Figure legends
Fig. 1. Disrupted peripheral differentiation of B cells in CCR2KO mice. Splenic mononuclear cells were isolated from mice and stained with corresponding antibodies. Flow cytometry (FCM) was performed. Organs extracted from mice were used for immunohistochemical and immunofluorescence experiments. (A-B ) B cell subsets derived from BM cells (n = 8). Pre-pro B cell (A), pro B cell (B), early-pre B cell (C), late-pre B cell (D), immature B cell (E) and recirculating mature B cell (F).(C) Quantitative analysis of BM subsets. (D-F)Peripheral B cell subsets including follicular B cell (FO), transitional type-1 B cell (T1), transitional type-2 B cell (T2), marginal zone B cell (MZ), and germinal center B cells (GCB) (n = 8). (G-K)Quantitative analysis of peripheral B subsets. (L) B-1a and B-1b B cells in the peritoneal cavity were counted (WT: n = 7, KO: n = 6). (M-N) Quantitative analysis of B1 B cell subsets.(O) Representative immunofluorescent analysis (60×, scale bar = 10 μm) of IgG deposits in glomeruli. (P) Serum anti-dsDNA Ab titer was quantified using ELISA (n = 7). (Q) Hematoxylin and eosin (HE) staining of lung anatomical structure (10×, scale bar = 25 μm). (R) Quantitative analysis of T-bet expression level in WT and CCR2 KO B220+ B cells. Quantitative analyses were shown as representative histograms with the specific populations, average percentages (± SEM), and cell number indicated. Each symbol represents a mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. 2. CCR2 KO mice exhibit enhanced BCR proximal signaling.Purified B cells were incubated with AF594-mB-F(ab’)2-anti-Ig (M + G) and activated at 37 °C for 5, 10, and 30 min, fixed and permeabilized before staining, confocal microscopy (CFm) was performed. Cells were incubated with soluble antigen (sAg) (biotin-conjugated F(ab’)2 anti-mouse Ig (M + G) and streptavidin), then activated at 37 °C for 5, 10, and 30 min, western blotting was performed. (A) CFm images of pCD19 and BCR localization. (B) Phosphorylated CD19 protein expression levels in B220+ B cells. (C)Quantitative analysis of pCD19 mean fluorescence intensity (MFI).(D) Colocalization of pCD19 and BCR. (E) CFm images of pY and BCR movement. (F) CFm images of pBtk and BCR movement.(G) Quantitative analysis of pY MFI. (H) Quantitative analysis of pBtk MFI. (I) The pBtk and pY protein expression levels in B220+ B cells. (J) Colocalization of pBtk and BCR. (K) Colocalization of pY and BCR. (L)CFm images of pSHIP and BCR localization. (M) Quantitative analysis of pSHIP MFI. (N) Colocalization of pSHIP and BCR.(O) Phosphorylated SHIP levels in B220+ B cells. (P) Intracellular Ca2+ flux in B cells following stimulation with 10 μg/ml biotin-conjugated F(ab’)2 anti-mouse Ig (M + G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. 3. CCR2 attenuates B cell metabolic process through the negative regulation of PI3K-Akt-mTORC1 signaling pathway. (A) pPI3K, PI3K, pmTOR, mTOR, pAkt, Akt, pFoxo-1, Foxo-1, pS6, and S6 protein expression levels in B cells activated with sAg. (B) pSHIP, pBtk, pCD19, pAkt, pFoxo-1, and pS6 protein expression levels in CCR2 KO B cells treated with 20 nM rapamycin. (C) Seahorse assay was performed to quantify the oxidative respiration of WT and CCR2 KO B cells. (D) Celltrace Violet (CTV) dilution of LPS-stimulated (5 μg/ml) B cell proliferation. (E) Quantification of in vitro proliferation and apoptosis of B cells following a 72-h stimulation with LPS (5 μg/ml). (F) Celltrace Violet (CTV) dilution of CPG-stimulated (5 μg/ml) B cell proliferation. (G) Quantification of in vitroproliferation and apoptosis of B cells following a 72-h stimulation with CPG (5 μg/ml). (H) HIF-1a and c-Myc protein expression levels. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. 