Rescue of HIV-1 Broad Neutralizing Antibody-Expressing B Cells in 2F5 VH × VL Knockin Mice Reveals Multiple Tolerance Controls

Abstract

The HIV-1 broad neutralizing antibody (bnAb) 2F5 has been shown to be poly/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 VH knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. Here, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 VHxVL KI mice, and find an even higher degree of tolerance control than observed in the 2F5 VH KI strain. Although B-cell development was severely impaired in 2F5 VHxVL KI animals, we demonstrate rescue of their B-cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B-cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B-cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 VH/VL expression by immature/transitional B-cells. Importantly, 7% of rescued B-cells retained 2F5 VH/VL-expression and secreted Env-specific mAbs with HIV-1 neutralizing activity. This “partial” rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B-cells in 2F5 VHxVL KI × Eμ-bcl2 tg mice, and significant (yet modest) enrichment of Env-specific B-cells and serum Igs. The rescued 2F5 mAb-producing B-cell clones in this study are the first examples of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs, and provide a starting point for design of strategies aimed at rescuing such B-cells.

An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

“…These traits suggest that the development of these types of BnAbs may be limited by immune tolerance controls that impede the required tortuous or polyreactive BnAb maturation pathways (20,21,24). This notion is supported by a number of observations in 2F5 V H ×V L knockin mice: targeted expression of the 2F5 V H DJ H / V L J L rearrangements triggered a near-complete B cell developmental blockade at the pre-B to immature B cell stage (25); even when clonal deletion mechanisms were circumvented, 2F5 V H DJ H / V L J L -expressing broadly neutralizing B cell rescue was limited by κ chain editing and an anergic phenotype (26); and MPER-specific serum neutralizing IgG responses were elicited in 2F5 knockin mice immunized with MPER-lipid complexes by rescuing the anergic self-reactive 2F5-expressing B cells that survived the B cell developmental blockade (26).…”

Polyreactivity and Autoreactivity among HIV-1 Antibodies

“…The threshold for autoreactivity, an MFI Ն500-fold over the MFI of the 151K control, was chosen empirically to be consistent with previous work (18) and, more significantly, the results of V(D)J knock-in animal studies (19)(20)(21). We have now identified three nonpolyreactive bNAbs, VRC01, 10E8, and 2F5, as significantly autoreactive.…”

“…3 and 4). The final measure of significant poly-and autoreactivity is the generation of V(D)J knock-in mice (19)(20)(21)(46)(47)(48), and additional knock-in strains will be necessary to determine the significance of off-target bNAb reactivities. However, if the 2F5 and 4E10 mice are reasonable guides, we expect that most, if not all, of the poly-and autoreactive bNAbs studied here will be subject to some degree of immunological control.…”

“…Our characterization of two MPER-binding bNAbs, 2F5 and 4E10, identified two human proteins, kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3), as autoantigens mimicked by the 2F5 and 4E10 HIV-1 epitopes (18). Mimicry of these conserved self-antigens by HIV-1 suggests that bNAbs to the 2F5 and 4E10 epitopes are proscribed by immune tolerance, a notion supported by impaired B-cell development in knock-in mice expressing the 2F5 or 4E10 V H DJ H and V L J L rearrangements (19)(20)(21). Further, immunization of opossums that naturally carry a KYNU mutation abrogating the shared 2F5 HIV-1 epitope elicited extraordinary Ab titers to the gp41 2F5 motif missing in opossums (18).…”

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity

“…Glycosylated BCRs may also be recognized by lectins on follicular dendritic cells or macrophages in the germinal center to deliver a survival advantage (26). A need for some preimmune antibodies to mutate away from self-reactivity provides an alternative explanation for the phenomenon of repertoire shift during the maturation of antibody responses, when certain antibody specificities that ultimately dominate responses to haptens (52)(53)(54)(55)(56)(57) or RhD alloantigens (16,20) or bind conserved neutralizing epitopes of viruses (58)(59)(60)(61)(62) are minimally represented in the primary wave of plasma cells but gradually emerge after repetitive stimulation by foreign antigen. Inherited defects that prevent hypermutation away from self-reactivity by antimicrobial antibodies may explain the surprising prevalence of autoantibodies in human AICDA deficiency, where GCs are formed but hypermutation is crippled (63).…”