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.