Faculty Bibliography

Amongst the many ploys used by microbial pathogens to interfere with host immune responses is the production of proteins with the properties of superantigens. These properties enable superantigens to interact with conserved variable region framework subdomains of the antigen receptors of lymphocytes rather than the complementarity determining region involved in the binding of conventional antigens. To understand how a B cell superantigen affects the host immune system, we infused protein A of Staphylococcus aureus (SpA) and followed the fate of peripheral B cells expressing B cell receptors (BCRs) with VH regions capable of binding SpA. Within hours, a sequence of events was initiated in SpA-binding splenic B cells, with rapid down-regulation of BCRs and coreceptors, CD19 and CD21, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, and DNA fragmentation. After exposure, B cell apoptotic bodies were deposited in the spleen, lymph nodes, and Peyer's patches. Although in vivo apoptosis did not require the Fas death receptor, B cells were protected by interleukin (IL)-4 or CD40L, or overexpression of Bcl-2. These studies define a pathway for BCR-mediated programmed cell death that is VH region targeted by a superantigen

A panel of novel recombinant single-chain variable fragment (scFv) antibody against human immunodeficiency virus type-1 (HIV-1) was isolated and characterized. We generated human scFvs using RNA harvested from cervical B lymphocytes of Kenyan prostitutes who are highly exposed to HIV-1, but remain persistently seronegative. The variable regions of the heavy (VH) and light (VL) chain antibody genes were selected as hybrids using guided-selection with the VL and VH, respectively, of a derivative of IgGb(12) using the phagemid vector pComb3X. IgGb(12) is a previously well-characterized HIV-1 neutralizing human monoclonal antibody (MAb). One of the hybrid scFv, IgA6/4L, neutralizes HIV-1 infectivity in in vitro cell culture assay. The cervical VH and VL chain antibody genes were connected by a DNA linker and subcloned in pComb3X. The cervical scFv clones were functional in recognizing HIV-1 gp120 by enzyme-linked immunosorbant assay (ELISA) and on cells in flow cytometry. Whole IgGb(12) does not inhibit binding of clones IgA6/5k nor IgA6/30lambda to gp120, which suggests that they bind different epitopes. Nucleotide sequence analysis of the cervical scFv show the clones are unique and reveal interesting characteristics of human cervical V gene pools. This work demonstrates, for the first time, cloning of a functional scFv MAb to a sexually transmitted disease pathogen from local cervical B-cell pools in exposed humans

B lymphocytes play several critical roles in the pathogenesis of rheumatoid arthritis. They are the source of the rheumatoid factors and anticitrullinated protein antibodies, which contribute to immune complex formation and complement activation in the joints. B cells are also very efficient antigen-presenting cells, and can contribute to T cell activation through expression of costimulatory molecules. B cells both respond to and produce the chemokines and cytokines that promote leukocyte infiltration into the joints, formation of ectopic lymphoid structures, angiogenesis, and synovial hyperplasia. The success of B cell depletion therapy in rheumatoid arthritis may depend on disruption of all these diverse functions

Impaired development of local Ab responses may predispose HIV-1-infected patients to an increased rate, severity, and duration of mucosal infections. We characterized the repertoire of Ig-producing cells in the intestinal effector compartment (the lamina propria) of HIV-1-infected (n = 29) and seronegative control (n = 27) subjects. The density of Ig-producing cells per area was similar in both groups. However, the proportions of IgA-producing cells were lower in both the duodenum and colon from HIV-1-infected patients compared with those of control subjects (p < 0.05), with compensatory increases in IgG-producing cells in the colon and IgM-producing cells in the duodenum. Similarly, among Abs in the lumen the proportions of IgA were also decreased and the proportions of IgG were increased among HIV-1-infected patients. On a molecular level, V(H) gene repertoire analyses by RT-PCR revealed comparable proportions of the V(H)3 family among duodenal IgA transcripts (50-53%) from both groups. V(H)3 expression was decreased only for IgM among patients with advanced HIV-1 disease (n = 6) compared with that of control subjects (n = 8) (48 +/- 8 vs 62 +/- 13%; p < 0.01). Moreover, the frequencies of individual IgM and IgA V(H)3 genes were comparable in each group, including rates of putative HIV-1 gp120-binding V(H)3 genes (V3-23, V3-30, V3-30/3-30.5). We conclude that, despite a decrement in local IgA producing cells, the density and molecular V(H) repertoire of mucosal plasma cells are relatively intact among patients with HIV-1 infection. These data suggest that HIV-1-infected patients use functional regulatory mechanisms to provide sufficient V(H) diversity and effective induction and differentiation of mucosal B cells

Recent reports have shown that protein A of Staphylococcus aureus (SpA) is a specific toxin for B cells by virtue of specific binding interactions with conserved sites on the V(H) region of the B-cell antigen receptor. The structural basis for these Fab-binding interactions has recently been revealed in crystallographic analyses, which have demonstrated many similarities with the interactions of T-cell superantigens. Investigations of the in vivo response to SpA have illustrated how a B-cell superantigen can be used to provide a window for examining fundamental principles that underlie the immunobiology of B lymphocytes

Using a panel of antibodies specific for H and L chain variable region subgroups, a panel of human monoclonal cold agglutinin (CA) and rheumatoid factor (RF) autoantibodies were analyzed. The vast majority of the two types of autoantibodies utilized VkIII L chains, many of which probably derive from the Humkv325 gene. However, while most RFs (77%) utilized VHI H chains, all the CAs used VHII subgroup H chains. These results are consistent with a model of autoantibody generation, wherein binding specificity is H chain defined in a set of antibodies that use a multipotential L chain

Rheumatoid factors (RFs) in humans have been studied intensively because of their association with autoimmune and lymphoproliferative diseases. Many human IgM-RFs express cross-reactive idiotypes (CRIs) and have homologous light chains, some of which are encoded by a single V kappa gene, termed V kappa 325. However, although antibody activity generally requires the interaction between heavy and light chain variable regions, much less is known about structural relationships among RF heavy chains. To delineate further the structural and genetic basis of RF autoantibody synthesis, we generated 'sequence-dependent' reagents specific for the human heavy and kappa light chain subgroups, and used them to analyze a panel of 27 monoclonal RFs. In addition, these proteins were tested for the expression of a heavy chain-associated CRI (G6), and a light chain-associated CRI (17.109). The results showed that most 17.109-reactive RFs contain heavy chains of the VHI subgroup, which bear the G6 idiotypic marker. However, among the 14 17.109-reactive RFs, two have heavy chains of the VHII subgroup, and another two contain heavy chains of the VHIII subgroup. Previously, we have shown that 17.109 is a phenotypic marker of the human V kappa 325 gene. Accordingly, these results demonstrate that the same human V kappa gene can combine with several VH genes from different VH gene subgroups to generate RF activity