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SVAVGSQFPFRYNYTI
ED1
ED2
Figure S1. Putative Structure of MS4a4B. MS4a4B is a transmembrane
protein containing 226 amino acid residues. The membrane-spanning
domains (TM) are predicted by TMHMM 2.0 program. The predicted
phosphorylation sites in the cytoplasmic regions are indicated by red dots
(Thr7, Ser26 and Tyr218). The first and second extracellular domains (ED)
are indicated as ED1 and ED2 respectively. The amino acid sequence (ED2)
selected for preparation of antibody is shown.
Clinical score
4
Ig control
Anti-MS4a4B
3
2
1
p<0.001 (N=7)
B
Clinical score
A
3
Ig control
Anti-MS4a4B
2
1
p<0.01 (N=6)
0
0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Days post immunization
Days post immunization
Figure S2. Anti-MS4a4B treatment ameliorates EAE. EAE in C57BL/6
mice were induced with MOG/CFA protocol. a. Mice were then treated
with anti-MS4a4B antibodies (or Ig as control)(1 mg/injection) on days 3,
6, 9 and 12 p.i. b. Mice were treated with anti-MS4a4B (or Ig control) on
day 10 (2 mg/mouse), day 13 (1 mg/mouse) and day 16 (1 mg/mouse)
p.i. The data are presented as Mean ± SE of clinical scores. Statistical
significance was determined by two-way ANOVA test. The representative
of two independently repeated experiments is shown.
Proliferation index
3.5
Ig
Anti-MS4a4B
3
*
2.5
2
1.5
1
0.5
0
0
5
10
20
Concentration of MOG (g/ml)
Figure S3. Anti-MS4a4B treatment decreases MOG-induced T cell
proliferation. Spleen cells from anti-MS4a4B-treated or control Ig-treated
EAE mice described in Figure 6 were cultured at 2 x 105/well in 96 well
plates and stimulated with MOG peptide. T cell proliferation was determined
by 3H-TdR incorporation as described in “Method”. The results are shown as
mean ± SD of triplicate proliferation index (Stimulated CPM/ Unstimulated
CPM. Statistical significance was determined by unpaired T-test. *p < 0.05.
IL-5 (pg/ml)
150
125
NS
Ig-treated
Anti-MS4a4B
100
75
50
25
0
MOG
IFN- (pg/ml)
2000
*
1500
Medium
Ig-treated
Anti-MS4a4B
1000
500
0
MOG
IL-17 (pg/ml)
3500
3000
*
Medium
Ig-treated
Anti-MS4a4B
2500
2000
1500
1000
500
0
MOG
Medium
Figure S4: Anti-MS4a4B treatment decreases MOG-induced IFN- and IL17 production. Spleen cells from anti-MS4a4B-treated or control-Ig treated
EAE mice described in Figure 6 were cultured at 1 x 106/ml in 24 well plates
and restimulated with 10 g/ml MOG peptide or without stimulation as control.
Supernatants were harvested from culture on day 3 of stimulation. Cytokine
levels in supernatants were measured by ELISA. The results are shown as
mean ± SD of duplicate. Statistical significance was determined by unpaired Ttest. NS: not significant; *p < 0.05.