In this study, we make two important observations regarding the regulation of B cells expressing the HIV-1 Env gp41 2F5 bnAb. Under normal selection conditions, we show in a novel murine line, 2F5 complete (VH/VL) KI mice, that pairing of the 2F5 H chain with its cognate L chain in vivo results in stringent tolerance controls. Secondly, under conditions where apoptosis of BM B cells is circumvented, we show that sIg+ B cells bearing self-reactive and functional 2F5 VH/VL pairs can be rescued. We also find that extensive L chain editing and anergy prevent efficient 2F5 VH/VL expression, thus revealing that additional tolerance mechanisms in 2F5 complete (VH/VL) KI mice control 2F5 mAb expression.
The profound reduction of Ig+ 2F5 bnAb-expressing-bone marrow B cells in 2F5 complete (VH+/+
) KI mice confirms that the predominant tolerizing mechanism/checkpoint of 2F5-expressing B cells is clonal deletion in the BM, and is similar to that seen in other KI models made with autoantibody Igs exhibiting high degrees of self-/polyreactivity in vivo
(reviewed in (32
)). Additionally, since this strain was engineered to produce a pre-antigenic B cell repertoire initially comprised of H/L chain pairs containing the original (mutated) 2F5 mAb’s VH
regions, this consolidates the notion that this Ig’s inherent self-reactivity triggered the profound developmental blockade, regardless of when in B cell ontogeny the original 2F5 H chain acquired somatic mutations, and irrespective of if/what extent ineffective pairing of mutated 2F5 H chains with surrogate or conventional L chains potentially occurred in the 2F5 VH
KI pre-antigenic repertoire. This study also defines the relative impact of 2F5 H and L chains in the development of B cells expressing them: the dominance of the 2F5 H chain is demonstrated in that its in vivo
expression, and not the L chain’s, results in profound loss of BM B cells. On the other hand, a definitive, albeit additive role for the 2F5 L chain is supported by the demonstration that 2F5 H+L chain co-expression in vivo
enhances the extent of clonal deletion/anergy in immature B cells. In this context, given the extensive amount of L chain editing in residual/rescued B cells from 2F5 complete KI mice, it will be of interest to explore the possibility that normal B cell development in 2F5 VL
KI mice is due to extensive editing of its 2F5 L chain, and if so, to determine if this occurs either in response to 2F5 L chain associations with self-reactive, endogenous H chains, or alternatively, results from general difficulties the 2F5 L chain has pairing with endogenous H chains.
Our study’s demonstration that B cells producing the HIV-1 bnAb 2F5 from the BM of 2F5 complete (VH xVL) KI mice, can be recovered in vitro in the absence of the stromal tolerizing environment, is critical for three reasons. First, it provides evidence, under normal selection conditions in vivo, that the absence of B cell precursors capable of expressing 2F5 VH/VL pairs with broadly neutralizing activity is due to their de novo synthesis prior to their clonal deletion, rather than not having been initially made. Secondly, it provides the first example of isolated in vivo-derived BM B cell precursors specifying a HIV-1 bnAb, and provides a starting basis to manipulate the immune system such that these desirable clones may be elicited. Thirdly, it reveals that under conditions where deletional controls are removed, two additional tolerance mechanisms remain that limit the efficient rescue of these bnAb-specific B cell precursors: extensive L chain editing, resulting in loss of MPER specificity and neutralization (, ), and anergy (, ).
With respect to the hurdle of overcoming anergy, our in vivo
studies of BM B cells from 2F5 VH
complete × Eμ-bcl2 mice confirm the above in vitro
studies, demonstrating the rescue of many 2F5-expressing B cells from central deletion, but not from anergy (), and is also consistent with previous studies of enforced B cell-specific overexpression of bcl2 in vivo
in certain autoreactive Ig transgenic systems such as the membrane-bound HEL tg model, where central deletion can be at least partially circumvented, but a large fraction of rescued autoreactive B cell clones remain phenotypically anergic (34
). However, the fact that we can rescue a significant MPER-specific IgG endpoint titer in naïve 2F5 complete KI × Eμ-bcl2 tg mice, relative to 2F5 complete KI mice () does also suggest that a subset of B cells can break anergy in vivo
, and in this context, it will be of particular interest to correlate rescued B cell subsets with serum MPER reactivity (and potentially neutralization titers) in immunized 2F5 complete KI × Eμ-bcl2 tg animals.
