T-cell-derived extracellular vesicles regulate B-cell IgG production via pyruvate kinase muscle isozyme 2

Abstract

Intercellular communication between lymphocytes plays a fundamental role in numerous immune responses. Previously, we demonstrated that hyperhomocysteinemia (HHcy) induced T cell intracellular glycolytic-lipogenic reprogramming and IFN-γ secretion via pyruvate kinase muscle isozyme 2 (PKM2) to accelerate atherosclerosis. Usually, B cells partially obtain help from T cells in antibody responses. However, whether PKM2 activation in T cells regulates B cell antibody production is unknown. Extracellular vesicles (EVs) are important cellular communication vehicles. Here, we found that PKM2 activator TEPP46-stimulated T-cell-derived EVs promoted B-cell IgG secretion. Conversely, EVs secreted from PKM2-null T cells were internalized into B cells and markedly inhibited B-cell mitochondrial programming, activation, and IgG production. Mechanistically, lipidomics analyses showed that increased ceramides in PKM2-activated T-cell EVs were mainly responsible for enhanced B cell IgG secretion induced by these EVs. Finally, quantum dots (QDs) were packaged with PKM2-null T cell EVs and anti-CD19 antibody to exert B-cell targeting and inhibit IgG production, eventually ameliorating HHcy-accelerated atherosclerosis in vivo. Thus, PKM2-mediated EV ceramides in T cells may be an important cargo for T-cell-regulated B cell IgG production, and QD-CD19-PKM2-null T cell EVs hold high potential to treat B cell overactivation-related diseases.-Yang, J., Dang, G., Lü, S., Liu, H., Ma, X., Han, L., Deng, J., Miao, Y., Li, X., Shao, F., Jiang, C., Xu, Q., Wang, X., Feng, J. T-cell-derived extracellular vesicles regulate B-cell IgG production via pyruvate kinase muscle isozyme 2.

Keywords: antibody; ceramide; lymphocyte.

Figure 1

EVs secreted from PKM2-activated T cells promote B-cell IgG production. TEVs were isolated by sequential centrifugation. A) Visualization of the EVs by TEM. B, C) Analysis of the size distribution of TEVs using the NAT and ζ potential distribution analyzer. D) The expression of EV-related protein markers CD63, CD9, Alix, and TSG101, the T cell marker CD3ε, and the EV-negative marker calnexin were measured by Western blot analysis. C57BL/6J mouse T cells were treated with TEPP46 (10 μM); after 48 h, TEVs were isolated and cocultured with normal B cells. E) The IgG level in the B cell culture supernatant was measured via ELISA. F) Blimp1 and IRF4 protein expression were analyzed via Western blot analysis. G) Gene expression of Prdm1 and Irf4 was measured via qPCR in B cells at 24 h. H) Gene expression of Aicda and postswitch transcripts (Iμ-Cγ1, Iμ-Cγ2b, and Iμ-Cγ3) was measured via qPCR in B cells at 72 h; n = 4, data are presented as the means ± sd. *P < 0.05 vs. Control (ctl)-TEVs group (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 2

EVs secreted from PKM2-null T cells decrease B-cell IgG production. TEVs were isolated from T-cell culture supernatants and cultured (TEVs 5 μg/ml, equal concentration was used between PKM2^fl/fl-TEVs and LckCrePKM2^fl/fl-TEVs) with 1 × 10⁷ primary splenic B cells purified from C57BL/6J mice seeded in a 6-well plate. A) B-cell culture supernatants at 72 h were assayed for the presence of IgG. B) Western blot analysis of B cells for Blimp1 and IRF4 protein expression. C) qPCR analysis of B-cell transcription factors at 24 h. D) Gene expression of Aicda and postswitch transcripts in B cells at 72 h. E, F) Metabolic parameters of TEV effect on B cells. OCR and ECAR were measured in real time after the addition of oligomycin (oligo), carbonyl cyanide p-trifluoromethoxyphenylhydrazone, antimycin A, and rotenone (AntiA/Rot); n = 4, data are presented as means ± sd. *P < 0.05 vs. the PKM2^fl/fl-TEVs group (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 3

