Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
TLR7 and IFN Influence B Cell Selection in the Germinal Center.
Moisini1, Ioana, Huang1, Weiqing, Marion3, Tony, Davidson2, Anne
Male NZW/BXSB.Yaa (W/B) mice express two copies of the TLR7 gene and develop pathogenic autoantibodies whereas females with only one copy of TLR7 have attenuated disease. VH3H9 is an autoreactive Ig heavy chain that can pair with a variety of light chains generating both autoreactive and non-autoreactive antibodies. Our goal was to analyze the regulation of the autoantibody response in male and female 3H9.W/B mice.
3H9.W/B mice were bred and monitored for proteinuria. Some female mice received IFNa adenovirus at 3 months of age. Sera were analyzed for autoantibodies by ELISA. Mice were sacrificed at 8, 24 and 56 weeks of age and single cell PCR and sequencing of heavy and light chain Ig genes performed for all B cell subsets. Selected germline light chains were coexpressed with the 3H9 heavy chain and transfectant supernatants screened for autoreactivity by ELISA.
IgM and IgG anti-CL antibodies appeared in the serum by 12 weeks of age in both males and females. Anti-dsDNA antibodies appeared in the serum of males at 12 weeks and most had high titer IgG anti-CL and anti-dsDNA antibodies and developed >300mg/dl proteinuria and thrombocytopenia by 8 months. Females had only low titer IgG anti-CL antibodies and none developed proteinuria by 1 year.
Using flow cytometry and single cell PCR of 3H9 associated light chains we showed that males had a much smaller marginal zone (MZ) with a repertoire that was distinct from the follicular repertoire, indicating that the loss of MZ B cells was not due to diversion to the follicular compartment. The germinal center (GC) repertoire was more diverse in males than in females. Vk545 and Vk548 were overrepresented in the GC repertoire of both males and females but the VJ junctions were different between males and females. For example Vk548 junctional diversity generated Leu at position 116 of the light chain in 88% of female sequences compared with only 22% of male sequences. In contrast there was a Phe at position 116 in 52% of male sequences but only 5% of female sequences (p < 0.0001). Germline 3H9/Vk548 with Phe116 had anti-cardiolipin, anti-histone and anti-dsDNA activity whereas 3H9/Vk548 with Leu116 did not bind dsDNA or cardiolipin and retained binding only to histones, a specificity associated with low renal pathogenicity. Administration of IFNa to female mice induced anti-cardiolipin and anti-DNA autoantibodies and proteinuria and was associated with a diverse GC repertoire and a male pattern of junctional diversity in Vk545 and Vk548.
Tolerance to cardiolipin is broken in W/B mice as they age and is regulated independently of anti-DNA reactivity. Analysis of the naïve repertoire suggests a shift in the threshold for negative selection in males resulting in deletion of MZ B cells. In contrast, selection into or expansion in the germinal center is a major checkpoint for regulation of autoreactivity, and female germinal centers are regulated more stringently than those of the males. Our studies are consistent with the hypothesis that TLR7 overexpression or exogenous IFNa relaxes the stringency for selection in the germinal centers resulting in increased autoreactivity of the antigen driven B cell repertoire.
To cite this abstract, please use the following information:
Moisini, Ioana, Huang, Weiqing, Marion, Tony, Davidson, Anne; TLR7 and IFN Influence B Cell Selection in the Germinal Center. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1410