Research ArticleIMMUNOLOGY

B cells polarize pathogenic inflammatory T helper subsets through ICOSL-dependent glycolysis

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Science Advances  09 Sep 2020:
Vol. 6, no. 37, eabb6296
DOI: 10.1126/sciadv.abb6296
  • Fig. 1 Identifying helper functions of B cells in generation of inflammatory TH subsets.

    (A) Confocal microscopy analysis of expression of CD4 (red, TH cells) and CD20 (green, B cells) in samples from RA synovial tissue (n = 8), HCC tissue (n = 11), and inflamed tonsils (n = 11). Scale bars, 50 μm. (B) FACS analysis of IFN-γ, IL-17, IL-22, IL-4, IL-9, and IL-10 in TH cells from paired blood and tissue samples from healthy donors (HD) and patients with RA, HCC, or inflamed tonsils (each n = 5). (C) Purified total B cells were left untreated or were stimulated with an anti-IgM antibody (αIgM), Pam3CysSK4 (Pam), LPS, or CpG-DNA ODN (CpG) for 18 hours and then cultured with autologous T cells for 9 days. Expression of Ki-67 and indicated cytokines in TH cells were detected by FACS (n = 5). (D to F) Purified T cells were cultured in medium or with autologous total B cells for 9 days (E and F) or indicated times (D) in the presence or absence of TCR triggering, as described in Materials and Methods. Expression of indicated transcription factors (D), Ki-67 (E), and inflammatory cytokines (F) in TH cells was detected by FACS (n = 5). MFI, mean fluorescence intensity. (G) Purified T cells were cultured in medium or with mature myeloid dendritic cells (mDCs) in the presence or absence of total B cells as described in workflow. FACS analysis was performed to illustrate expression of IFN-γ, IL-17, and IL-22 in TH cells (n = 5). Data are presented as means ± SEM of four independent experiments (C to E and G). ns, nonsignificant. *P < 0.05, **P < 0.01, and ***P < 0.001 [one-way ANOVA test for (C), (D), and (G); two-way ANOVA test for (E)].

  • Fig. 2 Naïve B cells are more potent B cell subpopulations in triggering memory inflammatory TH subsets.

    (A) Purified total B cells were cultured with T cells for 7 days in the presence of TCR triggering. The differentiation of B cells before and after coculture was analyzed by FACS (n = 3). (B to E) Purified T cells were cultured in medium or with naïve or memory B cells (B and C), or with naïve B cells or plasma cells (D and E) for 7 days as described in Materials and Methods. Expression of IFN-γ, IL-17, and IL-22 in TH cells was detected by FACS (each n = 4). (F to H) Purified naïve or memory T cells were cultured in medium or with autologous total B cells for 7 days (F) or indicated times (G and H), as described in Materials and Methods. Expression of inflammatory cytokines (F and G) and transcription factors (H) in CD4+ T cells was detected by FACS (n = 4 for each). (I) FACS analysis of IFN-γ, IL-17, and IL-22 in naïve (CD45RO) or memory (CD45RO+) TH cells from blood of healthy donors and RA patients (each n = 3). Data are presented as means ± SEM of four independent experiments (C, E, G, and H). *P < 0.05, **P < 0.01, and ***P < 0.001 [one-way ANOVA test for (C), (G), and (H); unpaired Student’s t test for (E)].

  • Fig. 3 Factors required for B cell–elicited polarization of inflammatory TH subsets.

    (A) Neutralizing indicated cytokines separately or together hardly affected B cell–mediated TH subset differentiation on day 7. (B) Effects of total B cells or conditioned medium from the culture of total B cells (B-CM) or of total B cells plus T cells (BT-CM) on TH subset differentiation on day 7. (C) Analysis of TH subsets after cultured for 7 days in medium or with total B cells directly or in a transwell chamber. (D and E) FACS analysis of ICOSL in indicated cells from blood of healthy donors (D) or in total B cells left untreated or stimulated with αIgM, Pam3CysSK4, LPS, or CpG for 18 hours (E). (F) FACS analysis of ICOS in indicated cells left untreated or stimulated with CD3–cross-linking antibodies for 24 hours. (G and H) Blocking (G) or silencing (H) ICOS in T cells abrogated B cell–mediated TH subset differentiation on day 7. (I and J) FACS analysis of CD40 in indicated cells from blood of healthy donors (I) or in total B cells left untreated or stimulated with αIgM, Pam3CysSK4, LPS, or CpG for 18 hours (J). (K) FACS analysis of ICOSL in indicated cells cultured for 18 hours in medium or with TCR-primed TH cells in the presence or absence of CD40-neutralizing antibody. (L) Blocking CD40 abrogated B cell–mediated TH subset differentiation on day 7. (M) Analysis of STAT activation in TH cells cultured alone or stimulated with total B cells, ICOS agonist, or conditioned medium from LPS-stimulated monocytes for 2 days (n = 3). Data are presented as means ± SEM (A to L). *P < 0.05, **P < 0.01, ***P < 0.001 [one-way ANOVA test for (A) to (G) and (I) to (L); two-way ANOVA test for (H)].

