Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) have been characterized in the context of malignancies. Here we show that PMN-MDSCs can restrain B cell accumulation during central nervous system (CNS) autoimmunity. Ly6G+ cells were recruited to the CNS during experimental autoimmune encephalomyelitis (EAE), interacted with B cells that produced the cytokines GM-CSF and interleukin-6 (IL-6), and acquired properties of PMN-MDSCs in the CNS in a manner dependent on the signal transducer STAT3. Depletion of Ly6G+ cells or dysfunction of Ly6G+ cells through conditional ablation of STAT3 led to the selective accumulation of GM-CSF-producing B cells in the CNS compartment, which in turn promoted an activated microglial phenotype and lack of recovery from EAE. The frequency of CD138+ B cells in the cerebrospinal fluid (CSF) of human subjects with multiple sclerosis was negatively correlated with the frequency of PMN-MDSCs in the CSF. Thus PMN-MDSCs might selectively control the accumulation and cytokine secretion of B cells in the inflamed CNS.
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RNA-seq data have been deposited in the European Nucleotide Archive under accession code PRJEB28339. All data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank all members of the Korn Group and especially V. Husterer for her skillful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB1054-B06 to T.K., TRR128 to T.K., SyNergy to T.K. and Kompetenznetz Multiple Sklerose KKNMS to B.K.), the German Ministry of Education and Research (BMBF, T-B in NMO to T.K.,) and by the ERC (CoG 647215 to T.K.). B.K. received intramural funding from the Technical University of Munich. L.A. is supported by a clinical scientist program provided by the Deutsche Forschungsgemeinschaft (Synergy). D.M. is supported by the Swiss National Science Foundation.
The authors declare no competing interests.
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Integrated supplementary information
a, Correlation of CD15+CD11bhiCD33loLOX1lo myeloid derived suppressor cells (MDSCs) and total CD15+ neutrophils with CD138+HLA-DR+ B cells in CSF of therapy-naive subjects with relapsing-remitting MS (RRMS) or clinically isolated syndrome (CIS); not significant (ns); Spearman’s r analysis. b, Demographic and disease specific features of subjects with RRMS or CIS who served as donors for PBMCs (PBMC cohort, n = 70), CSF (CSF cohort, n = 25) and healthy controls (n = 31); NA, data not available; mean ± s.d. c–e, Individual subject information of the RRMS/CIS subject cohort used for paired analysis of the frequency of PMN-MDSCs in blood samples during relapse and after recovery from relapse in a state of either active disease showing evidence of disease activity either by relapse and/or disease activity in MRI and/or worsening of EDSS score ≥ 1 point (no NEDA-3) (d) or in subjects with no signs of disease activity defined by the absence of relapse, absence of disease activity in MRI and stable EDSS values (no evidence of disease activity, NEDA-3) (e); fingolimod (Fingoli.), ocrelizumab (Ocreli.), natalizumab (Natali.), pegylated interferon β-1a (peg-IGN-β), teriflunomide (Teriflu), glatiramer acetate (GA); Wilcoxon matched-pairs signed rank test, *P < 0.05.
Supplementary Figure 2 Transcriptome (RNA-seq) of CNS-onset Ly6G+ cells and CNS-recovery Ly6G+ cells.
a,b, Waterfall plot of log2 fold change in gene expression comparing CNS-onset Ly6G−tdTomato+ cells versus spleen-onset Ly6G-tdTomato+ cells (a) and CNS-recovery Ly6G-tdTomato+ cells versus spleen-onset Ly6G-tdTomato+ cells (b). The positions of Nos2 (inducible NO synthetase) and Arg1 (arginase 1) are indicated. c,d, Heatmaps of gene expression in Ly6G-tdTomato+ subsets for top three significantly enriched gene ontology terms (GOrilla) in universally downregulated (c) and upregulated (d) genes in CNS-onset Ly6G-tdTomato+ cells versus all other Ly6G-tdTomato+ cell subsets (see Fig. 3a).
