CD19-targeting CAR T cells protect from ANCA-induced acute kidney injury

Abstract

Objectives: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are life-threatening systemic autoimmune diseases manifesting in the kidneys as necrotizing crescentic glomerulonephritis (NCGN). ANCA antigens are myeloperoxidase (MPO) or proteinase 3. Current treatments include steroids, cytotoxic drugs and B cell-depleting antibodies. The use of chimeric antigen receptor (CAR) T cells in autoimmune diseases is a promising new therapeutic approach. We tested the hypothesis that CAR T cells targeting CD19 deplete B cells, including MPO-ANCA-producing B cells, thereby protecting from ANCA-induced NCGN.

Methods: We tested this hypothesis in a preclinical MPO-AAV mouse model. NCGN was established by immunisation of MPO^-/- mice with murine MPO, followed by irradiation and transplantation with haematopoietic cells from wild-type mice alone or together with either CD19-targeting CAR T cells or control CAR T cells.

Results: CD19 CAR T cells efficiently migrated to and persisted in bone marrow, spleen, peripheral blood and kidneys for up to 8 weeks. CD19 CAR T cells, but not control CAR T cells, depleted B cells and plasmablasts, enhanced the MPO-ANCA decline, and most importantly protected from NCGN.

Conclusion: Our proof-of-principle study may encourage further exploration of CAR T cells as a treatment for ANCA-vasculitis patients with the goal of drug-free remission.

Keywords: Autoantibodies; Autoimmune Diseases; Granulomatosis with polyangiitis; T-Lymphocyte subsets; Vasculitis.

Figure 1

CD19 CAR T cells stably engraft in mice with anti-myeloperoxidase (MPO) induced glomerulonephritis. (A) Experimental procedure of anti-MPO induced glomerulonephritis. The disease was induced by immunisation of MPO^−/− mice with murine MPO and subsequent irradiation and wild-type bone marrow (BM) transplantation. CAR T cells (2×10⁶) were simultaneously injected with BM cells (1.5×10⁷). (B) Schematic of the anti-mouse CD19 chimeric antigen receptor (CD19 CAR). (C) Mouse splenic T cells were transduced with CD19 CAR or control SP6 CAR. CAR surface expression was detected by anti-CD8 and anti-IgG costaining and percentages of CAR⁺-T cells are indicated (CD8⁺ and CD8⁻). (D) Mouse splenic T cells (5×10⁴) were let untransduced or transduced with the mouse CD19 (CD19) CAR or GFP (the same viral vector backbone) and cocultured for 24 hours in a 1:1 ratio with primary murine Eµ-TCL tumour B cells (CD19⁺) or splenic B220⁺ B cells. As activation marker, IFNγ in the supernatant was quantified by ELISA. Data of n=2 independent experiments are shown. (E) Representative flow-cytometry analysis displaying engraftment of CAR T cells in the spleen of SP6 and CD19 CAR-treated mice at 2, 5 and 8 weeks detected by anti-IgG and anti-CD3 costaining. (F) Flow cytometric quantification of CAR T cells in different lymphoid compartments and the kidneys of SP6 and CD19 CAR-treated mice at 2, 5 and 8 weeks. (G) Real-time PCR analysis of CAR constructs in spleen and kidneys. Ct values as measure for CAR presence were depicted. Ct values higher than 30 are considered negative. Data in (D), (F) and (G) are represented as individual values and averages with SD. Difference between SP6 and CD19, *p<0.05, **p<0.01. Two independent experiments with a total of 5–7 mice per group were done. CAR, chimeric antigen receptor; CFA, complete Freund’s adjuvant; ic, intracellular; IFA, incomplete Freund’s adjuvant; L, Whitlow linker; LTR, long terminal repeat; Max, maximal IFNγ-secretion by CAR T cells induced by Phorbol-12-myristate-13-acetate/Ionomycin; Min, minimal IFNγ-secretion by CAR-T cells without target cells; mIgFc, hinge domain of murine immunoglobulin fragment crystallisable; SP, signal peptide; tm, transmembrane region; VH, variable heavy chain; VL, variable light chain.

Figure 2

CD19 chimeric antigen receptor (CAR) T cells deplete CD19⁺ B cells in mice with anti-myeloperoxidase-induced glomerulonephritis. (A) Representative flow-cytometry displaying gated CD19⁺ B cells in the spleen of controls, SP6 and CD19 CAR-treated mice at 2, 5 and 8 weeks. (B) Numbers of CD19⁺ B cells in different lymphoid compartments and (C) in the kidneys. (D) Quantification of CD19 and (E) CD20 mRNA by real-time PCR of spleen and kidney samples of control mice, SP6 and CD19 CAR-treated mice at 2, 5 and 8 weeks. Data in (B–E) are represented as individual values and averages with SD. Difference between SP6 and CD19, *p<0.05, **p<0.01. Two independent experiments with a total of 5–7 mice per group were done. Blood and lymph node samples at 2 weeks were only taken from 2 to 3 animals. At 5 weeks only from three animals per group enough cells could be isolated from blood and lymph nodes to analyse B cells.

Figure 3

CD19 chimeric antigen receptor (CAR) T cells deplete plasmablasts in mice with anti-myeloperoxidase-induced glomerulonephritis. (A) Representative flow-cytometry displaying detection of plasmablasts (CD138⁻ B220⁺, orange), plasma cells (CD138⁺ B220⁻, magenta) and intermediate plasma cells (CD138⁺ B220⁺, green) in the spleen at 2 weeks. (B) Numbers of plasmablasts, intermediate plasma cells and plasma cells in spleen, bone marrow and kidneys determined by flow cytometry. Data in (B) are represented as individual values and averages with SD. Difference between SP6 and CD19, *p<0.05, **p<0.01. Two independent experiments with a total of 5–7 mice per group were done.

Figure 4

CD19 chimeric antigen receptor (CAR) T cells protect from anti-myeloperoxidase (MPO)-induced glomerulonephritis. (A) Representative kidney histology (PAS reaction) of controls (w/o CAR), SP6 and CD19 CAR-treated mice at 2, 5 and 8 weeks. Glomerular damage with incipient crescent development and hypercellularity at 5 weeks and evident crescents and necrosis at 8 weeks in both control groups is displayed. Scale bar, 50 µm. (B) Quantification of crescent and necrosis formation in the kidneys indicating protection from glomerular damage in CD19 CAR T cell-treated mice. (C) Quantification of kidney-infiltrating neutrophils (CD45⁺ CD11b⁺ Ly6G⁺) and monocytes (CD45⁺ CD11b⁺ Ly6G⁻ Ly6C⁺) by flow-cytometry. (D) Quantification of albuminuria (albumin to creatinine ratio, ACR) and of urinary neutrophil gelatinase-associated lipocalin as markers of kidney damage, measured by ELISA. Urine samples of n=7 healthy MPO^−/− mice (HC, healthy control) are measured for comparison. (E) Determination of anti-MPO titre before irradiation and bone marrow transplantation (week 0) and during disease course (weeks 2, 5, 8) by ELISA. Data in (B–D) are represented as individual values and averages with SD. Data in (E) are represented as averages (circles) with SD. Linear regression analysis (lines) was performed to evaluate differences of slopes. Difference between SP6 and CD19, ^#difference between control and CD19, ^*/#p<0.05, ^**/##p<0.01. Two independent experiments with a total of 5–7 mice per group were done.