Precise Targeting of Autoantigen-Specific B Cells in Lupus Nephritis with Chimeric Autoantibody Receptor T Cells

Abstract

Despite conventional therapy, lupus nephritis (LN) remains a significant contributor to short- and long-term morbidity and mortality. B cell abnormalities and the production of autoantibodies against nuclear complexes like anti-dsDNA are recognised as key players in the pathogenesis of LN. To address the challenges of chronic immunosuppression associated with current therapies, we have engineered T cells to express chimeric autoantibody receptors (DNA-CAART) for the precise targeting of B cells expressing anti-dsDNA autoantibodies. T cells from LN patients were transduced using six different CAAR vectors based on their antigen specificity, including alpha-actinin, histone-1, heparan sulphate, or C1q. The cytotoxicity, cytokine production, and cell-cell contact of DNA-CAART were thoroughly investigated in co-culture experiments with B cells isolated from patients, both with and without anti-dsDNA positivity. The therapeutic effects were further evaluated using an in vitro immune kidney LN organoid. Among the six proposed DNA-CAART, DNA4 and DNA6 demonstrated superior selectively cytotoxic activity against anti-dsDNA⁺ B cells. Notably, DNA4-CAART exhibited improvements in organoid morphology, apoptosis, and the inflammatory process in the presence of IFNα-stimulated anti-dsDNA⁺ B cells. Based on these findings, DNA4-CAART emerge as promising candidates for modulating autoimmunity and represent a novel approach for the treatment of LN.

Keywords: B cell; CAART; anti-dsDNA; lupus nephritis.

Figure 1

DNA-CAART cell expression on primary human T cells from anti-dsDNA⁺ LN patients. (A) Schematic concept of the proposed DNA-CAART therapy for LN treatment. Six DNA-CAARTs were designed using different autoantigen protein sequences: DNA1 and DNA2-CAARs with sequence for α–actinin protein (αA1-2); DNA2 and DNA3-CAAR with sequence for heparan sulphate (HS1-2); DNA5-CAAR sequence for histone-1 (H1) and DNA6-CAAR sequence for C1q (C1q). Cross-reactivity of these antigens with BCRs for anti-dsDNA in B cells will induce their selective B cell depletion. Figure made in BioRender.com (accessed on 16 January 2024). (B) Primary human T cells were transduced with DNA1-6-CAAR lentivirus and CAAR expression was detected using anti-protein FITC antibody. CAAR⁺ transduction efficiency was determined in anti-dsDNA⁺ LN patients (N = 10). *** p < 0.001, one-way analysis of variance (ANOVA) was performed to analyse differences between six groups and t-test between two groups. (C) Gene expression of CAAR antigens (alpha-actinin, heparan sulphate, histone 1, and C1q) was evaluated in each DNA1-6-CAART cell from samples of anti-DNA⁺ LN patients (N = 10). Fold changes were calculated over control (non-transduced T cells). *** p < 0.001, one-way analysis of variance (ANOVA) was performed to analyse differences between six groups.

Figure 2

Evaluation of selective anti-DNA⁺ B cell depletion by DNA-CAART cells. (A) In vitro anti-dsDNA production of B cells isolated from anti-DNA⁺ LN patients (N = 10) was evaluated after being stimulated with IFNα or IFNβ (50 ng/mL) or in non-stimulated conditions (PBS buffer) for 72 h. One-way analysis of variance (ANOVA) was performed to analyse differences between three groups. *** p < 0.001. (B) Anti-dsDNA levels of IFNα-stimulated B cells isolated from anti-dsDNA⁺ or anti-dsDNA⁻ LN patients (N = 10 each group). Levels were measured in culture medium after 24 h of stimulation by ELISA. Student’s t-test. *** p < 0.001. (C) Cytotoxicity by DNA-CAAR T cells (effector cells) against anti-dsDNA⁺ or anti-dsDNA⁻-producing B cells (target cells) were measured at different E:T ratios after 24 h of co-culture. The percentage of lysed B cells was analysed by flow cytometry. Error bars represent the mean ± SEM from culture experiments (N = 10). Significant differences were calculated in comparison with the control (non-transduced T cells) in each E:T ratio using a paired t-test. * p < 0.05, ** p < 0.005 and *** p < 0.001.

