Background/Purpose

Systemic lupus erythematous (SLE) is characterized by production of highly pathogenic IgG autoantibodies (autoAbs).  While serum autoAb profiling is standard, it remains challenging to isolate authentic autoreactive B cells that give rise to these autoAbs.  Similar autoAbs are spontaneously produced in lupus-prone BXD2 mice that display apoptotic cell clearance defects and heightened type I interferon (IFN) signals.  The purpose of this study is to develop a comprehensive autoepitope and tetramer strategy to identify the dominant autoantigens (autoAg) and the underlying mechanisms promoting the development of autoreactive B cells in BXD2 mice.

Methods

A PEPperPRINT Autoimmunity Microarray covering 3,653 database derived, linear B-cell autoepitopes was probed with B6 and BXD2 serum to determine IgM and IgG binding reactivity.  Top peptides were selected for 3D crystal structural analysis.  A panel of over 50 peptide epitopes were verified by ELISA and ELISPOT.  Two epitopes from RNA-binding lupus La and 70 kDa U1snRNP were used to generate B cell tetramers for characterization of Ag-specific subpopulations of B cells.  

Results

The dominant epitopes recognized by BXD2 autoAbs are those commonly found in human SLE patients. Many immunodominant epitopes are located in cryptic regions of the native protein.  The strongest autoAb response was observed with nuclear peptides from RNA binding proteins (RBP) including lupus La_13-27  LEAKICHQIEYYFGD, U1snRNP_357-373SHRSERERRRDRDRDRD, and SmD1_97-111RGRGRGRGRGRGRG.  Other nuclear autoAgs include histones, centromere, and RNA polymerase III.  Tetramer analysis revealed a significant expansion in La⁺ and snRNP⁺ memory B cells and marginal zone precursor (MZ-P) B cells in BXD2 vs. B6 in both the circulation and the spleen.  Phenotype analysis of tetramer⁺MZ-P B cells showed upregulation of type I IFN inducible activation molecules CD69 and CD86 in BXD2 mouse spleens.

Conclusion

The bona fide presence of RBP cryptic autoAgs that were not predicted by Ag prediction programs is consistent with the previous finding that defective clearance of apoptotic blebs in BXD2 mice leads to production of autoepitopes that break B cell tolerance.  The potential activation of the TLR7 pathway by RBPs in combination with the upregulation of type I IFN response in Ag-specific MZ-P B cells further suggest that expanded tetramer⁺ MZ-P B cells may be a result of apoptotic cell clearance defects.  In light of our previous findings that MZ-P B cells are exceptional apoptotic Ag delivery MHCII^hi, CD86^hicells and can stimulate CD4 T cells, these results suggest that specific targeting on TLR7 and type I IFNs may be important to eliminate autoAg-specific B cells.

Grant support: NIH 1RO1 AI 071110, NIH 1RO1 AI083705, NIH P30 AR048311, Veterans Affairs Merit Review Grant 1I01BX000600, Rheumatology Research Foundation, Lupus Research Institute, and Arthritis Foundation Post-doctoral Fellowship

ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-cell-autoepitope-and-tetramer-analysis-reveals-expansion-of-apoptotic-autoantigen-la-and-snrnp-reactive-b-cells-in-bxd2-mice/