Novel enzyme-linked immunosorbent assays for the detection of anti-Fc gamma receptor autoantibodies.

There is a substantial interest in the role of antineutrophil antibodies since Fc gamma receptors (Fc gamma Rs) have been identified as the target for the majority of such autoantibodies. Antineutrophil antibodies have long been detected by an indirect immunofluorescence technique. Following optimization of the flow cytometric method of detection, we developed three enzyme-linked immunosorbent assays (ELISAs), each specific for autoantibodies against one of the three classes of human Fc gamma R. Fc gamma RI and Fc gamma RII were purified from cultured cells, and Fc gamma RIII was produced as a recombinant molecule. These were then used as capture agents in the respective ELISAs. When applied in parallel to a sizeable group of patients with primary Sjögren's syndrome, both methods established that anti-Fc gamma R autoantibodies were heterogeneous. This finding indicates that different populations of partly cross-reactive antibodies are detectable by these two methods.

Characterization of the CD45 molecule on murine intestinal intraepithelial lymphocytes.

The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.

Extrathymic selection of TCR gamma delta + T cells by class II major histocompatibility complex molecules.

The influence of MHC antigens on TCR gamma delta usage in CD8+ intraepithelial lymphocytes (IELs) was examined using a pan-reactive and V delta 4 region-specific MAb. While an average of 30% of IELs from the majority of mice of various MHC haplotypes were V delta 4+, a 2-fold or greater percentage of IELs from H-2k mice were V delta 4+. Analysis of IELs from F1 mice indicated that the increase in TCRs using V delta 4 was likely to be the result of positive selection. The V delta 4 usage patterns of IELs from recombinant inbred strains and from mice recombinant within H-2 revealed that the increase in V delta 4 usage mapped to H-2 and required I-E expression. Moreover, selection of TCRs using V delta 4 occurred in chimeric mice in the absence of a thymus. The results demonstrate an extrathymic selective mechanism for gamma delta TCRs of CD8+ IELs and suggest that these cells may exhibit MHC class II-restricted antigen recognition.

A monoclonal antibody (Po66) directed against human lung squamous cell carcinoma immunolocalization of tumour xenografts in nude mice.

Po66, a mouse IgG1 monoclonal antibody, was produced by immunization against a patient lung squamous cell carcinoma. The tissue reactivity of the antibody was measured by a radioimmunological assay with enzymatically dissociated cells, by an immunofluorescence test on frozen tissue sections and by peroxidase-staining of paraffin sections. The antibody bound to lung squamous cell carcinoma, oesophagus carcinoma and, inconsistently to lung adenocarcinoma but not to the other tumours tested. Some normal tissues also reacted positively, in particular bronchial serous glands, oesophagus epithelium and renal distal and collecting tubules. In normal and malignant tissues showing epithelioid differentiation, Po66 bound to the intermediate maturation area. The antigen immunoprecipitated by Po66 from lung squamous cell carcinoma appeared as a single band with a molecular weight 47,000 to 50,000 daltons. Purified monoclonal antibody Po66 and an unrelated IgG1 immunoglobulin were labelled with radioactive iodine and injected i.v. into nude mice bearing subcutaneous xenografts of human lung squamous cell carcinoma. The localization index in the tumour was 3.3. Antibody labelled with 131I allowed gamma-scintigraphic imaging of the xenografts which were clearly outlined by days 9 to 11.