Figure 3

Evaluation of selective anti-DNA⁺ B cells depletion by DNA-CAAR T cells. (A) Gene expressions of apoptotic genes (caspase 3, BIM, p53) were evaluated in DNA-CAAR T cell co-cultures from samples of anti-dsDNA⁺ or anti-dsDNA⁻ LN patients (N = 10). Fold changes were calculated over control (non-transduced T cells) by Student’s t-test. *** p < 0.001. (B,C) Enzyme-linked immunosorbent assay (ELISA) was performed in supernatants of DNA-CAAR T cells co-incubated with anti-dsDNA⁺ B cells (10:1 E:T, 24 h, experiments run using primary cells from different LN patients (N = 10)) to measure IFNγ (B) or anti-dsDNA levels (C). One-way analysis of variance (ANOVA) was performed for multiple comparisons. * p < 0.05, ** p < 0.005 and *** p < 0.001. (D) Standards of anti-dsDNA antibodies (0, 50, 100, 250, 500 UI/mL) were added to the co-culture of DNA4 or DNA6-CAART with IFN-α-stimulated anti-dsDNA⁺ B cells (ratio 10:1, E:T). After 24 h, the cytotoxicity was not altered in these conditions.

Figure 4

Visualization and evaluation of DNA4 and DNA6-CAART interaction with target B cells. (A) In vitro co-culture of PE-CD19 labelled B cells isolated from anti-dsDNA^+/− LN patients with DNA4 or DNA6-CAART (n = 10). Immunofluorescence images were captured after 24 h (ratio E:T, 10:1). Scale bar = 2 µm. One-way analysis of variance (ANOVA) was performed to analyse differences between three groups. *** p < 0.001. (B) Confocal imaging visualised DNA4 or DNA6-CAART (green)-mediated interaction of anti-dsDNA⁺ B cell (red) within 6 h of cell–cell contact. DNA-CAART were co-cultured with anti-dsDNA⁺ B cells in a effector-to-target ratio of 10:1. Image series illustrates the dynamics of DNA-CAART killing at 2, 6, and 24 h. Cell–cell contacts are marked with white arrow. * p < 0.05 and ** p < 0.005. Scale bar = 50 µm.

Figure 5

B cells from anti-DNA⁺ patients damage kidney organoid overtime. (A) Scheme of co-culture between anti-dsDNA⁺ B cells from LN patients and kidney organoid established from healthy donor cells. (B) Study of kidney organoid morphology after co-culture with anti-dsDNA+ IFNα-stimulated B cells for 48 h (n = 5). Scale bar = 50 µm. One-way analysis of variance (ANOVA) was performed to analyse differences between the groups. ** p < 0.005, *** p < 0.001. (C) Immunofluorescence image captured at 2, 4, 6, 18, 24, and 48 h using confocal microscopy to characterize the interaction between anti-dsDNA+ IFNα-stimulated B cells and kidney organoids. Arrows mark the positive PE-labelled anti-dsDNA⁺ B cells (red). Scale bar = 100 µm.

Figure 6

Therapeutic effect of DNA4 and DNA6-CAART on 3D immune-kidney organoid model damage. (A) In vivo imaging of 3D immune-kidney organoid with DNA4-CAART, where B cells were labelled with PE (red) and DNA4-CAART with FITC (green) two hours after the B cell-organoid co-culture DNA-CAART was added. Arrows indicate the killing of B cells by DNA4-CAART at 24 h and 48 h. Scale bar = 50 µm. (B) Morphology analysis of 3D immune-kidney organoid after 2, 24 and 48 h incubation with DNA4 or DNA6-CAART treatment or without treatment. Analyses of circularity, aspect ratio, roundness, and solidity were performed using Image J software version 1.51 (n = 5). Scale bar = 100 µm. Statistical analysis was performed between groups using two-way ANOVA. Statistical significance shown is in comparison with untreated control. ** p < 0.005, *** p < 0.001. (C) TUNEL assay was performed to quantify apoptotic cells (red) and protein levels of KIM-1 were quantified by immunofluorescence (green). Relative intensity of the fluorescence value is used as non-treatment conditions (control), and absolute values were obtained using Image J software version 1.51 (n = 5). Scale bar = 20 µm. One-way ANOVA was performed to analyse differences between the three groups. * p < 0.05. (D) To evaluate fibrosis, collagen IV and vimentin levels were measured and analysed via a one-way ANOVA test to obtain statistical significance between groups. Scale bar = 20 µm. * p < 0.05, ** p < 0.005.