Previously, it has been shown that the Eμ-bcl2 transgene not only increases the survival window of self-reactive bone marrow B cells but also allows them to concomitantly increase their frequency of κ and λ L chain editing events (35
). The anergic phenotype observed in our in vitro
cultured B cells was also associated with extensive (but largely ineffective) κ-specific L chain editing events under these rescue conditions. As expected, over-expression of the Eμ-bcl2 transgene in our studies increased L chain editing in WT mice (as demonstrated by decreased κ/λ ratios and increased κ+λ ratios), but interestingly had little effect on λ L chain editing in the context of 2F5 complete KI B cells (based on their unaltered κ/λ ratios), suggesting that either LC editing doesn’t occur, or, as seen in hybridomas derived from in vitro-
rescued 2F5 complete KI B cells, only κ-specific secondary rearrangement events can occur and/or are preferentially selected for. In support of this latter possibility, studies in 2F5 VH
KI mice suggest that the 2F5 H chain also imparts initial pairing constraints to restricted Vκ families and disfavors λ partners (manuscript in preparation). Regardless of if/what types of L chain editing events occur in bcl2-overexpressing 2F5 complete KI B-cells and the causal link these events have with anergy, these results reinforce our in vitro
findings that rescued 2F5 VH
-expressing B cells cannot reduce their self-reactivity sufficiently to overcome their anergic (functionally unresponsive) phenotype.
The unusually high extent of L chain editing observed in 2F5 complete KI mice could be influenced by two potential factors. One is compensation for the lack of observed VH
replacement events in hybridomas derived from our 2F5 KI models (, ). Consistent with this possibility is the fact that the original (mutated) 2F5 VH
bears “atypical” embedded/nonamer motifs (i.e. cryptic Recombination Signal Sequences (cRSS)) in the well-known VH
3′ FRW3 site, where cRSS have been experimentally linked with VH
replacement of the 3H9 H chain (36
). In particular, even though the 2F5 and 3H9 VH
3′ FRW3 regions share identical embedded heptamers, the 2F5 VH
3′ FRW3 lacks the consensus embedded nonamer found in the 3H9 3′ FRW3 region, as well as many other VH
genes examined (Table S2
). Another, likely possibility for the high rate of L chain editing in 2F5 complete KI mice relates to the largely unsuccessful nature of sequential κ L chain editing events in overcoming the threshold of sensitivity to anergy, for example in situations where increased attempts are permitted due to an increased survival window (, , ) and/or there is increased availability of endogenous κ L chain elements ().
The unsuccessful κ L chain editing pattern observed in rescued 2F5 complete KI B cells (in which only a highly-restricted set of κ L chain partners i.e. such as Vκ21-4 are used, and only partially mitigate 2F5 H chain reactivity), is analogous to the situation in KI mice bearing the dominant, high affinity anti-DNA H chain 3H9-76R, which, like 2F5, contains multiple HCDR3 positively-charged residues, and where almost all L chains are ineffective at vetoing H chain-encoded DNA reactivity. That the pattern of L chain editing in 2F5 complete KI mice appears to be κ-restricted however, is distinct from other situations where editing to the λ locus effectively vetoes H chain self-reactivity, and may reflect an inherent inability of λLCs to either mitigate 2F5 H chain self-reactivity, or alternatively, properly pair with 2F5 H chains). Further investigation using 2F5 VH and VL KI models into the contribution of the individual 2F5 H and L chains in inducing self-reactivity and H/L chain pairing constraints and the potential role of L chain editing in these two processes should be useful in this regard.
A final important finding of this study is that several specificities within the spectrum of 2F5’s overall polyreactivity likely contribute in its in vivo
tolerogenicity. The contribution of the original 2F5 L chain in specifying self-reactivity for a self-antigenic component mimicking the nominal 2F5 MPER epitope, is corroborated by several lines of evidence, including: a) 2F5 L+H chain co-expression conferring co-ordinate MPER specificity and enhanced degree of clonal deletion in immature BM B cells in vivo
, 2) many rescued secreting hybridoma lines derived from 2F5 complete KI BM B cells use endogenous L chain editors that completely eliminate MPER binding in vitro
, and 3) 2F5 complete KI BM B cell lines follow a hierarchy wherein higher frequencies of L chain editing events using putative MPER-complementing endogenous L chains, occur in non-secreting hybridomas, relative to secreting (potentially less anergic) ones (Table S1
). However, our study also implies an important role for 2F5 H chain-specific interactions with one or more self-antigens, since most 2F5 H chain-expressing-B cells in this study are only partially rescued from negative selection, even when “editor” L chains remove all measurable reactivities. Because such 2F5 H chain-specific interactions cannot be measured in our in vitro
reactivity assay, but may either be with the same MPER-mimicking self-antigen(s) recognized by the original 2F5 HC/LC, and/or to distinct self-ag(s) altogether, further studies aimed at comprehensively identifying the physiologically-relevant in vivo
self-antigen target(s) of 2F5 will be critical.