Origin of autoantibodies

Production of antinuclear antibodies (ANA) is a consistent manifestation of SLE (frequency ∼100%) and often the earliest. This is not due to a global loss of tolerance, as the antigenic targets are highly restricted. Indeed, autoantibodies against chromatin/DNA, U1 small nuclear ribonucleoproteins (snRNPs, recognized by anti-Sm and RNP antibodies), small cytoplasmic “Y” RNAs associated with Ro60, and other antigens (La, ribosomal P, RNA helicase A, and phospholipid-binding proteins) represent a serological signature of SLE. Anti-double-stranded (ds) DNA, anti-Sm, and anti-phospholipid antibodies are used diagnostically.

This chapter considers the origins of autoantibodies associated with SLE and their relationship to disease pathogenesis. We will focus on the origins of high-affinity (predominantly IgG) autoantibodies against the components of nucleic acid-protein complexes.

B cell tolerance in SLE

B cell activation depends on positive and negative signals transmitted through the B cell receptor (BCR) and co-receptors as well as competition for survival factors such as B cell activating factor (BAFF). The balance of these positive and negative signals, regulated by T cells, determines whether a B cell becomes activated or is tolerized. Genetic polymorphisms and mutations affecting these signaling pathways are associated with increased numbers of autoreactive B cells ( Table 26.1 ).

Table 26.1

Some genetic abnormalities causing increased numbers of autoreactive B cells.

Gene	Defect	Checkpoint(s)	Mechanism
PTPN22	C1858T	Central and peripheral	Reduced deletion/editing in BM; regulation of memory B cells ^a
MyD88 , IRAK4 , or UNC93B1	Mutation	Central	Abnormal regulation of TLR signaling resulting in increased numbers of polyreactive immature B cells without autoantibody production ^b
Btk	Transgene expression in mice	Central and peripheral	Alters threshold for B cell activation and negative selection of autoreactive B cells ^c
Lyn	B cell deficiency	Peripheral	Abnormal GC formation, increased BAFF, activation of transitional and follicular B cells with autoantibodies ^d
Fcgr2b	Deficiency in mice (knock-out)	Peripheral	Increased numbers of autoreactive GC B cells ^e
Tnfsf13b (BAFF)	Transgene expression in mice	Peripheral	Hyperactive NFκB2 signaling, expanded MZ B cells, increased GC formation ^f
AICDA (AID)	Deficiency (mutation)	Central	Abnormal Ig repertoire and increased serum autoantibodies (due to decreased SHM away from autoreactivity?) ^g

a Rhee I, et al. Nat Immunol 2012;13:439–447; Arechiga AF, et al. J Immunol 2009;182:3343–3347

b Fukui R, et al. Immunity 2011;35:69–81

c Kil LP, et al. Blood 2012;119:3744–3756

d Lamagna C, et al. J Immunol 2014;192:919–928

e Tiler T. J Exp Med 2010;207:2767–2778

f Liu Z, et al. Trends Immunol 2011;32:388–394; Thien M, Immunity 2004;20:785–798

g Meyers G, et al. Proc Natl Acad Sci USA 2011;108:11554–11559.

The immature BCR repertoire contains many receptors that bind DNA or other self-antigens. Additional autoreactive B cells are generated peripherally by somatic hypermutation (SHM) in secondary lymphoid organs. Self-tolerance is mediated at both central bone marrow (BM) and peripheral checkpoints. BCRs exhibiting high affinity for ubiquitous self-antigens generally are either deleted centrally or undergo receptor editing. In contrast, autoreactive B cells that do not encounter antigen in the BM or bind with low affinity to self-antigens are censored peripherally through deletion (at the transitional B cell stage), anergy (at the follicular B cell stage), or antigen-induced cell death. Regardless of origin, resting B cells do not secrete immunoglobulin until they differentiate into plasma cells. Thus, autoreactive B cells can circulate without producing autoantibodies. Most autoreactive B cells in the periphery are anergic, have the phenotype CD20 ⁺ CD27 ^- CD38 ^hi CD24 ^hi CD10 ⁺ , and express low levels of CD21.

