Kingsley Micklem

A human macrophage-associated antigen (CD68) detected by six different monoclonal antibodies

Summary. Antibodies grouped together by the Third Workshop on Leucocyte Differentiation Antigens on the basis of pan‐macrophage reactivity on tissue sections were analysed in immunoprecipitation experiments. Antibodies Y2/131. EBM11, Ki‐M6 and Ki‐M7 all precipitated antigens of Mr 110000 which were shown to be identical by preclearing experiments. In addition a recently produced antibody, KP1, which identifies macrophages in paraffin‐embedded tissue, was shown to recognize the same antigen. The antibodies were tested on murine cells transfected with two clones, which had been isolated by screening a cDNA library with antibodies Y1/82A and EBM11. Cells transfected with the longer cDNA clone, coding for a molecule of Mr 110000, reacted with antibodies Y2/131. EBM11. Y1/82A and Ki‐M6, whilst the shorter clone, encoding a molecule of Mr 70000 gave the same result except that it did not induce expression of the Ki‐M6 epitope. KP1 antibody did not recognize any transfectants, possibly because of differences in glycosylation by the transfected cell line compared with human tissue. Five of the six antibodies appear to recognize different epitopes (the sixth, Ki‐M7, not having been evaluated in this way). It was concluded that these six antibodies react with a macrophage‐associated antigen for which the gene has been cloned. This group of antibodies has recently been designated CD68 by the Fourth Workshop on Human Leucocyte Differentiation Antigens.

Double immunofluorescence labelling of routinely processed paraffin sections

Double immunoenzymatic labelling of routinely processed human tissues has been used in many histopathology laboratories to compare the expression patterns of pairs of antigenic markers. However, these techniques are time‐consuming, prone to background staining, and rarely suitable for detecting two antigens present at the same site, since one label tends to obscure the other. This paper reports the use of immmunofluorescence for double labelling of pairs of molecular markers in routinely processed tissue. The primary antibodies are either monoclonal reagents of differing isotype/subclass, or antibodies from different species, and labelling is visualized on a conventional fluorescence microscope equipped with a cooled CCD camera. Images can be captured and adjusted using personal computer hardware and software. This approach could be used for a wide range of tissue markers and only minimal tissue autofluorescence was observed. The procedure is more rapid than enzyme‐based techniques and avoids the problems of interpreting two antigens present at the same site. Its establishment involves relatively minor expenditure and it may represent the optimal technical approach to the co‐localization of pairs of antigens in routinely processed tissue samples. Copyright

Identification of the CD85 antigen as ILT2, an inhibitory MHC class I receptor of the immunoglobulin superfamily.

The CD85 molecule was originally defined at the Fifth Workshop on Leucocyte Antigens in 1993 by two monoclonal antibodies, VMP55 and GHI/75. This cell‐surface glycoprotein is expressed on B cells, monocytes, and subpopulations of T and natural killer (NK) cells, and particularly high levels are expressed by normal and neoplastic plasma cells and by hairy cell leukemia B cells. We affinity purified the CD85 antigen and obtained tryptic peptide sequence which indicated that this molecule might be ILT2, a recently described inhibitory major histocompatibility complex class I receptor of the immunoglobulin superfamily. This was confirmed by showing that both of the original anti‐CD85 mAbs stained ILT2 transfectants. The cell signaling role demonstrated for ILT2 is consistent with the previously reported involvement of CD85 in T cell activation. J. Leukoc. Biol. 65: 841–845; 1999.

Phosphorylated KDR can be located in the nucleus of neoplastic cells.

KDR (kinase insert domain receptor) phosphorylation induces several effects which lead eventually to cell proliferation and survival. The precise mechanisms by which KDR, once it is activated, communicates with the nucleus are starting to be understood but have not yet been completely unravelled. Two in vitro studies on animal cell lines reported in the literature have demonstrated that, following stimulation with VEGF, KDR is actually translocated within the nucleus. Our aim was to investigate whether this translocation occurs in human cells both in vitro and in vivo. Using laser scanning confocal microscopy, a variable nuclear localization of phosphorylated and total KDR in cell lines and tumour samples was found. In human neoplastic cell lines, hypoxic stimulation greatly increased the nuclear amount of total KDR but less so that of the phosphorylated form. Only after hypoxia and VEGF stimulation there was a comparably increased expression of phosphorylated and total KDR observed in the nuclei of these cells. We conclude that neoplastic cells show a variable expression of total and phosphorylated KDR in the nucleus. The precise functional meaning of nuclear location remains to be established.

