Research ArticleIMMUNOLOGY

STING couples with PI3K to regulate actin reorganization during BCR activation

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Science Advances  22 Apr 2020:
Vol. 6, no. 17, eaax9455
DOI: 10.1126/sciadv.aax9455
  • Fig. 1 STING deficiency impacts the differentiation of MZ and GC B cells, but not the development of BM B cells.

    Cells from BM of 6- to 8-week-old WT and Sting KO mice (n = 6) were labeled with Abs specific for surface markers of pre-pro (A), pro (B), early-pre (C), late-pre (D), immature (E), and recirculating mature B cells (F) and analyzed by flow cytometry. Shown are the average percentages (±SD) and numbers of total cells extracted from BM (A), and the average mean fluorescence intensity (MFI) of CD127 in B cell subsets (B). Splenic cells were stained with Abs specific for surface markers of T1, T2, FO (n = 6), MZ, and GC (n = 5) B cells. Samples were analyzed using flow cytometry. Shown are the average percentages (±SD) and numbers of subpopulations in spleen (C to G). Immunofluorescence staining of spleen sections from WT and Sting KO mice. Shown are representative follicles using pseudocolors: B220 (blue), CD4 (red), and GL7 (green); 20× objective; scale bars, 50 μm (H). H&E staining of spleen sections from 8-week-old WT and Sting KO mice. Scale bars, 200 μm (J). Quantification of the area of GCs and follicles from four mice (I and K). *P < 0.05; **P < 0.01; ****P < 0.0001.

  • Fig. 2 STING is involved in BCR activation.

    Purified splenic B cells from WT and Sting KO mice were labeled with AF546-mB-Fab-anti-Ig and activated by incubating with either streptavidin or the medium alone (0 min) as a control at 37°C for varying lengths of time. After fixation and permeabilization, samples of WT were stained with antibodies for STING or ER-Tracker Blue-White DPX dye, and samples of KO were stained with ER-Tracker Blue-White DPX dye. Then, samples were analyzed using confocal microscopy. Shown are representative images (A and D) and the correlation coefficients between the labeled BCR and STING (B) or ER (E) quantified using the ZEN 2.3 (blue edition) software. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were lysed, and then the lysates were probed with antibodies specific for STING (C). Enzyme-linked immunosorbent assay (ELISA) quantification of anti-dsDNA Ab in the serum of WT and Sting KO mice (n = 9). Dots represent individual mice (F). Immunofluorescence analysis of nephritic sections. Shown are representative glomeruli (G and H). 60× objective; scale bars, 25 μm. Flow cytometry analysis of GCB cells in spleens from aging (>5 months) and young (6- to 8-week-old) WT and Sting KO mice. Shown are representative dot plots of aging mice and the average percentages (±SD) and numbers of GCB cells in spleens of WT and Sting KO mice (I and J). H&E staining of kidney, lung, colon, and liver from WT and Sting KO mice. Shown are representative images and the pathological score from five mice (K and L). Each item was scored on a 5-point scale as follows: 0 = minimal damage, 1+ = mild damage, 2+ =moderate damage, 3+ =severe damage, and 4+ =maximal damage. For kidney, scale bar is 20 μm. For lung, colon, and liver, scale bars are 200 μm. *P < 0.05; ****P < 0.0001.

  • Fig. 3 STING deficiency up-regulates positive BCR signaling.

    Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab–anti-Ig and then without or with streptavidin to activate. Cells were fixed, permeabilized, and stained with Abs specific for pY, pBtk (A), and pCD19 (I). Shown are representative images and the correlation coefficients between the labeled BCR and pY/pBtk (B) and pCD19 (J) from three independent experiments. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’)2 anti-mouse Ig (M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS–polyacrylamide gel electrophoresis (PAGE) and Western blot and probed for pY, pBtk, Btk, pSyk, Syk (C), and pCD19 (K). Total protein or β-actin as controls. Shown are representative blots of three independent experiments and blots’ relative intensity (D to F and L). Western blot analysis of the level of pY in purified B cells from PBMCs of the healthy control and STING mutant patient (G and H). Flow cytometry analysis of MFI of CD19 in B220+ B cells from WT and Sting KO mice (M). *P < 0.05.

  • Fig. 4 STING deficiency down-regulates the activation of negative BCR signaling molecule, SHIP, and up-regulates the actin polymerization via enhancing the activation of WASP.

    Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab-anti-Ig and then incubated without or with streptavidin for activation. Cells were fixed, permeabilized, and stained with Abs specific for pSHIP, pWASP, and F-actin (A and F). Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’)2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pSHIP/SHIP, pSHP1/SHP1 (C). Total protein or β-actin was used as controls. Shown are representative images, blots, and the correlation coefficients between the labeled BCR and pSHIP (B), pWASP (G), as well as the relative intensity of bands from immunoblots (D and E) taken from three independent experiments. Splenic B cells were labeled with anti-B220 and stimulated with sAg for indicated times and then stained with phalloidin and antibody specific for pWASP for phos flow cytometry. MFI of pWASP and F-actin in B cells was quantified using FlowJo software (H and I). Shown are levels of pWASP and F-actin from one of three independent experiments. Scale bars, 2.5 μm. *P < 0.05; **P < 0.01.

  • Fig. 5 PI3K inhibition rescues the abnormal accumulation of F-actin in Sting KO B cells.

    Splenic B cells from WT and Sting KO mice, pretreated with or without PI3K inhibitor, were incubated with AF546-mB-Fab-anti-Ig tethered to lipid bilayers with varying lengths of time and then fixed, permeabilized, and stained with antibodies specific for pY and F-actin. Cells were analyzed using TIRFm. Shown are representative images (A to D), the average values of the B cell contact area (F), and the MFI of the pY (G), BCR (E), and F-actin (H) in the contact zone. Scale bars, 2.5 μm. Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’)2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pPI3K (p85)/PI3K (p85), pAtk/Akt, pS6/S6 (I), and pFoxO-1/FoxO-1 (J). Total protein or β-actin was used as controls. Shown are representative blots from three independent experiments and blots’ relative intensity (K to N). Western blot analysis the level of pPI3K in purified B cells from PBMCs of the healthy control and STING mutant patient (O and P). *P < 0.05; **P < 0.01.

  • Fig. 6 STING deficiency reduces the T-dependent Ab responses.

    Flow cytometry analysis of splenic cells from immunized WT and Sting KO mice (n = 4) for FO B, MZ B, T1 B, T2 B, GC B, MBCs, and PCs. Shown are representative dot plots, the average percentages (±SD) and numbers of subpopulations (A to L). ELISA analysis (n = 4) of NP-specific IgG1 and NP-specific IgM in the serum (M and N). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Materials

  • Supplementary Materials

    STING couples with PI3K to regulate actin reorganization during BCR activation

    Yukai Jing, Xin Dai, Lu Yang, Danqing Kang, Panpan Jiang, Na Li, Jiali Cheng, Jingwen Li, Heather Miller, Boxu Ren, Quan Gong, Wei Yin, Zheng Liu, Pieta K. Mattila, Qin Ning, Jinqiao Sun, Bing Yu, Chaohong Liu

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