Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

Abstract

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (∼7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgMhi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (∼15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igha (BALB/c) and Ighb (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igha locus. Mapping of MPER gp41 interactions with IgMa identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc CH regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgMa determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.

Efficient role of IgH 3′ regulatory region deficient B-cells in the development of oil granulomas

“…This result might be linked to a differential strength of signalling between IgM a BCR and IgM b BCR for proliferation/survival of peritoneal B-cells. Such specific interactions with IgM a (but not IgM b ) determinants have been already reported with the HIV-1 envelope gp41 membrane proximal external region [26]. Furthermore, the phenotype of mature B-cells differs between the various mouse substrains.…”

Immune perturbations in HIV-1–infected individuals who make broadly neutralizing antibodies

“…We previously demonstrated that two bnAbs (2F5, 4E10) directed at the HIV-1 Env gp41 neutralizing site near the viral membrane are autoreactive (13, 83), and in bnAb antibody heavy and light chain knock-in mice, both bnAbs were shown to be controlled by multiple immune tolerance mechanisms (21, 22, 24, 84, 85). The observations of bnAb autoreactivity prompted the hypothesis that patients with systemic lupus erythematosus (SLE) will be able to make bnAbs more readily than others during chronic HIV-1 infection (15).…”

“…Some bnAbs are restricted during early B-cell development at the first tolerance checkpoint in bone marrow due to germline B-cell receptor (BCR) autoreactivity, resulting in fewer bnAb precursors before vaccination (21, 22, 24, 84, 85), while other bnAb germline BCRs are not autoreactive, and autoreactivity is only acquired in the periphery during affinity maturation (90). BnAbs with long third heavy chain complementarity determining regions that do emerge in HIV-1 infection appear to be rare by virtue of tolerance mechanisms that reduce their precursor frequency (12, 26, 91).…”

Initiation of immune tolerance–controlled HIV gp41 neutralizing B cell lineages

“…Primary labeled mAbs used were Pacific Blue, allophycocyanin, or Texas Red-conjugated anti-B220 (clone RA3–6B2), phycoerythrin (PE)–Cy7 anti-CD19, fluorescein isothiocyanate (FITC)–conjugated anti-IgD (clone 11–26), FITC-conjugated anti-IgG2b (clone R12-3), FITC-, allophycocyanin-, or PE-Cy7–conjugated anti-IgM (clone 15F9), PE-conjugated anti-CD21, and PE-Cy7–labeled anti-CD23 (eBioscience). Flow cytometric analysis of B cell reactivity for the MPER 2F5 epitope was performed similarly using single-cell splenocyte suspensions from naïve and immunized 2F5 mature and 2F5 UA dKI mice that were stained with MPER (SP62) tetramers, also as previously described (17, 22, 48). IgG2b- and IgM-specific analysis was performed using an intracellular staining protocol based on the BD Cytofix/Cytoperm fixation/permeabilization method.…”