David Nemazee

Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors

Building Better Vaccines In the past few years, several highly potent, broadly neutralizing antibodies (bNAbs) specific for the gp120 envelope protein of HIV-1 have been discovered. The goal of this work is to use this information to inform the design of vaccines that are able to induce such antibodies (see the Perspective by Crowe). However, because of extensive somatic hypermutation, the epitope bound by these antibodies often does not bind to the germline sequence. Jardine et al. (p. 711, published online 28 March; see the cover) used computational analysis and in vitro screening to design an immunogen that could bind to VRC01-class bNAbs and to their germline precursors. Georgiev et al. (p. 751) took advantage of the fact that only four sites on the HIV viral envelope protein seem to bind bNAbs, and sera that contain particular bNAbs show characteristic patterns of neutralization. An algorithm was developed that could successfully delineate the neutralization specificity of antibodies present in polyclonal sera from HIV-infected patients. Structural knowledge of broadly neutralizing antibodies against HIV-1 guides the design of an immunogen to elicit them. Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1–infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens.

Receptor Editing in a Transgenic Mouse Model: Site, Efficiency, and Role in B Cell Tolerance and Antibody Diversification

Mice carrying transgenic rearranged V region genes in their IgH and Igkappa loci to encode an autoreactive specificity direct the emerging autoreactive progenitors into a pre-B cell compartment, in which their receptors are edited by secondary Vkappa-Jkappa rearrangements and RS recombination. Editing is an efficient process, because the mutant mice generate normal numbers of B cells. In a similar nonautoreactive transgenic strain, neither a pre-B cell compartment nor receptor editing was seen. Thus, the pre-B cell compartment may have evolved to edit the receptors of autoreactive cells and later been generally exploited for efficient antibody diversification through the invention of the pre-B cell receptor, mimicking an autoreactive antibody to direct the bulk of the progenitors into that compartment.

Developmental Regulation of B Lymphocyte Immune Tolerance Compartmentalizes Clonal Selection from Receptor Selection

B lymphocyte development is a highly ordered process that involves immunoglobulin gene rearrangements, antigen receptor expression, and a learning process that minimizes the development of cells with reactivity to self tissue. Two distinct mechanisms for immune tolerance have been defined that operate during early bone marrow stages of B cell development: apoptosis, which eliminates clones of cells, and receptor editing, which spares the cells but genetically reprograms their autoreactive antigen receptors through nested immunoglobulin L chain gene rearrangements. We show here that sensitivity to antigen-induced apoptosis arises relatively late in B cell development and is preceded by a functionally distinct developmental stage capable of receptor editing. This regulation compartmentalizes clonal selection from receptor selection.

BCR Ligation Induces Receptor Editing in IgM+IgD− Bone Marrow B Cells In Vitro

The ability of BCR cross-linking to stimulate receptor editing was analyzed in vitro using bone marrow B cells from immunoglobulin (Ig) transgenic (Tg) and non-Tg mice. In cultured Ig-Tg cells, BCR ligation induced receptor editing as measured by up-regulation of RAG gene expression, light chain gene DNA rearrangements, and expression of lambda-light chain protein in cells that previously expressed kappa. In the culture conditions used, BCR ligation induced light chain rearrangements in most immature IgM+IgD- bone marrow B cells in the absence of significant cell death or cell growth. Receptor editing in non-Tg B cells was also documented in cultures treated with anti-immunoglobulin. These results provide direct evidence for the ability of BCR ligation to stimulate immunoglobulin light chain gene rearrangements in immature B cells.

Clonal Deletion of Autospecific B Lymphocytes

Using mice transgenic for functional, rearranged immunoglobulin heavy and light chain genes, it can be demonstrated that B lymphocytes reactive with cell surface-bound class I MHC antigen can be controlled by clonal elimination. Even low-affinity cell-bound ligands can induce deletion. Deletion can occur in the pre-B to B cell transitional stage or after the B cells exist the bone marrow, depending on where the cells first encounter autoantigen. IgD appears to play no role in protecting cells from deletion. It is argued that defects in B-cell tolerance alone may be sufficient to lead to systemic autoimmunity.

