Nico J. Stam

Biochemical complexity of serum HLA class I molecules.

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around Mr 44 000, 40 000, and 35 000–37 000. HLA-A24-positive individuals are distinguished by higher serum levels of Mr 44 000 and 40 000 class I-like molecules than those found in HLA-A24-negative individuals. The Mr 44 000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The Mr 35 000 and 37 000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum Mr 44 000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The Mr 40 000 molecules do not contain a TM region. Mr 40 000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.

An analysis of class I antigens of man and other species by one-dimensional IEF and immunoblotting

A procedure for the molecular identification of MHC class I products based on 1-D IEF and subsequent immunoblotting is described. Optimal conditions for 1-D IEF, the electrophoretic transfer of proteins out of denaturing, nonionic detergent-containing gels to nitrocellulose, and the requisite antibodies, both polyclonal and monoclonal, for the visualization of class I heavy chains have been established. Cross-reactivity of antibodies has enabled the biochemical analysis of class I heavy chains in the dog. The procedure reported here requires modest amounts of cells and allows a rapid molecular characterization of class I heavy chain polymorphisms in man and other species without the need for radiochemical methods.

Allele-specific down-regulation of MHC class I antigens in Burkitt lymphoma lines

We have reported that Burkitt lymphomas (BL) that arise in HLA-A11 positive individuals are resistant to lysis by HLA-A11-specific and HLA-A11-restricted CTLs(10,11). Here we show that this phenomenon can be explained by a selective loss of the HLA-A11 polypeptide. The HLA-A11 negative phenotype is due to a regulatory phenomenon, rather than a structural defect, as proven by the ability to rescue expression of HLA-A11 in in vitro Epstein-Barr virus (EBV)-converted sublines of EBV negative BLs.

Characteristics of HLA Class I Antigens in Human Sera

Class I antigens deriving from classical or nonclassical major histocompatibility complex class I genes have been detected in sera of many different species (1). Those present in human serum — as inferred from microcytotoxicity-inhibition assays (2) — have not been analyzed biochemically in detail yet.