Contact-dependent suppression of FVIII-specific memory B cells by BAR-Tregs

Abstract

Abstract Anti-drug antibody formation poses tremendous obstacles for optimal treatment of hemophilia A (HA). In this study, we sought to utilize chimeric receptor-modified natural regulatory T cells (Tregs) to target FVIII-specific memory B cells, which are responsible for persistent anti-FVIII neutralizing antibodies (inhibitors) in HA patients. Thus, CD4+CD25hiCD304+ natural Tregs were FACS sorted from naïve C57BL/6 mice and retrovirally transduced to express a chimeric B-cell antibody receptor (BAR) containing the immunodominant A2 domain of FVIII. Plasmablast-depleted (CD138neg) splenocytes from FVIII/IFA immunized FVIII-knockout HA mice served as the source for FVIII-specific memory B cells, which were then specifically stimulated in vitro with FVIII and enumerated in B-cell ELISPOT assays. Adding A2-BAR Tregs (1 per 150 splenocytes), but not conventional T cells, to the CD138− splenocytes significantly suppressed the formation of anti-FVIII producing cells, compared to the non-relevant OVA-BAR Tregs control group. Transwell experiments confirmed that the suppression was contact-dependent. Interestingly, even in the presence of antibody to FVIII (so-called inhibitors), similarly prepared CD4+CD25hiCD127low A2-BAR human natural Tregs completely suppressed polyclonal anti-FVIII Elispots formation, indicating potential by-stander suppression by A2-BAR Tregs on all FVIII-specific B cells in the local milieu. In conclusion, we demonstrated in vitro that FVIII domain-expressing BAR Tregs could efficiently target and suppress FVIII-specific memory B cells. Supported by a Bayer Hemophilia Grant [AHZ] and NIH HL126727 [DWS].