We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire.

A cardinal feature of systemic lupus erythematosus (SLE) is the development of autoantibodies. The first autoantibodies described in patients with SLE were those specific for nuclei and DNA, but subsequent work has shown that individuals with this disease produce a panoply of different autoantibodies. Thus, one of the constant features of SLE is a profound breakdown in tolerance in the antibody system. The appearance of self-reactive antibodies in SLE precedes clinical disease, but where in the B cell pathway tolerance is first broken has not been defined. In healthy humans, autoantibodies are removed from the B cell repertoire in two discrete early checkpoints in B cell development. We found these checkpoints to be defective in three adolescent patients with SLE. 25–50% of the mature naive B cells in SLE patients produce self-reactive antibodies even before they participate in immune responses as compared with 5–20% in controls. We conclude that SLE is associated with abnormal early B cell tolerance.

More than half of the nascent B cells in humans initially express autoreactive antibodies. However, most of these autoantibodies are removed from the repertoire at two checkpoints before maturation into naive B cells. A third checkpoint excludes remaining autoantibodies from the antigen-experienced IgM(+) memory B cell pool. Nevertheless, low-affinity self-reactive antibodies are frequently found in the serum of normal humans. To determine the source of these antibodies, we cloned and expressed antibodies from circulating human IgG(+) memory B cells. Surprisingly, we found that self-reactive antibodies including anti-nuclear antibodies were frequently expressed by IgG(+) memory B cells in healthy donors. Most of these antibodies were created de novo by somatic hypermutation during the transition between mature naive and IgG(+) memory B cells. We conclude that deregulation of self-reactive IgG(+) memory B cells may be associated with autoimmunity.

Filoviruses cause lethal hemorrhagic disease in humans and nonhuman primates. An initial target of filovirus infection is the mononuclear phagocytic cell. Calcium-dependent (C-type) lectins such as dendritic cell-or liver/lymph node-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as the hepatic asialoglycoprotein receptor, bind to Ebola or Marburg virus glycoprotein (GP) and enhance the infectivity of these viruses in vitro. Here, we demonstrate that a recently identified human macrophage galactose-and N-acetylgalactosamine-specific C-type lectin (hMGL), whose ligand specificity differs from DC-SIGN and L-SIGN, also enhances the infectivity of filoviruses. This enhancement was substantially weaker for the Reston and Marburg viruses than for the highly pathogenic Zaire virus. We also show that the heavily glycosylated, mucin-like domain on the filovirus GP is required for efficient interaction with this lectin. Furthermore, hMGL, like DC-SIGN and L-SIGN, is present on cells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggesting a role for these C-type lectins in viral replication in vivo. We propose that filoviruses use different C-type lectins to gain cellular entry, depending on the cell type, and promote efficient viral replication.

Persistent autoantibody production in patients with systemic lupus erythematosus (SLE) suggests the existence of autoreactive humoral memory, but the frequency of self-reactive memory B cells in SLE has not been determined. Here, we report on the reactivity of 200 monoclonal antibodies from single IgG؉ memory B cells of four SLE patients. The overall frequency of polyreactive and HEp-2 self-reactive antibodies in this compartment was similar to controls. We found 15% of IgG memory B cell antibodies highly reactive and specific for SLE-associated extractable nuclear antigens (ENA) Ro52 and La in one patient with serum autoantibody titers of the same specificity but not in the other three patients or healthy individuals. The germ-line forms of the ENA antibodies were non-self-reactive or polyreactive with low binding to Ro52, supporting the idea that somatic mutations contributed to autoantibody specificity and reactivity. Heterogeneity in the frequency of memory B cells expressing SLE-associated autoantibodies suggests that this variable may be important in the outcome of therapies that ablate this compartment.autoimmunity ͉ B cell ͉ repertoire ͉ self-tolerance