Cutting Edge: DNase II deficiency prevents activation of autoreactive B cells by double-stranded DNA endogenous ligands

Abstract

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.

Figure 1. DKO mice make RNA-biased ANAs through a TLR-dependent mechanism

A. Monoclonal autoantibodies (mAb) reactive with dsDNA (a) or RNA (c) or sera from BALB Fas-deficient (BALB/lpr, b) or RNA (c), DKO (d–f), Het (g), and TKO (h–i) mice were evaluated for ANA staining on HEp-2 coated slides by immunofluorescence. White arrows indicate examples of mitotic plates. (B) ANA staining patterns of serum from BALB/lpr (n=12) and DKO (n=23) mice were classified as nuclear homogenous or nuclear speckled and/or cytoplasmic.

Figure 2. Splenomegaly is TLR-dependent

(A–B) Spleen weights from DNase Het, DKO and TKO mice were determined at 10 weeks of age. Each dot represents 1 mouse, n=12 for all groups. (C) Percentage of B220⁺ cells in total spleen cells. (D) Representative FACS plots of spleen cells stained for B220 and AA4.1 and (E) average total number of splenic B cells and % mature B cells (grey portion of the bar and inserted number) n=8 mice for all groups. (F) Representative FACS plots of spleen cells stained for Ter119 vs side scatter (SSC). (G) Percentage of splenic Ter119⁺ cells in Het, DKO, and TKO mice. n=8 mice. N.S. is not significant, P>0.05; *, P < 0.005; and **, P < 0.00005 by Student’s t-test.

Figure 3. DVD-Ig^™ molecules bind both IgM and DNA and induce non-Tg B cells to proliferate through a TLR9-dependent mechanism

(A) Schematic diagram of bifunctional DVD-Ig^™ molecules. (B) IgM-binding ELISA of representative DVD-Ig^™ molecules, with anti-IgM domain as V1 (DVD3756, blue), or with the anti-IgM domain as V2 (DVD3751, red; DVD3754, green), compared to the original anti-IgM antibody. (C) ANA staining patterns of the original anti-DNA mAb compared to the DVD-Ig molecules depicted in B. (D) Composite plot of EC50 and ANA score with capacity of each DVD-Ig molecule to activate B cells, as determined by ³H-thymidine incorporation, indicated by the size of the circle; (proliferation index, large circle >20-fold; medium circle 10–20 fold; small circle <10-fold). The color of the circle corresponds to the DVD-Ig molecules depicted in B and C, additional DVD-Ig molecules depicted as open black circles, and original mAbs indicated by filled black circles. (E) WT or Tlr9^−/− B cells were stimulated with the DVD3754 or anti-DNA mAb for 24hr and compared to medium control. Proliferation was assessed by ³H-thymidine incorporation. The data represent the average of 3 separate experiments +/− the SEM.

Figure 4. B cell response to DNA ICs requires DNase II

(A–B) B cells from Het (circle), DKO (square), and TKO (triangle) mice were stimulated with anti-IgM, ODN 1826 or DVD3754 for 24 hr and proliferation was determined by ³H-thymidine incorporation. Results in (A) are representative of 3 independent experiments and (B) are average of separate experiments include 8 mice/group and are summarized as CPM +/− SEM.