Supplementary Figure 3 Depletion efficiency of Ly6G-tdTomato+ cells using a monoclonal antibody to Ly6G.
a, Fraction of SSChi Ly6G-tdTomato+ cells in peripheral blood of naive Ly6gCre/WT mice one day after i.p. application of 400 μg monoclonal antibody to Ly6G (clone 1A8) or 400 μg of a rat-IgG2a control antibody (2A3); gate on peripheral blood cells after erythrocyte lysis. b, Fraction of SSChi Ly6G-tdTomato+ cells in peripheral blood of Ly6gCre/WT mice during EAE; mice were treated with 400 μg anti-Ly6G (Ly6G Ab, n = 5) or 400 μg rat-IgG2a control antibody (rat IgG2a, n = 5) every other day starting on day 5 after immunization; symbols depict mean ± s.d. of biological replicates; two-way ANOVA with Bonferroni’s multiple comparison test; ***P < 0.001. c, Fraction of CD45.2intermedLy6G-tdTomato+ cells purified from brain of Ly6gCre/WT mice at early EAE recovery (day 22) treated with 400 μg monoclonal antibody to Ly6G i.p. (clone 1A8, Bio X Cell) or 400 μg of a rat-IgG2a control antibody (clone 2A3) every other day starting at disease onset; representative plot of ten biological replicates from each group, repeated in three independent experiments; gate on live CD45.2+CD11b+ cells.
Supplementary Figure 4 The T cell and myeloid compartments are unaltered in the absence of Ly6G-tdTomato+ cells during recovery from EAE.
a, Total cell count of CD45+ cells, CD4+ T cells, Ly6G-tdTomato+ cells, CD45hiCD11bhiLy6G− monocytes and CD45dimCD11bhi microglia, purified from the spinal cord of Ly6gCre/WT mice at early EAE recovery (day 21) treated with control antibody (rat IgG2a, clone 2A3, n = 5) or anti-Ly6G (Ly6G Ab, clone 1A8, n = 5) starting on day 12 after immunization; for gating see s. b, Cell counts of immune cell subsets purified from the spinal cord of Ly6gCre/WT mice at early EAE recovery (day 21) treated with 200 μg kg–1 G-CSF (n = 5) or 5% glucose (control, n = 5) i.p. every other day, starting on day 12 after immunization; for gating see d. c, Analysis of cytokine production by intracellular cytokine staining in CD4+ T cells purified from spinal cord of Ly6gCre/WT mice treated with control antibody (rat IgG2a, n = 4) or anti-Ly6G (Ly6G Ab, n = 4) at early EAE recovery (day 23) and stimulated ex vivo with PMA/ionomycin. Symbols depict mean ± s.d. of biological replicates; Mann-Whitney U-test; **P < 0.01 (a–c). d, Gating strategy to identify different cell subsets purified from spinal cord of Ly6gCre/WT mice at early EAE recovery (day 21); representative plot of >30 mice, gate on analyzed single cells.
Supplementary Figure 5 Effect of Ly6G conditional ablation of Il6ra and Il6st (gp130) on the disease course of EAE.
a, Course of EAE in Ly6gCre/WT control (n = 11) and Il6raΔLy6G (n = 11) mice; symbols depict mean ± s.d. of EAE scores in individual mice; two-way ANOVA with Bonferroni’s multiple comparison test; ns, not significant; representative disease course. The experiment was repeated four times. b, Course of EAE in Ly6gCre/WT (n = 13) and Il6stΔLy6G (n = 19) mice; symbols depict mean ± s.d. of EAE scores in individual mice; two-way ANOVA with Bonferroni’s multiple comparison test; *P < 0.05; representative disease course; the experiment was repeated two times.
