9G4+ Autoantibodies Are an Important Source of Apoptotic Cell Reactivity Associated With High Levels of Disease Activity in Systemic Lupus Erythematosus

Abstract

Objective To determine the prevalence anti-apoptotic cell antibodies with the 9G4+ idiotype (9G4+) and the relationship between this reactivity and other known 9G4+ specificities and disease activity. Methods Sera from 60 SLE patients and 40 healthy control donors were incubated with apoptotic Jurkat cells and assayed for binding of 9G4+ antibodies by flow cytometry. These samples were also tested for 9G4+ reactivity against naïve B cells and total IgG and IgM anti-apoptotic cell antibody (AACA) reactivity. Results 9G4+ antibodies bound late apoptotic cells in a majority of SLE patients (60%) but only 2 healthy control sera. Among samples with global IgM or IgG AACA, samples with 9G4+ AACA predominated. Patients with high levels of 9G4+ AACA were more likely to have active disease and this remained true even in patients with IgG AACA, or anti-dsDNA. Patients with lupus nephritis were also more likely to have 9G4+ AACA. While 9G4+ reactivity to apoptotic cells often coincided with anti-B cell reactivity, some samples had distinct anti-apoptotic cell or anti-B cell reactivity. Conclusion 9G4+ antibodies represent a major species of anti-apoptotic cell antibodies in SLE serum and this autoreactivity is associated with disease activity. The anti-apoptotic cell reactivity of 9G4+ antibodies can be separated from the germline VH4-34 encoded anti-B cell autoreactivity. Our results indicate that apoptotic cells are an important antigenic source in SLE that positively select B cells with intrinsic autoreactivity against other self-antigens. This selection of 9G4+ B cells by apoptotic cells may represent an important step in disease progression.

Molecular Basis of 9G4 B Cell Autoreactivity in Human Systemic Lupus Erythematosus

“…In vivo, 9G4 BCB is prominent in active SLE and correlates with naive B cell lymphopenia possibly due to a direct lytic activity of these Abs (9,23). Yet, 9G4 Abs may also react with ssDNA, dsDNA (11,(24)(25)(26), as well as apoptotic cells as shown own by our studies of SLE sera (27)(28)(29) and by chronic lymphocytic leukemia studies (30). The latter reactivity is of great interest because apoptotic cells accumulate in SLE GC (31) and may induce antiapoptotic cell Abs (APCA) with pathogenic functions (32)(33)(34)(35)(36)(37)(38)(39)(40).…”

“…It is important to emphasize that in addition to studying a number of mAbs that compare favorably with other studies, the focus on a single VH gene considerably enhances the power to understand the structural features that underlie the participation of 9G4 Abs in several SLE-related autoreactivities. It should be noted that although our mAbs were derived from only two patients, their autoreactivity mirrors the 9G4 serum autoreactivity of large numbers of SLE patients tested in our laboratory and is therefore representative of this disease population at large (9,28,29). Accordingly, we slanted our experimental design toward the analysis of large numbers of original Abs, mutants, and L chain hybrids rather than increasing the number of donors, as this design is better suited to the strength of any structural correlates.…”

“…The ensuing APCA may play a powerful effector pathogenic role not only through conventional type III hypersensitivity reactions but also through their ability to induce type I IFN production by dendritic cells (40). Again, 9G4 cells represent a highly informative model as they are specifically expanded in the GC of SLE patients but not in other autoimmune diseases (12), and 9G4 APCB binding is strongly present in SLE serum and correlates with disease activity population at large (28,29). Indeed, we observed enhanced APCB in a significant fraction (31%) of SLE 9G4 memory cells in the context of substantially lower levels of BCB relative to SLE and HC naive cells.…”

“…Our initial recognition of 9G4 APCB in SLE (9,27,28) has been recently consolidated by extensive serological analysis in our laboratory showing that such autoreactivity is present in 60% of unselected SLE patients, is present in the vast majority of patients with elevated serum 9G4 Ab titers (80% of 9G4 + ), and correlates with disease activity and active lupus nephritis (29). A summary of these data are provided in Supplemental Fig.…”

Rationale for B cell targeting in SLE

“…SLE autoantibodies include disease-specific ones such as anti-dsDNA/chromatin/nucleosomes, anti-Sm and autoantibodies encoded by VH4-34 (recognized by the 9G4 anti-idiotype and accordingly referred to as 9G4 antibodies)[66,67]. Of great interest, with the exception of anti-Sm, SLE-specific autoantibodies tend to fluctuate with disease activity [68–70,66], thereby suggesting that they are produced by short-lived PB generated by ongoing immune responses, a feature of significant importance for the design and understanding of outcome of B cell therapies. The importance of B cell in SLE is also illustrated by multiple B cell abnormalities [71,67,58,72–75]; the concentration of SLE susceptibility genes on B cell pathways [76,77]; and the efficacy of B cell therapies [78,79,52,80].…”