Secondary perturbation of lipid metabolites in EVs secreted from PKM2-null T cells. HPLC-MS/MS analysis of lipid metabolites in EVs isolated from T-cell culture supernatants. A) PCA scatter plot of the lipid metabolites in TEVs. B) VIP scatter plot identified by PCA analysis showing the top 10 lipid metabolites in PKM2^fl/fl-TEVs and LckCrePKM2^fl/fl-TEVs. A: PKM2^fl/fl-TEVs, B: LckCrePKM2^fl/fl-TEVs. C) Heat map of changes in the TEV ceramide profile. D) The relative levels of ceramide metabolites, normalized to TEV protein levels, are presented. TEV protein levels were normalized to lysate total protein. E) IgG levels in the B-cell culture supernatants after 18:1 ceramide and 16:0 ceramide stimulation for 72 h. Ctl, control. F) Ceramide synthesis pathways: the de novo, sphingomyelin hydrolysis, and salvage pathways. G–I) The mRNA levels of enzymes required for de novo ceramide synthesis (G), sphingomyelin hydrolysis (H), and the salvage pathway that recreates ceramide from sphingosine (I) in T cells of PKM2^fl/fl mice and LckCrePKM2^fl/fl mice; n = 4, data are presented as means ± sd. *P < 0.05 vs. the PKM2^fl/fl-TEVs group (D, G–I) or control group (E) (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 4

Adhesion and internalization are required for TEV–mediated B-cell IgG secretion. Primary splenic B cells (1 × 10⁷) pretreated with RGD peptide and/or Dynasore were treated with TEVs, and cells and cell lysates were used to evaluate EV-mediated signaling. A, B) ImageStream flow cytometry and flow cytometry were performed to evaluate the fluorescence intensity of TEVs internalized into cells. C) IgG levels in the B-cell culture supernatants at 72 h. D) Western blot analysis of B-cell protein levels. E) qPCR analysis of B-cell gene expression at 24 h. F) qPCR analysis of B cell gene expression at 72 h; n = 3–5, data are presented as means ± sd. *P < 0.05 vs. the PKM2^fl/fl-TEV group (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 5

QDs packaged with PKM2-null TEVs specifically target and inhibit B-cell activation in vitro. QDs labeled with anti-CD63 and anti-CD19 monoclonal antibodies and combined with TEVs labeled using PKH26 membrane dye targeted B220-positive B cells. A) Confocal micrographs of increased entry of CD19-QD-CD63-TEVs into B cells (blue box) relative to QD-CD63-TEVs (white box). B) Quantitative analysis of the results of A. C–F) CD19-QD-CD63-TEVs (LckCrePKM2^fl/fl-TEVs) targeted and impaired B-cell activation, including IgG levels in culture supernatants at 72 h (C), Blimp1 and IRF4 protein expression and quantification in B cells at 24 h (D), gene expression of Prdm1 and Irf4 in B cells at 24 h (E), and gene expression of Aicda and postswitch transcripts after treatment for 72 h (F). B cells stained by B220 in blue, TEVs stained by PKH26 in red, and QDs are shown in green; n = 3–5, data are presented as means ± sd. *P < 0.05 vs. the QD-CD63-TEV group (B) or PKM2^fl/fl-TEV group (C–E) (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 6

Nanomaterial packaging with TEVs modulates B-cell IgG production in vivo. A, B) Animals administered tail-vein injections of the nanocomposites CD19-QD-CD63-TEVs or QD-CD63-TEVs (50 μg of TEVs/mouse) in C57BL/6J mice. Representative micrographs of the specificity of the CD19-QD-CD63-TEVs (TEVs labeled with PKH26) in mouse spleen tissues (A) and splenic B220-positive B cells (B). C) Schematic flowchart of C57BL/6J mice that were injected intraperitoneally with 100 ng of NP-LPS or 100 ng of NP-OVA antigen; after 30 min or 1 d, mice were given tail-vein injections of CD19-QD-CD63-TEVs (LckCrePKM2^fl/fl-TEVs vs. PKM2^fl/fl-TEVs). D) Plasma IgG levels induced by NP-LPS antigen were measured via ELISA. E) Plasma IgG levels induced by NP-OVA antigen were measured via ELISA; n ≥ 5, data are presented as means ± sd. *P < 0.05 vs. the control (CTL) group, ^#P < 0.05 vs. the NP-LPS + PKM2^fl/fl-TEVs group (D) or NP-OVA + PKM2^fl/fl-TEV group (E) (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 7

