Description: Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic ("knock-in") mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol . 191: 3186–3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.

Description: A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated knock-in mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the companion article (Ota et al. 2013. J. Immunol. 191: 3179–3185), 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms, including receptor editing, clonal deletion, and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1 –/– background, 4E10HL mice had virtually no serum Ig of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of a membrane proximal external region–directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly described bNAb 10E8.

Description: Challenge studies following passive immunization with neutralizing Abs suggest that an HIV vaccine could be efficacious were it able to elicit broadly neutralizing Abs (bNAbs). To better understand the requirements for activation of B cells producing bNAbs, we generated cell lines expressing bNAbs or their germline-reverted versions (gl-bNAbs) as BCRs. We then tested the abilities of the bNAb-expressing cells to recognize HIV pseudovirions and vaccine candidate proteins by binding and activation assays. The results suggest that HIV envelope (Env) Ag-expressing, infection-competent virions are poorly recognized by high-affinity bNAb-expressing cells, as measured by the inability of Ags to induce rapid increases in intracellular calcium levels. Other Ag forms appear to be highly stimulatory, in particular, soluble gp140 trimers and a multimerized, scaffolded epitope protein. Virions failed to efficiently activate bNAb-expressing B cells owing to delayed or inefficient BCR recognition, most likely caused by the low density of Env spikes. Importantly, B cells carrying gl-bNAb BCRs were not stimulated by any of the tested vaccine candidates. These data provide insight into why many HIV immunogens, as well as natural HIV infections, fail to rapidly stimulate bNAb responses and suggest that bNAb-expressing cell lines might be useful tools in evaluation of vaccine Ags for infectious diseases. Because soluble Env trimers or multimerized scaffolded epitopes are best at activating B cell-expressing bNAbs, these antigenic forms should be considered as preferred vaccine components, although they should be modified to better target naive gl-bNAb B cells.

Description: Invariant NKT cells (iNKT) are potent immunoregulatory T cells that recognize CD1d via a semi-invariant TCR (iNKT-TCR). Despite the knowledge of a defective iNKT pool in several autoimmune conditions, including rheumatoid arthritis (RA), a clear understanding of the intrinsic mechanisms, including qualitative and structural changes of the human iNKT repertoire at the earlier stages of autoimmune disease, is lacking. In this study, we compared the structure and function of the iNKT repertoire in early RA patients with age- and gender-matched controls. We analyzed the phenotype and function of the ex vivo iNKT repertoire as well as CD1d Ag presentation, combined with analyses of a large panel of ex vivo sorted iNKT clones. We show that circulating iNKTs were reduced in early RA, and their frequency was inversely correlated to disease activity score 28. Proliferative iNKT responses were defective in early RA, independent of CD1d function. Functional iNKT alterations were associated with a skewed iNKT-TCR repertoire with a selective reduction of high-affinity iNKT clones in early RA. Furthermore, high-affinity iNKTs in early RA exhibited an altered functional Th profile with Th1- or Th2-like phenotype, in treatment-naive and treated patients, respectively, compared with Th0-like Th profiles exhibited by high-affinity iNKTs in controls. To our knowledge, this is the first study to provide a mechanism for the intrinsic qualitative defects of the circulating iNKT clonal repertoire in early RA, demonstrating defects of iNKTs bearing high-affinity TCRs. These defects may contribute to immune dysregulation, and our findings could be exploited for future therapeutic intervention.