Background/Purpose: B-cell depletion therapy (BCDT) is a
promising therapy for autoimmune diseases but relapse can occur.  In rheumatoid
arthritis, patients who did not respond well to rituximab exhibited higher
levels of type I interferon (IFN) signature gene expression in PBMCs but the
underlying mechanisms remain unknown. Spleen marginal zone macrophages (MZMs)
are essential for tolerogenic clearance of apoptotic cells (ACs). Maintenance
of MZMs requires MZ B cells. The purpose of this study is to determine if
anti-CD20 depletion of MZ B cells promotes the loss of MZMs, leading to excessive
uncleared ACs and stimulation of autoreactive B cells by type I IFN in
autoimmune BXD2 mice.

Methods: BXD2 mice and normal B6 mice were administered anti-CD20
(clone 5D2, IgG2a) or isotype control (250 µg/single dose iv). B-cell depletion
and repopulation were determined over 9 weeks of time. The distribution of MZMs
in the spleen was examined by confocal microscopy. The expression of type I IFN
signature genes, Usp18 and Oasl, in repopulated B cells was
determined by quantitative PCR. The development of autoantibody titers and
autoAb producing B cells was determined by ELISA and ELISPOT assays.

Results:
Administration of anti-CD20 resulted in nearly complete depletion of B cells in
the PBMCs but there was less complete depletion of B cells in the spleen. In
the spleen, the deepest depletion occurred at week (wk) 2 and this was
associated with a dramatic decline in MZMs of both strains but uncleared ACs
were observed in only BXD2 but not B6 spleen. By wk 5, the spleen B-cell
repopulation rate was 69% in BXD2 but was only 26% in B6 mice and MZM and AC
clearance defects were found for both strains. By wk 9, there was >85%
repopulation of spleen B cells in both strains. In BXD2 mice, at wk 5, there
was a 1.7-fold increase in the number of PNA⁺Fas⁺ CD19⁺
spleen germinal center (GC) B cells and an increased number of IgG anti-DNA and
anti-histone producing B cells in the BCDT group, compared to the control
group. This is associated with an increased expression of Usp18 and Oasl
in repopulated MZ precursor (MZ-P) B cells in the BCDT group, compared to the
control group. At wk 9, serum titers of IgG autoantibodies against DNA,
histone, and malondialdehyde, a lipid peroxidation product associated with
apoptotic blebs, were markedly increased in the BCDT group, compared to the
control group. Interestingly, despite the relatively slower repopulation of B
cells and MZMs in anti-CD20 treated B6 mice, there was no elevation in GC B
cells and autoantibody titers when B cells were repopulated.

Conclusion: Although BXD2 mice responded to BCDT, anti-CD20 induced
repopulation of GC and autoreactive B cells. The relapse was associated with
the functional loss of tolerogenic AC clearance. This is consistent with our
previous findings that normal MZ B cells are required to maintain normal
tolerogenic function of MZMs. The results suggest that measurement of AC
by-production and type I IFN responses should be considered as biomarkers for
therapeutic efficacy and timing of repeated BCDT (This
worked was supported by the NIH (RO1-AI-071110, RO1-AI-083705, P30-AR-048311
and P30-AI-027767) and the VA Merit Review 1I01BX000600-01. Anti-CD20 and
isotype control are generous gifts from Genentech Inc.).