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, 6 (23), 1802219
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CD4 + T Cell-Released Extracellular Vesicles Potentiate the Efficacy of the HBsAg Vaccine by Enhancing B Cell Responses

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CD4 + T Cell-Released Extracellular Vesicles Potentiate the Efficacy of the HBsAg Vaccine by Enhancing B Cell Responses

Jian Lu et al. Adv Sci (Weinh).

Abstract

T cells secrete bioactive extracellular vesicles (EVs), but the potential biological effects of CD4+ T cell EVs are not clear. The main purpose of this study is to investigate the effects of CD4+ T cell-derived EVs on B cell responses and examine their role in antigen-mediated humoral immune responses. In this study, CD4+ T cell EVs are purified from activated CD4+ T cells in vitro. After immunization with the Hepatitis B surface antigen (HBsAg) vaccine, CD4+ T cell EVs-treated mice show stronger humoral immune responses, which is indicated by a greater Hepatitis B surface antibody (HBsAb) level in serum and a greater proportion of plasma cells in bone marrow. In addition, it is found that EVs released from activated CD4+ T cells play an important role in B cell responses in vitro, which significantly promote B cell activation, proliferation, and antibody production. Interestingly, antigen-specific CD4+ T cell EVs are found to be more efficient than control EVs in enhancing B cell responses. Furthermore, it is shown that CD40 ligand (CD40L) is involved in CD4+ T cell EVs-mediated B cell responses. Overall, the results have demonstrated that CD4+ T cell EVs enhance B cell responses and serve as a novel immunomodulator to promote antigen-specific humoral immune responses.

