Construction and characterization of CD19-targeting NK-92-derived extracellular vesicles.

(A) Construction of the CD19-targeting CD19scFv-LAMP2B-pLVX plasmid (CD19 plasmid). The CD19-targeting receptor gene was generated by fusing an anti-CD19 single-chain variable fragment (scFv) to the N-terminus of the exosomal membrane protein LAMP2B and linking it to the N-terminus of GFP. The fusion gene sequence was cloned into the multiple cloning site of the plasmid. (B) Fluorescence microscopy image of NK-92 cells 72 hours after transfection with the lentivirally packaged CD19 plasmid (V-CD19). Scale bar = 200 µm. (C) Western blot analysis of exosomal markers in V-CD19-Exo. The exosomes positively expressed CD63 (55 kDa), CD9 (25 kDa), and CD81 (24 kDa), while the endoplasmic reticulum protein Calnexin (100 kDa) was negative. (D) Transmission electron microscopy (TEM) image of extracellular vesicles (V-CD19-Exo) isolated from the supernatant of NK-92 cells after V-CD19 plasmid transfection, negatively stained. Arrows indicate cup-shaped vesicles. Scale bar = 100 nm. (E) Nanoparticle tracking analysis (NTA) of the particle size distribution and concentration of V-CD19-Exo. The exosomes exhibited a unimodal size distribution with a peak diameter of approximately 100 nm and a corresponding concentration of approximately 6.0×1010 particles/mL.

V-CD19-Exo retains NK-92 cytotoxicity against tumor cells and selectively targets CD19+ B cells in vivo.

(A) Cytotoxic effect of V-CD19-Exo on A549 tumor cells. Tumor cells were seeded in 6-well plates containing DMEM + 10% FBS (1x106 A549 cells/2 mL/well), cultured for 24 hours, then V-CD19-Exo was added, mixed, and cultured for another 24 hours. (B) Effect of V-CD19-Exo on B cells in MRL/lpr mice. 16-week-old female MRL/lpr mice were intraperitoneally injected with V-CD19-Exo (109 particles/mouse/time). Mice were euthanized 3 weeks later, spleens were collected to prepare single-cell suspensions, and splenic B cell populations were analyzed by flow cytometry using anti-CD20-FITC and anti-CD19-APC antibodies. NC, control group; NK92-EXO, MRL/lpr mice injected with NK92 cell exosomes; CD19scfv-NK92-Exos, MRL/lpr mice injected with V-CD19-Exo exosomes. The dotted box indicates CD20 and CD19 double-positive B cells. (C) Quantitative analysis of CD20 and CD19 double-positive B cells (cells within the circular gate in Figure B) in the spleens from mice treated with NC, control, and NK92-EXO groups, corresponding to Figure B.

Effects of V-CD19-Exo on body weight, proteinuria, and renal histopathology in MRL/lpr mice.

Treatment and detection timelines are as indicated. (A) Drugs were injected according to the experimental design timeline, or mouse urine and serum were collected to monitor urinary protein, urinary creatinine, and serum autoantibody levels. Mice were euthanized at the experimental endpoint, and kidneys were collected for pathological analysis. Experimental data analysis is shown as follows: (B) Dynamic changes in mean body weight of mice in each group, (C) Kidney function indicator - urinary protein concentration, (D) Urinary protein to urinary creatinine ratio. Data presented as mean ± SEM. **p < 0.01, ***p < 0.001 using two-way ANOVA analysis. (E) Representative images of renal histopathology at the experimental endpoint (H&E staining, scale bar = 100 μm), showing glomerular necrosis (black arrows) and lymphocyte infiltration (white arrows). (F) Kidney injury score, **p<0.001, n=5; **** p<0.0001, n=5.

V-CD19-Exo treatment alleviates splenomegaly and systemic inflammation in MRL/lpr mice.

(A) Representative images of spleens at the treatment endpoint (week 19). Spleens from healthy control C57BL/6J mice, untreated MRL/lpr mice, control NK92-Exo treatment group, and V-CD19-Exo treatment group. Scale bar: 1 cm. (B-E) Quantitative analysis of spleen weight and serum inflammatory cytokines at the treatment endpoint. (B) Spleen weight. (C) Serum total IgE concentration. (D) Serum IL-17A concentration. (E) Serum IFN-γ concentration. Data are presented as mean ± standard error of the mean (n=X per group). Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant.

Autoantibody levels and survival during V-CD19-Exo treatment in MRL/lpr mice.

(A) Serum anti-double-stranded DNA IgG antibody levels, and (B) Anti-nuclear antibody titers (n=5). (C) Kaplan-Meier survival curve of MRL/lpr mice over a 30-week observation period (n=10).