Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).CD81 is a member of the tetraspanin family. Tetraspanins are membrane proteins containing four transmembrane domains, short intracellular domains and two extracellular loops (8). Tetraspanins have been shown to be involved in cell activation, proliferation, motility, and metastasis, as well as in cell fusion (14). Although CD81 is widely expressed, its level of expression varies in specific cell lineages and during differentiation. Moreover, its association with cell surface proteins differs in cell types of various lineages. In B cells CD81 is a component of the CD19-CD21-CD81-Leu-13 molecular complex, which plays a role in B-cell activation (2), whereas in T cells the molecule is associated with T-cell-specific molecules, including CD4 and CD8 (5, 15). In addition to its association with lineage-specific proteins, CD81 is associated with integrins and other tetraspanins (8). As a consequence of such protein associations, it is possible to activate multiple adhesion/signaling pathways in different cell types by engaging CD81 at their surface. For example, treatment of B-cell lines with a monoclonal antibody (MAb) specific for CD81 induces changes in cell adhesion and inhibits proliferation, whereas treatment of T-cell lines affects cell adhesion but not proliferation (10).CD81 was recently reported to interact with the hepatitis C virus (HCV) envelope glycoprotein (gp) E2 and hypothesized to act as a putative viral receptor (12). We have confirmed this observation and have shown that cell surface-expressed human CD81, but not murine or monkey (Chlorocebus aethiops, African green monkey [AGM]) CD81, binds E2 661 , a truncated soluble version of the E2 gp. Furthermore, interaction of E2 661 with the B-cell line Daudi had effects on cell adhesion and proliferation similar to the effects induced by anti-CD81 antibodies (3). This observation is of particular interest since in addition to causing acute and chronic liver disease, HCV is the major cause for mixed cryoglobulinemia...

Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2‐fold more lambda+ B cells than wild‐type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda‐expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3′ kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.