Supplementary Figure 6 STAT3 deficiency in Ly6G+ neutrophils does not alter the T cell and myeloid compartments during recovery from EAE.
a, Total cell count of CD45+ cells, CD4+ T cells, Ly6G-tdTomato+ cells, CD45hiCD11bhiLy6G− monocytes and CD45dimCD11bhi microglia, purified from the spinal cord of Ly6gCre/WT (n = 6) or Stat3ΔLy6G mice (n = 10) at EAE recovery (day 25); for gating strategy see Supplementary Fig. 4. b, Analysis of cytokine production by intracellular cytokine staining in CD4+ T cells, purified from spinal cord of Ly6gCre/WT (n = 4) or Stat3ΔLy6G mice (n = 5) at EAE recovery (day 24) and stimulated ex vivo with PMA/ionomycin. Symbols depict mean ± s.d. of biological replicates; Mann-Whitney U-test (a,b).
Supplementary Figure 7 Characterization of B cells in spleen and CNS of mice lacking functional Ly6G+ MDSCs during recovery from EAE.
a, Histologic analysis showing co-staining of CD3, CD19 and B220 in control mice (Ly6gCre/WT) and Stat3ΔLy6G mice during early recovery (day 21). Scale bar, 20 μm. b, Flow cytometric analysis of live CD19+ B cell compartment in spleen and CNS of control mice (Ly6gCre/WT), Stat3ΔLy6G mice and Ly6G-tdTomato+ MDSC-depleted mice (Ly6G Ab) during early recovery from EAE. CD1+CD5+ regulatory B cells (Breg cells) are not increased upon MDSC loss of function; representative plots of n = 6 individuals per group; gate on CD19+B220+ cells. c, Analysis of cytokine production by intracellular cytokine staining in CD19+B220+ B cells purified from CNS of Ly6gCre/WT (n = 6), Stat3ΔLy6G mice (n = 6) and Ly6gCre/WT mice treated with anti-Ly6G depleting antibody (Ly6G Ab, n = 6) at EAE recovery (day 21) and stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A; symbols depict mean ± s.d. of biological replicates; Kruskal-Wallis test with Dunn’s post test; *P < 0.05, ***P < 0.001. d, Representative cytograms of live CD19+ B cells isolated from the CNS of Ly6gCre/WT control mice or Stat3ΔLy6G mice (with dysfunctional MDSCs) and stimulated ex vivo with PMA/ionomycin in the presence of brefeldin A for intracellular cytokine staining (double staining for IL-6 and IL-10).
Supplementary Figure 8 Microglial cells adopt a ‘neurotoxic’ phenotype during EAE in the absence of functional Ly6G+ MDSCs.
a,b, CD11bintCD45intLy6G− microglia was sorted from Ly6gCre/WT mice (n = 4) and Stat3ΔLy6G mice (n = 4) at early disease recovery (day 22). Relative RNA abundance (RQ) of Clec7a, Gpnmb, Trem2 and ApoE, which are signature genes of a ‘neurotoxic’ microglial state (a) (Krasemann et al. 2017) and Tmem119, P2ry12, Sall1 and Spi1, which are signature genes of a ‘homeostatic’ microglial state (b) (Krasemann et al. 2017); symbols depict biological replicates (bars, mean ± s.d.); Mann-Whitney U-test; *P < 0.05. Ref: Krasemann, S. et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neuro-degenerative diseases. Immunity 47, 566–581.e9 (2017).
Supplementary Figures 1–8
Genes differentially expressed in CNS onset-Ly6G+ cells when compared to all other Ly6G+ cell subsets
Gene ontology (GO) terms significantly enriched in genes universally downregulated in CNS onset-Ly6G+ cells
Gene ontology (GO) terms significantly enriched in genes universally upregulated in CNS onset-Ly6G+ cells
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Knier, B., Hiltensperger, M., Sie, C. et al. Myeloid-derived suppressor cells control B cell accumulation in the central nervous system during autoimmunity. Nat Immunol 19, 1341–1351 (2018). https://doi.org/10.1038/s41590-018-0237-5
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