Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus

Abstract

Dysregulated B-cell activation plays pivotal roles in systemic lupus erythematosus (SLE), which makes B-cell depletion a potential strategy for SLE treatment. The clinical success of anti-CD19 CAR-T cells in treating B-cell malignancies has attracted the attention of researchers. In this study, we aimed to investigate the feasibility of applying anti-CD19 CAR-T cell therapy to SLE treatment in a mouse disease model. We constructed murine anti-CD19 CARs with either CD28 or 4-1BB as the intracellular costimulatory motif and evaluated the therapeutic function of the corresponding CAR-T cells by infusing them into MRL-lpr mice. Furthermore, anti-CD19 CAR-T cells were transferred to MRL-lpr mice before the onset of disease to determine their role in SLE prevention. According to our observations, compared with antibody treatment, the adoptive transfer of our anti-CD19 CAR-T cells showed a more sustained B-cell-depletion effect in MRL-lpr mice. The transfer of syngeneic anti-CD19 CAR-T cells not only prevented disease pathogenesis before the onset of disease symptoms but also displayed therapeutic benefits at a later stage after disease progression. We also tried to optimize the treatment strategy and found that compared with CAR-T cells with the CD28 costimulatory motif, CAR-T cells with the 4-1BB costimulatory motif showed better therapeutic efficiency without cell enrichment. Taken together, these results show that anti-CD19 CAR-T cell therapy was effective in the prevention and treatment of a murine model of SLE, indicating its potential for clinical use in patients.

Keywords: Autoimmune disease; B cells; Systemic lupus erythematosus; T cells; Treatment.

Conflict of interest statement

The authors declare no competing interests.

© 2020. CSI and USTC.

Figures

Fig. 1

CAR-T cell construction. a Flow cytometry showing mCherry-CAR expression on mouse primary T cells 2 days after transduction. b–d Effector cells, sorted CAR-T cells cultured for another 3–5 days; target cells, freshly sorted splenocytes labeled with CellTrace Violet. After coculture at the indicated ratios for 4 h, the percentage of lysed B cells (b) and levels of intracellular IFNγ expressed by effector cells (c) were analyzed by flow cytometry. d The supernatant IFNγ concentration after 24 h of coculture was analyzed by ELISA. The data are representative of at least two independent experiments with similar results

Fig. 2

Efficacy of CAR-T cells in an SLE mouse model. a Working scheme of CAR-T cell manufacture and adoptive transfer in a mouse experiment. The percentage of blood CD19+ cells at 1 week (b, c) or 9 weeks (d) after transfer was analyzed by flow cytometry; the percentage of blood CD138+ cells at 1 week (e) or 9 weeks (f) after transfer was analyzed by flow cytometry. Live CD45+ cells were gated and plotted (b–f). g Effector cells, lymphocytes isolated from TBI + CAR-T cell-treated mice 9 weeks after transfer; target cells, fresh splenocytes labeled with CellTrace Violet; the percentage of lysed B cells was analyzed by flow cytometry after 24 h of coculture. The data are representative of at least two independent experiments with similar results. The data were analyzed by Student’s t test, and significance is indicated by *P < 0.05, ****P < 0.0001, and ns no significant differences

Fig. 3

CAR-T cell treatment shows therapeutic benefits in mouse SLE. Mice received different treatments at 13 weeks of age and were sacrificed at 22 weeks of age for subsequent examinations. a The survival rate of MRL-lpr mice that received different treatments; the difference in survival between the TBI + PBS group and TBI + CAR-T group was compared by logrank (Mantel–Cox test) calculation. b The mouse skin lesions were scored, and the scores were plotted. c The ratios of spleen weight to total body weight were determined. d Representative hematoxylin and eosin (H&E)-stained sections of glomerular areas of the kidneys. Black arrow, immune complex formation; red arrow, infiltration of numerous lymphocytes. Original magnification, ×400. Bars represent 50 µm. e Statistical histology scores of H&E-stained kidney sections. f C3 and IgG deposition in the glomerulus was detected by immunofluorescence and is presented in a merged manner; frozen sections of the kidneys were stained with anti-mouse-C3-APC, anti-mouse-IgG-FITC, and DAPI. Original magnification, ×400. Bars represent 50 µm. The data are representative of at least two independent experiments with similar results. b, c The data were analyzed by Student’s t test, and significance is indicated by *P < 0.05

Fig. 4

1D3-4-1BB CAR-T cells partially deplete B cells. a Diagram of the DNA encoding 1D3-4-1BB CAR. It encodes the antibody light chains (blue boxes), including their native signal peptides (white box), joined by a flexible linker (blue bar) to the VH and fused to the transmembrane region of CD8a (navy blue), signaling domains of 4-1BB (red box) and cytosolic domain of CD3ζ (orange box). The construct also contains IRES-driven EGFP to allow the detection of transduced cells. b Flow cytometry of GFP+ expression on mouse primary T cells after lentiviral transduction. Cells were FVD− TCRβ+ gated. c Unsorted 4-1BB CAR-T cells (effector) and CellTrace Violet-labeled splenocytes (target) were cocultured for 4 h and analyzed by flow cytometry. Blood was collected from the femoral arteries of MRL-lpr mice at 1 week (d, f) or 9 weeks (e, g) after transfer, and CD19+ and CD138+ percentages among FVD− CD45+ cells were analyzed by flow cytometry. CD4/CD8 ratios in the blood were also analyzed by flow cytometry at 1 week (h) or 9 weeks (i) after transfer. The data are representative of at least two independent experiments with similar results. The data were analyzed by Student’s t test, and significance is indicated by *P < 0.05

Fig. 5

Therapeutic effects of 4-1BB CAR-T cell treatment. Mice that received low-dose TBI as preconditioning and adoptive transfer with 4-1BB CAR-T cells or PBS at 13 weeks of age were sacrificed at 22 weeks of age for subsequent examinations. a The survival rate of the mice; the difference in survival between the two groups was compared by logrank (Mantel–Cox test) calculation. b Total body weight and spleen weight. Sera were isolated from MRL-lpr mice; anti-dsDNA antibody (c) and ANA (d) levels were detected by ELISA. e Urine protein to creatinine ratios were analyzed by a chemistry analyzer. f Representative H&E-stained sections of glomerular areas of the kidneys. Black arrow, immune complex formation; red arrow, infiltration of numerous lymphocytes. Original magnification, ×400. Bars represent 50 µm. g C3 and IgG deposition in the glomerulus was detected by immunofluorescence and is presented in a merged manner; frozen sections of the kidney were stained with anti-mouse-C3-APC, anti-mouse-IgG-FITC, and DAPI. Original magnification, ×400. Bars represent 50 µm. The data are representative of at least two independent experiments with similar results. b, d The data were analyzed by Student’s t test, and significance is indicated by *P < 0.05