Altered BCR signaling in lupus

Altered BCR signaling is associated with increased numbers of autoreactive B cells ( Table 26.1 ). Polymorphism of the protein tyrosine phosphatase PTPN22 decreases B cell responsiveness causing defective central censoring of autoreactive B cells and autoimmunity. Heterozygous mutations of the TNFRSF13B gene encoding TACI, a protein that interacts with TLR7 and TLR9, also impair central censoring of autoreactive B cells leading to ANAs. However, in contrast to SLE patients, these individuals do not accumulate autoreactive mature naïve B cells, consistent with defective peripheral tolerance.

Over-expression of the Bruton’s tyrosine kinase ( Btk ) gene causes spontaneous germinal center (GC) formation, hyper-responsiveness to BCR stimulation, altered peripheral censoring of autoreactive B cells, ANAs, and lupus-like disease. Deletion of the tyrosine kinase Lyn in B cells enhances signaling in transitional and follicular B cells, leading to anti-Sm and anti-dsDNA autoantibodies, glomerulonephritis, poor GC formation, and increased B1a cells. The autoimmune phenotype is reversed by deleting MyD88, suggesting that toll-like receptors (TLRs) have a role in B cell tolerance. Decreased LYN expression also is reported in human SLE.

Properties of lupus autoantibodies

DNA sequencing of the V _H and V _L regions expressed by autoantibody-producing hybridoma cells from lupus mice reveals SHM. A single somatic mutation can transform an antibody against a non-self antigen to an autoantibody. Moreover, most lupus autoantibodies are derived from B cells that have undergone class switch recombination (CSR) to IgG, suggesting that they are products of T cell driven responses generated within GCs. This is further supported by evidence that autoantibody levels are maintained by long-lived plasma cells, usually derived from post-GC B cells. However, other studies show that T cell-independent, class-switched, somatically-mutated autoantibodies also can be generated extrafollicularly. There is debate over the relative importance of each pathway.

GC versus extrafollicular origin of autoantibodies

Mature B cells consist of three subsets: follicular (FO), marginal zone (MZ), and B-1, a self-renewing population that localizes near serosal surfaces. All three subsets are implicated in autoantibody formation. FO B cells are involved in T cell-dependent (TD) formation of GCs in response to protein antigens. In contrast, MZ B cells respond to particulate antigens in a T cell-independent (TI) manner. They respond to TI-1 antigens (e.g., lipopolysaccharide, which engages TLR4) and TI-2 antigens (e.g., pneumococcal polysaccharide, which consists of highly repetitive epitopes), although they also can become GC B cells in response to TD antigens. B-1 cells are the primary source of polyreactive “natural” IgM antibodies, which opsonize bacteria and like MZ B cells respond to TI-1 and TI-2 antigens. In general, FO B cells are more highly subject to SHM and CSR than MZ or B-1 cells. However, SHM and CSR can occur in all three subsets. Likewise, although memory B cell responses are considered a property of FO B cell responses to TD antigens, memory also can develop to TI-1 and TI-2 antigens.

Role of GC

Tolerance of anti-Sm B cells is critically dependent on censoring at the FO B cell and pre-plasma cell stages. There is considerable evidence in mice that post-GC B cells mediate anti-Sm and anti-dsDNA responses. Key features of the GC reaction include a requirement for T follicular helper (T _FH ) cells and production of antibodies that have undergone SHM and CSR. Evidence for the role of GCs comes from the near-simultaneous development of anti-dsDNA autoantibodies and lupus-like disease in sanroque mice. Homozygyosity for a mutation of the ubiquitin ligase Roquin in sanroque mice causes accumulation of T _FH cells, spontaneous GC formation, autoantibodies, and glomerulonephritis. B cell activation in GCs reflects competition for limited numbers of T _FH cells, which provide positive and negative selection signals via CD40L-CD40 and Fas-FasL signaling, respectively. Fas mutations increase GC and memory B cells following immunization with TD antigens and cause autoantibody production in B6/ lpr mice and lupus in MRL/ lpr mice. Fas deficiency promotes abnormal localization of autoreactive B cells to the T cell zone, where censoring may be ineffective. Whereas Fas regulates negative selection of autoreactive GC B cells, Roquin affects positive selection by increasing T _FH cells in GCs.

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