each animal 8 slices were examined, which equals 6 cm2 in total. a. The number of infiltrated cells per mm2 is given for the spinal cord. The amount of demyelination is given for the spinal cord (b), optic nerve (c) and brain white matter (d). A significant treatment effect was only observed for the spinal cord of anti-BLyS treated animals. *p < 0.05 Mann-Whitney U test, antibody treated group vs. control group
Fig. 5
Fig. 5
Modulation of T cell proliferation in anti-BlyS and anti-APRIL treated monkeys. a. PBMC were isolated at the indicated time points (psd; post sensitization day) and assayed for the response to stimulation with rhMOG or 23-mer synthetic peptides containing previously identified dominant MOG T cell epitopes, i.e. MOG14-36, MOG24-46 and MOG34-56. The read-out was incorporation of [3H]-thymidine during the final 18 h of 3 days culture. Data on the left panel show the stimulation index (SI). Grey shaded boxes indicate the period of treatment. T cell proliferation increased after immunization, and was significantly enhanced during the anti-BLyS treatment, but was reduced during anti-APRIL treatment. b. At necropsy the incorporation of [3H]-thymidine was measured as a read-out for proliferation in PBMC, spleen, ALN, ILN and LLN against the same MOG antigens as described under (A). c. Proliferated T cells in blood and lymphoid organs were phenotyped using the CFSE diluting assay. Data are shown of MNC derived from spleen, as a representative example. Stimulation with MOG34-56 and rhMOG are depicted. The y-axes indicates the percentage proliferated cells; CD4+, CD8+ and double positive for CD4 and CD8 were first gated within the CD3+ proliferated population, next for CD4 + CD56+, CD8 + CD56+ and CD4 + CD8 + CD56+, respectively. Data are presented as mean ± SEM. *p < 0.05 Mann Whitney U test, antibody treated group vs. control group
Fig. 6
Fig. 6
Altered cytokine expression with anti-BLyS and anti-APRIL. a. At necropsy MNC were collected from blood and lymphoid organs and cultured for 48 h with a panel of MOG peptides and rhMOG. Levels of IL-17A (left panel), TNF-α (middle panel) and IFN-γ (right panel) were measured in culture supernatants with ELISA (mean ± SEM). Data is only shown of rhMOG stimulated MNC, since no high levels of cytokines were measured in the MOG peptide stimulated cells. b. Cytokine mrRNA transcripts of IL-17A, TNF-α, IFN-γ, IL-7 and IL-10 were measured in PBMC, spleen and ALN. Data was normalized to the household gene ABL (mean ± SEM). Mann Whitney U test was performed as statistical calculation, but no significant differences were observed
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