Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.

Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT-PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.

Evidence for an antigen-driven humoral immune response in medullary ductal breast cancer.

A minority of breast cancers is characterized by lymphoplasmacytic infiltrates that have been correlated with improved patient survivals. The association of improved prognosis with plasmacytic infiltrates has been classically linked with the rare medullary carcinoma subtype but is also evident in the smaller infiltrated fraction of the more abundant nonmedullary (not otherwise specified) tumors. It is our hypothesis that these plasma cell (PC) infiltrates represent a host humoral response driven by one or more tumor-derived neoantigens. As the index study group, two primary medullary carcinoma tumors were examined. Immunophenotyping confirmed a large number of IgG PCs in contradistinction to normal breast, which typically contains a lesser number of mainly IgA isotypes. IgG heavy and light chains were expressed as combinatorial phage Fab libraries. VH and VL sequences showed a preponderance of clonal groups in both patients, as identified by germ-line gene usage and junctional mutation patterns. Panning of phage Fab libraries against purified antigens excluded Her2/neu and p53 as the eliciting antigen, and failure of clonal enrichment by cell panning suggested that the neoantigen was not membrane expressed or was expressed at low levels. Cognate, original VH+VL pairs were obtained by single cell PCR of tumor PCs, which showed overlap with the pooled IgG libraries. Tumor-derived IgG V genes exhibited mutational patterns that were consistent with antigenic selection and affinity maturation. Where examined, IgG1 was the predominant isotype, consistent with a T-dependent (i.e., protein) antigen. From these data, we infer that the breast tumor PC infiltrates of the medullary carcinoma subtype are compatible with an autogenic tumor neoantigen-driven humoral immune response.

Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products.

Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties. We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation. 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity. This correlation indicates that in adult Drosophila 70 +/- 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1. The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g. GSTA4-4). The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life.