Figure legend
Figure1.
PMN-MDSCs and B cells expanded abnormally in CIA models.
Joint and spleen cells were stained for CD11b, Ly6G, and Ly6C 7 weeks
after the induction of CIA. Naïve DBA/1J mice not treated with
CFA-collagen were used as controls. (A, B ) Representative
contour plots (A ) and percentages (B ) of PMN-MDSCs
(CD11b+ Ly6G+Ly6C-) in control (n=4) and CIA (n=4) mice. (C ) Correlation between
the frequency of PMN-MDSCs in the Joints and the clinical score (n=15)
of the corresponding joints. (Spearman’s r analysis.) (D )
Comparison of plasma IgG concentrations between control (n=4) and CIA
(n=4) groups. (E, F ) The percentage and counts of
TNF-𝛂+ B and Ki67+ B cells in the
joints (E ) and spleens (F ) of control and CIA group.
*, P < 0.05; **, P < 0.01; ***,P < 0.001; ****, P < 0.0001; ns, not
significant
Figure
2. PMN-MDSCs depletion alleviated the severity of CIA.
(A, B )Control and Ly6G Ab group were CIA mice treated with
isotype IgG(n=4) or Ly6G antibody (n=3) i.p. every other day
(highlighted by arrows). The clinical arthritis score of joints
(A ) and the representative image of joints and HE (B )
between Control and Ly6G Ab group.
Scale
bar,
10𝛍m.
(Two-way
ANOVA with Bonferroni’s.) (C ) The plasma IgG and TNF-𝛂 were
assayed by ELISA in control (n=3) and Ly6G Ab (n=3) group.
(D-H ) Flow-cytometry analysis of frequency and absolute number
of TNF-𝛂 and Ki67 in CD19+ B cells from the joints and
spleens. *, P < 0.05; **, P < 0.01; ns,
not significant.
Figure
3. PMN-MDSCs administration enhanced disease severity.
Control (n=4) and PMN-MDSC transfer groups (n=4) were treated with PBS
and PMN-MDSCs (5*10^6/mouse) respectively (highlighted by arrows).
(A ) The clinical arthritis score of CIA mice increased after
adoptive transfer of PMN-MDSCs. (Two-way ANOVA with Bonferroni’s
correction). (B ) Representative joints and HE staining of the
control and PMN-MDSC transfer groups. Scale bar, 10𝛍m. (C ) The
IgG concentration in the plasma increased after PMN-MDSCs transfer.
(D-H ) The representative flow chart (D ) depicting the
frequency and count of TNF-𝛂+ B cells and
Ki67+ B cells from the joints and spleens between
control and PMN-MDSCs transfer group. *, P < 0.05; **,P < 0.01; ***, P < 0.001; ****,P < 0.0001; ns, not significant.
Figure
4. PMN-MDSCs facilitated TNF-𝛂 expression of B cells and proliferation,
and hindered apoptosis in vitro.
(A, B ) The expression of TNF-𝛂 (A ) and
Ki67(B ) in CD19+ B cells upon coculture with
PMN-MDSCs (1:1) from CIA spleen. (C, D ) The concentrations of
IgG (C ) and TNF-𝛂 (D ) in the supernatants were assayed
by ELISA. (E, F ) The percentage of TNF-𝛂+(E ) and Ki67+ (F ) in B cells when
cocultured with PMN-MDSC from the CIA spleen at different ratio.
(G-H ) The apoptosis of CD19+ B cells was
assessed when coculture with PMN-MDSC (1:1) from spleen of CIA mice.
(I-K ) The analysis of different effects on B cells of PMN-MDSCs
from CIA and DBA/1J mice spleen.
(ANOVA
with Tukey’s post-hoc.) . **, P < 0.01; ***,P < 0.001; ****, P < 0.0001; ns, not
significant.
Figure
5. BAFF derived from PMN-MDSC-CIA mediated the enhancement of B cells’
function.
(A ) PMN-MDSCs isolated from the spleens of control DBA/1J and
CIA mice were assayed by RNA-seq. The differentially expressed genes are
shown in the heatmap, indicating that Tnfsf13b (also known asBaff ) was up-regulated in the CIA group. (B ) The mRNA
expression of Baff increased in PMN-MDSCs from CIA mice (n=7)
compared to control DBA/1J mice (n=7). (C ) anti-BAFF antibody
decreased the concentration of TNF-𝛂 not IgG in the supernatant. (ANOVA
with Tukey’s post-hoc.) (D ) The flow chart depicting TNF-𝛂 and
Ki67 expression in B cell when coculture with PMN-MDSCs from CIA mice
with or without anti-BAFF antibody. (E ) The PMN-MDSC mediated
enhancement of TNF-𝛂 and Ki67 in B cells decreased in the presence of
anti-BAFF antibody.
(ANOVA
with Tukey’s post-hoc.) (F-G ) The PMN-MDSCs reduced B cells’
apoptosis, and anti-BAFF antibody counteracted PMN-MDSCs’ effect on B
cells. The representative flow chart and statistical analysis were
shown. (ANOVA with Tukey’s post-hoc.). *, P < 0.05; **,P < 0.01; ***, P < 0.001; ns, not
significant.
Figure
6. BAFF promoted TNF-𝛂 secretion of B cell through BTK/NF-𝛋B signaling
pathway.
(A ) BAFF promoted the TNF-𝛂 expression in B cells. (B )
The phosphorylation level of BTK, I𝛋B𝛂, p65 in B cells were shown in 5,
30 and 60 min after stimulation with BAFF. (C ) Ibrutinib
inhibited the effects of BAFF on TNF-𝛂+ B cells in a
dose dependent way. (ANOVA with Tukey’s post-hoc.) (D ) The
phosphorylation of BTK, I𝛋B𝛂, p65 in B cells were reduced by Ibrutinib.
*, P < 0.05; **, P < 0.01; ***,P < 0.001; ns, not significant.
Figure
S1 . The frequency of M-MDSC in the joint (A ) and spleen
(B ) (gated in CD11b+ cells) in control
(DBA/1J mice) and CIA group. ns, not significant.
Figure S2 . (A-B ) The frequency of PMN-MDSC decreased
after administrating Ly6G Ab in the joint and spleen (gated in
CD11b+ cells). The relative expression of TNF-𝛂in SP
(C )and joint (D) in control and Ly6G Ab groups. *,P < 0.05. ***, P < 0.001.