PKM2-null TEVs capsulated in QDs inhibit HHcy-induced B cell overactivation. T cells from PKM2^fl/fl or LckCrePKM2^fl/fl mice were isolated and then treated with Hcy (100 μM) in vitro. After 48 h, TEVs were purified and subsequently cocultured with equal concentrations of PKM2^fl/fl-TEVs or LckCre PKM2^fl/fl-TEVs with normal B cells for an additional 72 h. A) B cell IgG production. Metabolites in these purified TEVs were analyzed using an HPLC-MS/MS system. B) PCA scatter plot of the metabolites in TEVs. C) Heat map of changes in the ceramide metabolites from TEVs given via tail-vein injection of the nanocomposites CD19-QD-CD63-TEVs or QD-CD63-TEVs (TEVs 50 μg/mouse) in C57BL/6J mice. D–G) TEVs isolated from LckCrePKM2^fl/fl T-cell culture supernatants combined with QDs and CD19 mAb showed targeted inhibition of the HHcy-mediated increase in B cell IgG secretion (D), Blimp1 and IRF4 protein levels (E), Prdm1 and Irf4 gene expression levels (F), and Aicda, Iμ-Cγ1, Iμ-Cγ2b, and Iμ-Cγ3 gene expression levels (G). Ctl, control; n = 4, data are presented as means ± sd. *P < 0.05 vs. ctl-PKM2^fl/fl-TEVs (A) or ctl group (D–G), ^#P < 0.05 vs. Hcy-PKM2^fl/fl-TEVs group (A) or HHcy + PKM2^fl/fl-TEV group (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 8

Nanomaterial packaging with PKM2-null TEVs inhibits HHcy-accelerated atherosclerosis. A) Schematic flowchart of ApoE^−/−mice tail-vein injected with CD19-QD-CD63-TEVs (LckCrePKM2^fl/fl-TEVs vs. PKM2^fl/fl-TEVs) twice per week; 3 d after the first injection, HHcy-accelerated atherosclerosis was induced by giving mice drinking water supplemented with 1.8 g/L Hcy. B) Oil Red O staining of aortic roots for quantitative lesion size (left panel). Quantification of the mean atherosclerotic lesion area is shown (right panel). C) Representative confocal images of T-cell infiltration (positive CD3 staining). D) Representative confocal micrographs of the infiltration of macrophages/macrophage [macrophages/monocytes antibody (MOMA2) staining positive] in lesion areas; there were no obvious changes in the α-SMA-stained area. E) The gene expression of Icam-1, Vcam-1, Ifn-γ, Tnf-α, Il-2, and CCL2 in thoracic aortas isolated from mice was measured via qPCR; n = 5–8, data are presented as means ± sd. *P < 0.05 vs. control (ctl) group;. ^#P < 0.05 vs. HHcy-PKM2^fl/fl-TEVs group (1-way ANOVA followed by Tukey’s test for multiple comparisons).

Figure 9

Proposed model of the study. Schematic representation of the proposal that EVs secreted from PKM2-activated T cells promote B-cell pathogenic IgG production. EVs derived from PKM2-null T cells with low ceramide components, especially 16:0 ceramide, were internalized into B cells and then inhibited B-cell IgG antibody production. Antibodies against CD19 and CD63 were loaded onto QDs and combined with PKM2-null TEVs; we fabricated the bionanocomposite QD-EVs and demonstrated their prominent effects as B-cell–targeted nanovectors on HHcy-accelerated pathologic IgG production and atherosclerosis.