Autoreactivity in an HIV-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance

Abstract

We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (V(H)DJ(H)) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 V(H)DJ(H) insertion supported development of large- and small pre-B cells that expressed the chimeric human/mouse Igmu chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 V(H)DJ(H) knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 V(H)DJ(H) knock-in mice support reduced numbers of residual splenic B cells with low surface IgM density, severely diminished serum IgM levels, but normal to elevated quantities of serum IgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery.

Fig. 1.

The chimeric, recombinant 2F5 antibody (m2F5) is functionally equivalent to the original human 2F5 mAb (h2F5) in vitro. (A and B) Functional comparison of m2F5 with h2F5 by SPR analysis demonstrating that antigen binding specificity and lipid reactivity are preserved in the recombinant m2F5 antibody. (A) m2F5 bound to the HIV-1 gp41 membrane-proximal external region (MPER) peptides gp41_652–671 MPER (Top) and gp41_656–683 MPER (Bottom), which include the 2F5 binding epitope (ELDKWAS). The binding of m2F5 (solid line) to MPER peptides was comparable to those of h2F5 (dashed line). The CD4i gp120 mAb 17b (dotted line) was used as a negative control. (B) Lipid reactivity was retained in m2F5 antibody, and binding to liposomes with either phosphatidylserine (dashed line) or cardiolipin (solid line) was observed with both h2F5 and m2F5 mAbs. (C) m2F5 and h2F5 antibodies react with comparable avidity and specificity to mouse NIH/3T3 cell nuclear antigens. (D) m2F5 and h2F5 antibodies have comparable reactivities to human HEp-2 cell nuclear antigens. In addition, m2F5 and h2F5 were tested in the TZM-bl HIV Env pseudovirus neutralization assay, and both antibodies neutralized HIV-1 strains B.BG1168, B.SF162, B.QH0692, B.JRFL, and C.92UG0237, but not C.TV-1. IC₅₀ neutralization titer values for m2F5 and h2F5 ranged from 0.35 to 11.9 μg/mL and from 0.06 to 1.8 μg/mL, respectively (Table S1).

Fig. 2.

Targeted replacement of the J_H cluster with the 2F5 V_H gene. Genomic structure of the 2F5 V_H targeting construct, the endogenous Ig HC locus, the targeted allele after homologous recombination, and the targeted allele after Cre-mediated neo cassette deletion. The 2F5 V_H expression cassette comprises a J558 H10 family V_H promoter (p), an H10 split leader sequence (L), and the prerearranged 2F5 V_HDJ_H segment (2F5 V_H). Exons are represented as closed boxes, the Igh intronic enhancer (E) is represented by a circle, the 2F5 V_H, neo, and CAG-DTA cassettes are represented by shaded boxes, and loxP sites are depicted as triangles. The indicated restriction fragment sizes are indicated for WT and targeted loci. The 5′ and 3′ probes used to verify homologous recombination events at the 5′ and 3′ regions of the J_H-Eμ region, respectively, are shown as black bars. Also indicated are PCR primers to identify (i) homologous recombinant clones (black arrows), (ii) removal of the neo marker (gray arrows), and (iii) germline-transmitted heterozygous (2F5 V_H^+/−) and homozygous (2F5 V_H^+/+) mice (colored arrows). B, Bam HI; RV, EcoRV; n, Nde I.

Fig. 3.

Flow cytometric analysis of B cell development in the BM of C57BL/6 (WT), 2F5 V_H^+/−, and 2F5 V_H^+/+ mice. Left: Representative dot plot histograms, with numbers indicating the percentage of cells within total BM B cell populations (gated as singlet, live, lin⁻ cells; lin = Ter-119, Gr-1, CD11b, CD4, and CD8). BM cells were isolated from 9–12-week-old female mice. Right: Statistical analysis of BM B cell subset frequencies with each black, open, and gray circle representing an individual WT, 2F5 V_H^+/−, and 2F5 V_H^+/+ mouse, respectively; horizontal lines represent averages for each group. Significance values were determined by a two-tailed Student's test: *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.0001; NS, not significant. Populations were defined as follows: pro/large pre-B (fractions A–C’; B220^loCD43⁺), small pre-B (fraction D; B220^loCD43⁻), immature B (B220^int/loIgM^loIgD⁻), transitional, T1+T2 (B220^intIgM^int/hiIgD^lo), and BM mature (B220^hiIgM^intIgD^hi).

Fig. 4.

Flow cytometric analysis of WT and 2F5 V_H transgenic HCs expressed on the surface of 2F5 V_H^+/− B cells. (A) Representative contour histograms of splenic B cell populations from WT IgH^b/WT IgH^a and 2F5 V_H IgH^b/WT IgH^a F1 mice, with numbers indicating the percentage of IgM^a+ and IgM^b+ cells (bearing endogenous and 2F5 V_H transgene-bearing HCs, respectively) within the singlet, live, total B (B220⁺CD19⁺) cell gate. (B) Frequencies of IgM^a positive cells within IgM⁺ BM and splenic fractions shown for five and six mice per F1 group, respectively.

Fig. 5.

Flow cytometric analysis of splenic B cell development in C57BL/6 (WT), 2F5 V_H^+/−, and 2F5 V_H^+/+ mice. Representative dot plot histograms using the Allman classification scheme (32), with numbers in Top showing the number of total B cells (B220⁺) that were CD93 (AA4.1)⁺ transitional or CD93⁻ (mature + MZ) B cells, and further gated to allow for enumeration of transitional T1–T3 populations within the B220⁺CD93⁺ fraction (Middle) or enumeration of MZ and mature B cell subsets within the B220⁺CD93⁻ fraction (Bottom). Mean fluorescence intensities for staining of WT, 2F5 V_H^+/−, and 2F5 V_H^+/+ mature B cell subsets with PE-labeled anti-IgM were 756, 506, and 342, respectively.

Fig. 6.

Total or autoantigen-specific serum Ig levels and gp41 MPER-specific serum Ig or B cell reactivity in WT, 2F5 V_H ^+/−, and 2F5 V_H^+/+ mice. (A) Total IgM and IgG serum antibody levels. Each dot represents an individual mouse; horizontal lines represent mean serum antibody levels. Significance values were determined by a two-tailed Student's test: *P ≤ 0.05; ***P ≤ 0.0001; NS, not significant. (B) Total serum Ig reactivity against plate-bound 2F5 nominal epitope (gp41_652–671 MPER) peptide, nuclear antigens, and cardiolipin. For gp41 MPER, ANA, and cardiolipin reactivity assays, MRL/lpr serum was used as a positive control. Each dot represents an individual mouse; horizontal lines represent mean serum antibody levels.