  • Fig. 4 Glycolysis is significantly up-regulated in T cells polarized by B cells.

    (A) Volcano plot showing changes in genes in TH cells cultured together with total B cells (B cell–treated T cell) for 7 days versus TH cells cultured alone (T cell) for 7 days. Each gene was symbol-coded according to its adjusted P value generated using DESeq2 with a Benjamini-Hochberg false discovery rate (FDR) correction. (B) Functional annotation analysis using DAVID tool listing differential gene expression in (A). The top 10 enrichment GO terms are shown. (C and D) GSEA of glucose catabolic process (C) and fatty acid beta oxidation (D) in TH cells cultured together with total B cells for 7 days versus TH cells cultured alone for 7 days. NES, normalized enrichment score. (E) Heat map showing expression of genes associated with glycolysis and pentose phosphate pathway (PPP).

  • Fig. 5 ICOSL-ICOS axis–elicited glycolysis is involved in T cell polarization by B cells.

    (A) Process and key rate-limiting enzymes during glycolysis. (B to E) Purified T cells were cultured in medium or with total B cells in the presence or absence of isotype antibody or ICOS-neutralizing antibody for 7 days, as described in Materials and Methods. Expression of key rate-limiting glycolytic enzymes, ECAR, and capabilities of glucose incorporation (Glut1+ or 2-NBDG intensity) were determined using real-time PCR (B, n = 3), a Seahorse Extracellular Flux XF-24 analyzer (C, n = 3), and FACS (D and E, n = 5 for each), respectively. (F) TCR-primed T cells were left untreated or were stimulated with ICOS agonist antibody for 1 hour. Activation of the indicated signals was detected by immunoblotting (n = 4). (G and H) Using rapamycin to inhibit the mTOR signal impaired B cell–mediated up-regulation of key rate-limiting glycolytic enzymes (G, n = 3) and the incorporation of glucose in TH cells (H, n = 4). DMSO, dimethyl sulfoxide. (I) Using 2DG, rapamycin, and ICOS-neutralizing antibody to inhibit glycolysis, the mTOR signal, and the ICOS signal, respectively, suppressed B cell–meditated inflammatory TH subset differentiation on day 7 (n = 5). Data are representative of three independent experiments (B to I). Data are presented as means ± SEM (B to E and G to I). *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA test).

  • Fig. 6 Depleting B cells attenuates CD4+ T cell inflammation and glycolysis in SLE and RA patients.

    (A to E and G) FACS analysis of ICOS expression (A), Glut1 (D), incorporated 2-NBDG (E), and inflammatory cytokines (G) in circulating TH cells from healthy donors (n = 7), SLE patients (n = 7), RA patients (n = 15), and RA patients treated with rituximab (n = 5). Correlations between ICOS expression and inflammatory cytokines in circulating TH cells are shown in (B); correlations between ICOS expression and pathological parameters in circulating TH cells from RA patients are presented in (C). P and R values were calculated based on the analysis of Pearson’s correlation. (F) Purified T cells from blood of healthy donors (n = 5) or patients with SLE (n = 4) or RA (n = 5) were cultured for 12 hours in the presence or absence of 2DG. Expression of IFN-γ, IL-17, and IL-22 in TH cells was detected by FACS. (H and I) Purified CD69+ effector TH cells were cultured in medium or with blood total B cells from patients with SLE (H, n = 3) or RA (I, n = 6) for 7 days in the absence or presence of ICOS-neutralizing antibody or 2DG. Expression of IFN-γ, IL-17, and IL-22 in TH cells was detected by FACS. Data are representative of six independent experiments (F, H, and I). Data are presented as means ± SEM (A, D, E, and G to I). *P < 0.05, **P < 0.01, and ***P < 0.001 [one-way ANOVA test for (A), (D), (E), and (G) to (I); two-way ANOVA test for (F)].

Supplementary Materials

  • Supplementary Materials

    B cells polarize pathogenic inflammatory T helper subsets through ICOSL-dependent glycolysis

    Qiu-Hui Zeng, Yuan Wei, Xiang-Ming Lao, Dong-Ping Chen, Chun-Xiang Huang, Qian-Yi Lin, Min He, Yuan Liao, Limin Zheng, Bo Li, Guang-Bo Zhang, Yun Chen, Dong-Ming Kuang

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