True epithelial hyperplasia in the thymus of early-onset myasthenia gravis patients: implications for immunopathogenesis

The early-onset myasthenia gravis (EOMG) thymus shows characteristic medullary epithelial bands (MEB), greatly expanded perivascular infiltrates and fenestrations of the intervening basement membranes. We now compare epithelial expression of epidermal growth factor receptor (EGFR) and many integrins in EOMG and control samples. The main differences are striking/consistent thickening (in MEB) of what is normally a monolayer of perivascular epithelium, with focal protrusion into the infiltrates. This evidently hyperplastic epithelial subpopulation also strongly expresses EGFR and certain integrins. We suggest that its enhanced interactions with the locally increased extracellular matrix protein deposits may play an important role in autosensitization.

Antibody By 114 is selective for the 90 kD PI‐linked component of the CD66 antigen: a new reagent for the study of paroxysmal nocturnal haemoglobinuria

Summary This paper describes a monoclonal antibody which reacts with transfected cells carrying a gene (NCA‐50/90) which has been shown to encode the human CD66 antigen. However, antibody By 114 recognizes only a single 90 kD polypeptide from human neutrophils, whereas the antibodies which originally defined the CD66 antigen also recognize a larger 180‐200 kD protein. We conclude that antibody By 114 is selective for the smaller of the two CD66 gene products. which is a surface membrane phosphatidylinositol (PI)‐linked molecule. The reactivity of antibody By 114 on peripheral blood cells (positive on neutrophils, weak or negative on eosinophils, and negative on basophils, monocytes and lymphocytes) and myeloid precursor cells was identical to those of a reference CD66 antibody, as was the staining of leukaemic cells. However, the reactions of the two antibodies differed on kidney, liver and pancreas, and in cases of myeloma, Waldenströms macroglobulinaemia and lymphoma, indicating that By 114 represents a new CD66 sub‐specificity. Granulocytes from a case of paroxysmal nocturnal haemoglobinuria (PNH) were negative with antibody By114, indicating that it may be of value in detecting the defect in PI‐linked surface molecules characteristic of this condition. Antibody By114 also stained formalin fixed paraffin embedded tissues and may therefore be of use in routine diagnostic histopathology.

Novel leukaemia markers

Using synthetic peptide or recombinant protein as immunising antigens we have produced monoclonal antibodies and polyclonal antisera directed against targets of particular interest in leukaemia diagnosis. In this way we have prepared reagents which recognise all T or all B lymphocytes in routinely fixed paraffin sections which are unique in this respect. We have also produced monoclonal antibodies to molecules potentially involved in specific neoplastic transformations, implicated by virtue of the involvement of their genes in chromosomal defects in these neoplasms. In particular, we have produced antibodies recognising bcl-2, involved in follicular lymphoma, tal-1, involved in T-cell acute leukaemias and HRX involved in a variety of hematologic disorders. The application of these reagents to diagnosis has so far proved useful. In addition their use outside the field of leukaemia diagnosis has proved to be even more important in some cases.

ZB51 : a monoclonal antibody reactive with human plasma cells

Summary. Several monoclonal antibodies have been raised in the past which react with human plasma cells but they have all shown additional reactions with other cell types. In this paper we describe a new monoclonal antibody, ZB51, which recognizes an intracellular antigen in normal plasma cells in cryostat tissue sections and cell smears, and which also reacts with neoplastic cells in most cases of myeloma and with plasma cell lines. The antibody shows minimal reactivity with a few eosinophils and myelocytes in bone marrow and stains a myeloid cell line. Whilst normal epithelium is not labelled, antibody ZB51 stains two carcinoma cell lines. Although it was not possible to characterize the target antigen in terms of molecular weight, the reactivity of antibody ZB51 with normal and neoplastic plasma cells makes it a useful new immunocytochemical reagent.