V(D)J recombinase induction in splenic B lymphocytes is inhibited by antigen-receptor signalling

In lymphocytes, DNA recombinations that generate the antigen-receptor genes can sometimes be reinduced in receptor-bearing cells in a process called receptor editing, which modifies the specificity of the receptor for antigen. In immature B lymphocytes, B-cell antigen receptor (BCR) signalling stimulates immune tolerance by receptor editing. More mature splenic B cells can also be induced to undergo V(D)J recombination, which generates diversity in the immune system, either by immunization with foreign proteins or by stimulation in vitro with interleukin-4 and lipopolysaccharide. Here we show that immune tolerance is unlikely to induce V(D)J recombination in mature B cells, because BCR ligation actively inhibits V(D)J recombination induced by interleukin-4 and lipopolysaccharide. Furthermore, immunization of immunoglobulin transgenic mice with ligands of varying avidities for the BCR showed that low-avidity antigen could induce strong V(D)J recombination, whereas non-binding or high-avidity ligands could not. These data suggest that V(D)J recombination induced during the immune response modifies the antigen receptors of B cells with weak, but not strong, reactivity to antigen, potentially rescuing cells with improved receptor affinity and promoting their contribution to the immune response. Thus BCR signalling regulates V(D)J recombination in both tolerance and immunity, but in strikingly different ways.

Visualization of the Genesis and Fate of Isotype-switched B Cells during a Primary Immune Response

The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment.

ΔBAFF, a Splice Isoform of BAFF, Opposes Full-Length BAFF Activity In Vivo in Transgenic Mouse Models

ΔBAFF is a novel splicing isoform of the regulator B cell-activating factor (BAFF, BLyS), a TNF family protein with powerful immunoregulatory effects. Overexpression of BAFF leads to excessive B cell accumulation, activation, autoantibodies, and lupus-like disease, whereas an absence of BAFF causes peripheral B cell immunodeficiency. Based on the ability of ΔBAFF to multimerize with full-length BAFF and to limit BAFF proteolytic shedding from the cell surface, we previously proposed a role for ΔBAFF in restraining the effects of BAFF and in regulating B lymphocyte homeostasis. To test these ideas we generated mice transgenic for ΔBAFF under the control of human CD68 regulatory elements, which target expression to myeloid and dendritic cells. We also generated in parallel BAFF transgenic mice using the same expression elements. Analysis of the transgenic mice revealed that ΔBAFF and BAFF had opposing effects on B cell survival and marginal zone B cell numbers. ΔBAFF transgenic mice had reduced B cell numbers and T cell-dependent Ab responses, but normal preimmune serum Ig levels. In contrast, BAFF transgenic mice had extraordinarily elevated Ig levels and increases in subsets of B cells. Unexpectedly, both BAFF and ΔBAFF appeared to modulate the numbers of B-1 phenotype B cells.

ΔBAFF, an Alternate Splice Isoform That Regulates Receptor Binding and Biopresentation of the B Cell Survival Cytokine, BAFF

The tumor necrosis family member BAFF is limiting for the survival of follicular B lymphocytes, but excessive BAFF signaling can lead to autoimmunity, suggesting that its activity must be tightly regulated. We have identified a conserved alternate splice isoform of BAFF, called ΔBAFF, which lacks 57 nt encoding the A–A1 loop and is co-expressed with BAFF in many mouse and human myeloid cells. Mouse ΔBAFF appears on the plasma membrane, but unlike BAFF it is inefficiently released by proteolysis. ΔBAFF can associate with BAFF in heteromultimers and diminish BAFF bioactivity and release. Thus, alternative splicing of the BAFF gene suppresses BAFF B cell stimulatory function in several ways, and ΔBAFF may promote other functions as well.

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.