Molecular characterization of human anti-hinge antibodies derived from single-cell cloning of normal human B cells.

Anti-hinge antibodies (AHAs) are an autoantibody subclass that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart. AHAs have been postulated to exacerbate chronic inflammatory disorders such as inflammatory bowel disease and rheumatoid arthritis. On the other hand, AHAs may protect against invasive microbial pathogens and cancer. However, despite more than 50 years of study, the origin and specific B cell compartments that express AHAs remain elusive. Recent research on serum AHAs suggests that they arise during an active immune response, in contrast to previous proposals that they derive from the preexisting immune repertoire in the absence of antigenic stimuli. We report here the isolation and characterization of AHAs from memory B cells, although anti-hinge-reactive B cells were also detected in the naive B cell compartment. IgG AHAs cloned from a single human donor exhibited restricted specificity for protease-cleaved F(ab')2 fragments and did not bind the intact IgG counterpart. The cloned IgG-specific AHA-variable regions were mutated from germ line-derived sequences and displayed a high sequence variability, confirming that these AHAs underwent class-switch recombination and somatic hypermutation. Consistent with previous studies of serum AHAs, several of these clones recognized a linear, peptide-like epitope, but one clone was unique in recognizing a conformational epitope. All cloned AHAs could restore immune effector functions to proteolytically generated F(ab')2 fragments. Our results confirm that a diverse set of epitope-specific AHAs can be isolated from a single human donor.

IL-6 signaling via the STAT3/SOCS3 pathway: functional analysis of the conserved STAT3 N-domain.

The conserved N-domain of the STAT proteins has been implicated in several activities crucial to cytokine signaling including receptor recruitment and STAT activation, cooperative DNA binding and STAT-dependent gene expression. We evaluated the role of the STAT3 N-domain in the IL-6 signal transduction pathway leading to Socs3 gene expression, an essential mechanism that controls the quality and magnitude of IL-6-dependent transcriptional responses. Based on the model for STAT N-domain function in cooperative gene expression and the presence of tandem STAT binding motifs in the murine Socs3 promoter, we anticipated that stabilizing interactions between adjacent STAT3 dimers via N-domain sequences might be essential for Socs3 gene expression. This was underscored by the tight conservation in the location and sequence of the tandem STAT binding sites between the murine and human Socs3 promoters. Using reconstitution into Stat3-/- mouse embryonic fibroblasts (Stat3-/- MEFs), we find that a STAT3 N-domain deletion mutant (Delta 133STAT3) is activated by tyrosine phosphorylation in response to IL-6 and then undergoes dephosphorylation with kinetics similar to full-length STAT3. These results highlight important differences compared to other STATs where the N-domain has been shown to mediate activation (STAT4) or dephosphorylation (STAT1). STAT3 binds predominantly to a single STAT consensus site in the Socs3 promoter, despite the presence of an adjacent STAT motif. Significantly, Delta 133STAT3 stimulates expression of the endogenous Socs3 gene in Stat3-/- MEFs upon IL-6 treatment with an activity similar to reconstituted STAT3, demonstrating that the N-domain is dispensable for Socs3 gene expression. We propose that the Socs3 gene in its chromosomal context is activated by the IL-6/STAT3 pathway independent of STAT3 N-domain sequences.