Cross-Reactivity to Kynureninase Tolerizes B Cells That Express the HIV-1 Broadly Neutralizing Antibody 2F5

2F5 is an HIV-1 broadly neutralizing Ab that also binds the autoantigens kynureninase (KYNU) and anionic lipids. Generation of 2F5-like Abs is proscribed by immune tolerance, but it is unclear which autospecificity is responsible. We sampled the BCR repertoire of 2F5 knock-in mice before and after the first and second tolerance checkpoints. Nearly all small pre-B (precheckpoint) and 35-70% of anergic peripheral B cells (postcheckpoint) expressed the 2F5 BCR and maintained KYNU, lipid, and HIV-1 gp41 reactivity. In contrast, all postcheckpoint mature follicular (MF) B cells had undergone L chain editing that purged KYNU and gp41 binding but left lipid reactivity largely intact. We conclude that specificity for KYNU is the primary driver of tolerization of 2F5-expressing B cells. The MF and anergic B cell populations favored distinct collections of editor L chains; surprisingly, however, MF and anergic B cells also frequently expressed identical BCRs. These results imply that BCR autoreactivity is the primary determinant of whether a developing B cell enters the MF or anergic compartments, with a secondary role for stochastic factors that slightly mix the two pools. Our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1 and supports activation of anergic B cells as a potential method for HIV-1 vaccination.