4. CCR2 KO mice exhibit increased accumulation of F-actin through the enhancement of the Mst1-mTORC1-Dock8-WASP pathway. (A) CFm images of pWASP, F-actin, and BCR localization. (B)Quantitative analysis of F-actin MFI. (C) Quantitative analysis of F-actin and BCR colocalization. (D) Quantitative analysis of pWASP MFI. (E) Quantitative analysis of pWASP and BCR colocalization. (F-G) Phosflow detection of pWASP and F-actin levels in B220+ B cells. (H) Dock8, pMst1, Mst1, pWASP, WASP, and pEzrin protein expression levels. (I)Filopodia dilatation visualized using electron microscopy (scale bars = 5 μm). (J-K) Quantitative analysis of number and length of the filopodia. (L) Dock8, pMst1, pSHIP, pBtk, pCD19, pMTOR, pAkt, pPi3k, pFoxo-1, and pS6 protein expression levels following XMU-MP-1 treatment on CCR2 KO B cells. (M) Dock8, pMst1, and pWASP protein expression levels following rapamycin treatment on CCR2 KO B cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. 5. CCR2 depletion promotes spatial aggregation of F-actin which is accompanied by the enhancement of early BCR activation.Purified WT and CCR2 KO B cells were stimulated with membrane antigen (mAg) (AF546–mB-Fab–anti-Ig tethered to lipid bilayers) for 3, 5, and 7 min, total internal reflection fluorescence microscopy (TIRFm) was performed. (A) Representative TIRFm images of F-actin and pWASP (scale bars = 2.5 μm). (B-D) Quantitative analysis of B cell area in the contact zone and the MFI of F-actin as well as pWASP.(E) Representative TIRFm images of pCD19 (scale bars = 2.5 μm).(F) Quantitative analysis of pCD19 MFI. (G)Representative TIRFm images of pY and pBtk (scale bars = 2.5 μm).(H-I) Quantitative analysis of pY and pBtk MFIs.(J) Representative TIRFm images of pSHIP (scale bars = 2.5 μm).(K) Quantitative analysis of pSHIP MFI. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. 6. CCR2 attenuates the effect of STAT1 activation in BCR signaling pathway. (A) Representative CFm images of pSTAT1 (scale bars = 2.5 μm). (B-C) Quantitative analysis of the pSTAT1 MFI and its colocalization with BCR. (D) Representative CFm images of pNF-κB (scale bars = 2.5 μm). (E-F) Quantitative analysis of the pNF-κB MFI and its colocalization with BCR. (G)Representative CFm images of pSTAT5 (scale bars = 2.5 μm).(H-I) Quantitative analysis of the pSTAT5 MFI and its colocalization with BCR. (J) Protein expression levels of pSTAT1, STAT1, pSTAT5, STAT5, pNF-κB NF-κB, pIKKB, and IKKB in WT and CCR2 KO B cells. (K-M) Protein expression levels of the markers mentioned in panel J following fludarabine, XMU-MP-1, or rapamycin treatment of CCR2 KO B cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. 7. CCR2 KO mice generate fewer plasma cells and produce less antibodies during T cell dependent immune responses. WT and CCR2 KO mice were injected intraperitoneally (i.p.) with T cell dependent antigen 4-hydroxy-3-nitrophenylacetyl conjugated keyhole limpet hemo-cyanin (NP-KLH, 40 µg/mouse). Boost mice with the same reagents 14 days post-prime. (A-B) FCM gating strategy of peripheral B cell subsets after immunization. (C-F) Quantitative analysis of the proportion and cell number of FO, MZ, T1, and T2 groups. (G)FCM gating strategy of GC B cells. (H) Quantitative analysis of the proportion and cell number of GC B cells. (I-L) FCM gating strategy of PBC, PC, and MBC. (J-M) Quantitative analysis of the proportion and cell number of PBC, PC, and MBC. (N-O) ELISA was performed on serum extracted from blood, and NP-specific IgM as well as IgG1 levels were quantified post-prime and post-boost. (P)Images of HE stained anatomical spleens (scale bars = 200 μm). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.
Fig. S1. CCR2 deficiency cause intrinsic peripheral B cell differentiation impairment. B cell subsets in WT (n = 5) and CCR2 KO (n = 3) mice after bone marrow chimeras were determined using FCM.(A) Gating strategy. (B-D) Representative histogram of peripheral B cell subsets with the specific populations. (E-I)Quantitative analysis of the proportion and cell number of peripheral B cell subset from WT and CCR2 KO B mice. (J) The procedure of mice immunization. Each symbol represents a mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no statistical significance.