Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.

The presence of anergy within iglb12 heavy chain transgenic B cells suggests that immunogens targeting this population would need to overcome these functional defects. Before performing immunization experiments, we modified IB2 to ensure optimal murine responses. Since IB2 was generated in a mouse, it would likely generate a poor response if injected unmodified, since tolerance mechanisms would be expected to eliminate most sources of T cell help targeting it. To increase possible CD4 + T cell help, the heavy and light chain constant regions used by this anti-idiotype were humanized (Tiller et al., 2009 ). Since multimerization can overcome anergic responses (Cooke et al., 1994; Gautam et al., 2016; McGuire et al., 2016) , humanized IB2 antigen-binding fragment (Fab) was fused to a modified multimerization domain of the chicken C4b-binding protein (huIB2-C4b), which self-assembles into a multimer containing up to seven humanized IB2 Fab domains (Ogun et al., 2008; Hofmeyer et al., 2013) . To further garner T cell help, the 2W epitope (Rees et al., 1999) was added to the C-terminus of C4b separated by a cathepsin consensus sequence in order to promote endosomal processing and MHC presentation (Schneider . For immunization experiments, 4 × 10 5 B cells from CD45.2 + iglb12 heavy chain transgenic mice were labeled with CTV and transferred into CD45.1 + WT recipient mice. 1 d after transfer, the mice were immunized with huIB2-C4b or an irrelevant humanized isotype control-C4b fusion (control-C4b). 5 or 14 d following the injection of the control-C4b, CD45.2 + donor B cells accounted for an average of ∼0.0006% of total B cells in control recipient animals, which we detected by enriching for CD45.2 + cells before analysis (Fig. 9, A and B) . As expected of resting cells that had not encountered antigen and are therefore not proliferating, these cells largely retained high levels of CTV (Fig. 9 , C and D). In contrast, 5 d following injection of huIB2-C4b, donor B cells increased twofold to an average of ∼0.001% of total B cells (Fig. 9, B-D) . Approximately half of the donor B cells had low levels of CTV, indicating that proliferation accounted for the increased frequency of donor cells (Fig. 9, B-D) . In addition to expansion, ∼33% of transgenic B cells in huIB2-C4b-immunized mice bound GL7 and down-regulated CD38 (Fig. 9 , E and F), a phenotype of germinal center B cells (Allen et al., 2007; Kurosaki et al., 2010; Victora et al., 2010; Vinuesa et al., 2010; Kitano et al., 2011) . In these experiments, IB2binding germinal center B cells derived from both the transgenic donor and recipient animals could be detected using IB2 tetramers (Fig. 10 , A-C). This analysis revealed that transgenic CD45.2 + iglb12 B cells accounted for 5.8% of the IB2binding germinal center B cells (Fig. 10 D) . This frequency represented an increase from the 1-2% of transgenic cells found in cells outside the germinal center in both huIB2-C4b and control-C4b injected animals (Fig. 10 D) . Together, these results indicate that anti-iglb12 idiotype antibodies can be used as immunogens that can overcome anergy and induce B cell activation and entry into germinal centers.