Keywords: B cells; CD4+ T cells; CD40L; HBsAg vaccine; extracellular vesicles.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Inhibition of EVs release impairs the function of CD4+ T cells in vitro. A) Bicinchoninic acid (BCA) protein assay of total protein (top) and immunoblot analysis of CD63 (bottom) in purified EVs from supernatants of CD4+ T cells treated with a control vehicle (dimethyl sulfoxide (DMSO)) or 10, 20, or 40 × 10−6 m GW4869 for 48 h. B) A schematic of the Transwell coculture model with B cells in the upper chamber and CD4+ T cells in the lower chamber of the well. A porous (0.4 µm) membrane allows the transfer of EVs but precludes direct cell contact. C) Flow cytometry analysis of CD86 and MHCII expression on the surface of B cells. D) Total IgG levels in B cell supernatants were analyzed by enzyme‐linked immunosorbent assay (ELISA). E) A schematic of the utilization of anti‐CD63 microbeads to remove EVs in CD4+ T cell supernatant. F) The removal efficiency of CD63‐positive EVs in the supernatant was evaluated by western blotting. G) CD86 and MHCII expression on the surface of B cells cultured with different supernatants for 48 h was analyzed by flow cytometry (FCM). H) B cells were cultured with different supernatants for 72 h, and the total IgG in the supernatant was analyzed by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t‐test). The data are from three independent experiments (A (top), C, D, F (left), G, and H; mean and s.e.m.) or are representative of three independent experiments (A (bottom) and F (right)).
Figure 2
Figure 2
Characterization of EVs released from activated CD4+ T cells. A) CD4+ T cells from wild‐type mice were activated with plate‐bound anti‐CD3 (2 µg mL−1) and soluble CD28 (2 µg mL−1) for 24 h, stained with fluorescent conjugated anti‐CD40L and ICOS mAbs or isotype‐matched mAbs and then analyzed by flow cytometry. B) Electron microscopy analysis of CD4+ T cell EVs. Representative electron micrographs of EVs are shown. Scale bar: 100 nm. C) Nanoparticle tracking analysis (NTA) of CD4+ T cell EVs. D) For the analysis of EVs surface molecules, EVs were first incubated with 2 µL 4 µm diameter aldehyde/sulfate latex beads, then stained with a panel of mAbs (solid lines) or isotype‐matched mAbs (dotted lines) and analyzed by flow cytometry. E) Western blot analysis of CD4+ T cell lysates and their EVs using the previously indicated mAbs. 50 µg CD4+ T cell EVs were exposed to F) 0.05% trypsin or G) 25 µg mL−1 proteinase K, samples were subjected to immunoblot analysis using antibodies against CD63 and CD40L.*P < 0.05 (Student's t‐test). The data are representative of three independent experiments (A)–(G).
Figure 3
Figure 3
CD4+ T cell EVs stimulate the production of HBsAb in HBsAg‐vaccinated mice. A) A schematic of the mouse treatments. The mice were injected with HBsAg vaccine (i.m.) together with HB‐T‐EVs/OVA‐T‐EVs/WT‐T‐EVs or PBS treatment (i.v.), and serum was collected on days 16, 23, 30, 40, and 50. B–D) The absorbance of HBsAb IgG, IgG2a, and IgG1 in serum at different time points of treatment was quantified by ELISA. E) The proportion of spleen Th1 cells, Th2 cells, B cells, and plasma cells were analyzed by flow cytometry at day 50. F) Flow cytometry analysis of bone marrow plasma cells by CD19 and CD138 staining (gate on bone marrow lymphocytes). Representative dot plots of bone marrow cells are shown. *P < 0.05 and **P < 0.01 (Student's t‐test). The data are from two independent experiments with five mice per group (B, C, D, E, and F (right); mean and s.e.m.) or are representative of two independent experiments with five mice per group (F (left)).
Figure 4
Figure 4
CD4+ T cell EVs bind to B cells and are taken up by B cells. A) A total of 5 × 105 spleen cells from OVA‐immunized mice were incubated in 50 µg PKH26‐labeled OVA‐T‐EVs for 4 h at 37 °C (5% CO2). The cells were then stained with fluorescent conjugated anti‐CD3 and CD19 mAbs for 30 min. The percentage of PKH26‐positive cells and the mean fluorescence intensity (MFI) of PKH26 in T and B cells were analyzed by flow cytometry. B) Confocal microscopy analysis of CD19+ B cells (red) incubated with PKH67‐labeled OVA‐T‐EVs (green) for 4 h at 37 °C. **P < 0.01 (Student's t‐test). The data are from three independent experiments (A; mean and s.e.m.) or are representative of three independent experiments (B).
Figure 5
Figure 5
CD4+ T cell EVs promote B cell activation, proliferation and antibody production in vitro. A) A total of 5 × 105 B cells isolated from OVA‐immunized mice were incubated with 50 µg WT‐T‐EVs (control)/WT‐T‐EVs or OVA‐T‐EVs (control)/OVA‐T‐EVs at 37 °C for 48 h and then incubated with fluorescent conjugated anti‐CD86, CD80, CD40, and MHCII mAbs. The data were analyzed by FCM. B) B cells isolated from OVA‐immunized mice were incubated with different doses of OVA‐T‐EVs or WT‐T‐EVs for 48 h. The activation of B cells was evaluated by the expression of CD86, CD80, CD40, and MHCII. C) A total of 5 × 105 CFSE‐labeled B cells were incubated with 50 µg OVA‐T‐EVs or WT‐T‐EVs for 4 d, and the proliferation of B cells was analyzed by microscopy and flow cytometry. Furthermore, the culture supernatant was collected, and the total D) IgG and E) OVA‐specific IgG antibodies were analyzed by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t‐test). The data are from three independent experiments (A, B, D, and E; mean and s.e.m.) or are representative of three independent experiments (C).
Figure 6
Figure 6
Activated CD4+ T cell EVs highly express CD40L. A) CD3/CD28‐stimulated (black solid lines) or control (black dotted lines) CD4+ T cells from OVA‐immunized mice and wild‐type mice were stained with a fluorescent conjugated anti‐CD40L mAb and then analyzed by flow cytometry (top). Total CD40L expression in OVA‐CD4 T/WT‐CD4 T and OVA‐CD4 T (control)/WT‐CD4 T (control) were compared by western blotting (bottom). B) The expression of CD40L in OVA‐T‐EVs/WT‐T‐EVs and OVA‐T‐EVs (control)/WT‐T‐EVs (control) were compared by western blotting. One representative experiment of three experiments is shown.
Figure 7
Figure 7
CD40L is involved in T cell EVs‐mediated B cell responses in vitro. A) Different CD40L shRNA recombinant plasmids were transfected into EL‐4 cells using Lipofectamine 2000 transfection reagent, positive clones were screened by G418, CD40L interference efficiency was analyzed by western blotting, and the most efficient plasmid was filtered out. B) Surface (top) and total CD40L (bottom) expression of EL‐4CD40L shRNA and EL‐4NC shRNA cells were analyzed by FCM. C) CD40L expression of EVs released from EL‐4CD40L shRNA and EL‐4NC shRNA cells were analyzed by western blotting. The interference efficiency was indicated by the gray level ratio of CD40L/β‐actin. A total of 5 × 105 CFSE‐labeled B cells isolated from wild‐type mice were incubated with 50 µg EL‐4CD40L shRNA EVs or EL‐4NC shRNA EVs for 72 h. The proliferation of B cells was evaluated by D) microscopy and E) FCM. For the analysis of B cell activation, 5 × 105 B cells were incubated with 50 µg EL‐4CD40L shRNA EVs or EL‐4NC shRNA EVs for 48 h; the expression of CD86 and MHCII was analyzed by F) FCM, and G) the total IgG in culture supernatant was analyzed by ELISA. *P < 0.05 and **P < 0.01 (Student's t‐test). The data are from three independent experiments (B (right), F, and G; mean and s.e.m.) or are representative of three independent experiments (A, B (left), C(top), D, and E).

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