The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p.

The JC12 monoclonal antibody recognizes a previously unknown nuclear protein that showed a restricted distribution in normal tonsil and was also overexpressed in a subset of diffuse large B-cell lymphomas. Using this reagent, we expression cloned cDNAs encoding its antigenic target and identified this protein as a novel putative transcription factor, FOXP1. The FOXP1 protein sequence contains predicted domains characteristic of transcription factors, including a winged helix DNA-binding motif, a second potential DNA-binding motif, a C(2)H(2) zinc finger, nuclear localization signals, coiled-coil regions, PEST sequences, and potential transactivation domains. The FOXP1 gene has been mapped to chromosome 3p14.1, a region that commonly shows loss of heterozygosity in a wide range of tumors and which is reported to contain a tumor suppressor gene(s). Using tissue arrays and immunohistochemistry, we demonstrate that both the FOXP1 mRNA and protein are widely expressed in normal tissues. The levels of FOXP1 mRNA were compared in paired normal and tumor tissues (from the same patient) using a tissue array containing cDNAs extracted from 68 samples taken from kidney, breast, prostate, uterus, ovary, cervix, colon, lung, stomach, rectum, small intestine, and from nine cancer cell lines. Differences in FOXP1 mRNA expression between normal and tumor samples were observed in 51% of cases. Most striking was the comparative loss of expression in 73% of colon tumors and comparative overexpression of FOXP1 mRNA in 75% of stomach tumors. Analysis of the FOXP1 mRNA expression in normal tissues (not taken from cancer patients) indicated that loss of FOXP1 expression may occur in some histologically normal tissues adjacent to tumors. Immunohistochemical analysis of FOXP1 protein expression was performed on 128 solid tumors, including 16 renal, 9 breast, 12 lung, 20 colon, 21 stomach, 10 head and neck, 35 prostate, and 5 pancreatic cases. Complete loss of expression, increased expression, and cytoplasmic mislocalization of the predominantly nuclear FOXP1 protein were frequently observed in neoplastic cells. Our study identifies FOXP1 as a new candidate tumor suppressor gene localized to the chromosome 3p14.1 region.

Description of a sequential staining procedure for double immunoenzymatic staining of pairs of antigens using monoclonal antibodies.

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies.

The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best immunohistological labeling reactions, being stable on storage and compatible with a wide range of human monoclonal antibodies. Gel filtration revealed that monoclonal PAP is of lower molecular weight than conventional PAP complexes (fulfilling theoretical predictions based on the monospecificity of monoclonal antibodies).

Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").

Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence.

AIMS: To evaluate whether cytotoxic/suppressor T cells can be detected in paraffin wax embedded human tissue samples using antibodies to a synthetic CD8 peptide sequence. METHODS: Polyclonal and monoclonal antibodies were raised against a 13 amino acid peptide sequence from the cytoplasmic portion of the alpha chain of the human CD8 molecule. RESULTS: These antibodies specifically detected the native form of the CD8 polypeptide when tested by immunoprecipitation with radiolabelled T cells, and gave the expected staining pattern for cytotoxic/suppressor T cells in cryostat sections. Being raised in rabbits, the polyclonal antibodies were also useful for double labelling for CD8 in conjunction with monoclonal antibodies. CD8 positive cells could also be detected in paraffin wax embedded tissues. This was achieved without prior treatment of the sections if the tissue had been fixed in Bouin's fixative. When tissues had been exposed to conventional formalin fixation, preliminary microwave treatment was required. CONCLUSIONS: These findings provide further evidence that antibodies against leucocyte associated antigens, capable of reacting on paraffin wax embedded tissue, can be produced by immunisation with synthetic peptide sequences.

Monoclonal antibody (Y1/82A) with specificity towards peripheral blood monocytes and tissue macrophages.

A new monoclonal antibody, Y1/82A, was raised against phytohaemagglutinin activated peripheral blood mononuclear cells. Using an immunohistochemical technique it was shown that Y1/82A reacts against peripheral blood and bone marrow monocytes and resident macrophages from essentially all human tissues. Y1/82A bound to determinants present in leukaemic cells from patients with acute myelomonocytic leukaemia and acute monocytic leukaemia, but not to neoplastic cells from patients with malignant lymphoproliferative disorders or malignant epithelial tumours. Y1/82A failed to react with other cell types, with the exception of osteoclasts and megakaryocytes. Analysis by Western blotting showed that the antigen detected by antibody Y1/82A was associated with intracellular granules in macrophages. Monoclonal antibody Y1/82A may be useful in the diagnosis of monocytic leukaemias and histiocytic neoplasms and in the identification of macrophages in tissues from various inflammatory and neoplastic conditions.

Monoclonal antibody JC1: new reagent for studying cell proliferation.

AIM: To characterise a newly developed mouse monoclonal antibody JC1 which recognises a nuclear antigen present in proliferating cells in normal tissues and neoplastic lesions, and which is absent in resting cells. METHODS: The methodology was established using a representative range of frozen sections from normal tissues and from certain tumours which were immunostained with antibodies Ki67 and JC1. The molecular weight of the antigen recognised by JC1 was obtained by western blot analysis and this was compared with that of Ki67. IM-9 cell lysates containing Ki67 derived plasmids were also tested with JC1 antibody. RESULTS: Biochemical investigation indicated that the antigen recognised by JC1 gives two molecular weight bands of 212 and 123 kilodaltons, which is distinct from the well characterised anti-proliferation monoclonal antibody Ki67 (395-345 kilodaltons). In addition recombinant Ki67 protein is not recognised by JC1. Immunohistological reactivity was seen in areas known to contain numerous proliferating cells such as lymphoid germinal centres, splenic white matter, cortical thymocytes and undifferentiated spermatogonia. In tumours many cells from adenocarcinomas, oat cell carcinomas, squamous cell carcinomas of lung, and seminomas were labelled by JC1 with a distribution and proportion similar to that seen with Ki67. In normal tissues the only apparent difference was in testis where JC1 stained a considerably greater number of cells than Ki67. In all cases studied the new antibody showed nuclear reactivity only. JC1 did not show any cytoplasmic crossreactivity with squamous cells as is frequently seen with Ki67. CONCLUSION: Antibody JC1, which recognises a nuclear antigen present in proliferating cells, should provide a useful adjunct to Ki67 as a marker of proliferation especially in those cases such as squamous cell carcinomas where a Ki67 index cannot be determined.

JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections.

A new monoclonal antibody, JC70, raised against a membrane preparation from a spleen affected by hairy cell leukaemia, recognises a membrane bound glycoprotein identical with that of the CD31 group of monoclonal antibodies. The antibody stains a fixation resistant epitope on endothelial cells in benign and malignant conditions in a wide variety of paraffin wax embedded tissue. JC70 stained malignant endothelial cells in 10 angiosarcomas with more consistency than monoclonal or polyclonal antibodies to factor VIII related antigen (FVIII-Rag). In four cases of Kaposi's sarcoma the antibody stained malignant endothelial cells but not spindle cells. It is concluded that antibody JC70 is of value for studying benign and malignant human vascular disorders in routinely processed tissue.

Cellular distribution of human leucocyte adhesion molecule ICAM-3.

AIMS: To describe the distribution of the recently cloned human leucocyte adhesion molecule ICAM-3 in normal and neoplastic tissues and cell lines. METHODS: A panel of four monoclonal antibodies to ICAM-3 were used to stain cell lines and sections of human lymphoid tissues using the alkaline phosphatase-anti-alkaline phosphatase immunocytochemical method (APAAP). RESULTS: In peripheral blood ICAM-3 was detected on monocytes, granulocytes, and most lymphocytes. In sections of human lymphoid tissue the antigen was also found on most lymphocytes, but many of the proliferating B cells found in the germinal centres of secondary lymphoid follicles were ICAM-3 negative. ICAM-3 was also found on neoplastic white cells (in chronic lymphocytic leukaemia, hairy cell leukaemia, acute and chronic myeloid leukaemia, and multiple myeloma) with the exception of Reed-Sternberg cells in Hodgkin's disease, many of which were negative. ICAM-3 was consistently absent from cells and tissues of non-haemopoietic origin. Endothelium (which expresses ICAM-1) was negative for ICAM-3, with the exception of vessels in some neoplastic lymphoid samples which showed variable staining for ICAM-3. CONCLUSIONS: These findings suggest that ICAM-3 is essentially restricted to the haemopoietic system and is reciprocal in its expression to ICAM-1, in that it is present on resting cells and its level falls as a result of cell activation.

Detection of T and B cells in many animal species using cross-reactive anti-peptide antibodies.

A wide range of lineage-specific Ag are detectable in the human lymphoid system using mAb, but only a few such markers are detectable in animal species. In this paper, we have investigated the interspecies reactivity of antibodies raised against intracytoplasmic peptide sequences from two T cell Ag (CD3 and CD5) and two B cell markers (the Ig-associated polypeptides encoded by the mb-1 and B29 genes). Immunocytochemical labeling of tissue sections showed that these antibodies cross-react widely between different species (including ungulates, rodents, and marsupials), staining B or T cell areas selectively in lymphoid tissue. The specificity of these antibodies for the animal homologues of the human T and B cell markers was confirmed for the rat by Western blotting analysis. The broad cross-reactivity of these antibodies appears to be due to the fact that they were raised against intracytoplasmic peptide sequences that are highly conserved between humans and rodents, i.e., 80% for mb-1, 85% for CD5, and 100% for CD3 and B29. This strategy should, in the future, widen the range of lineage-associated markers detectable in experimental animals.

Relative expression of surface IgM, IgD and the Ig-associating alpha(mb-1) and beta(B-29) polypeptide chains.

Membrane immunoglobulins are associated with a transmembrane disulphide-linked heterodimer composed of an alpha-chain (mb-1) and a beta-chain (B-29). The relative surface expression of all of the polypeptide chains comprising the Ig-alpha beta complex has been investigated using surface labelling coprecipitation analysis and two-colour flow cytometric analysis. The main conclusions are that mb-1 and B-29 are B-cell surface markers on immature and mature B cells, and that all components of the surface Ig-alpha beta complex are expressed in stoichiometrically equivalent amounts. Thus the complex was quantitatively precipitated from digitonin lysates of 125I-surface-labelled cells with anti-B-29, anti-mb-1 or anti-Ig. Secondly, by two-colour FACS analysis there was a proportionality between the relative amounts of cell surface mb-1 or B-29 and surface IgM or IgD, but not other B-cell markers (class II, B220, FcR gamma, FcR epsilon). Finally there was an insignificant number of B cells expressing membrane Ig without alpha- and beta-chains, and vice versa. Thus there appears to be a closely controlled relative synthesis and surface expression of all components of the B-cell receptor complex.

CD79 alpha/CD79 beta heterodimers are expressed on pro-B cell surfaces without associated mu heavy chain.

During B cell development, the surface expression of CD79 alpha/CD79 beta heterodimers had been thought to begin in the pre-B cell stage where the heterodimers constitute pre-B cell receptors together with mu heavy and surrogate light chains. Thereafter, in mature B cells, CD79 alpha/CD79 beta associates with surface Ig to form B cell antigen receptors. In this study, we revealed by using newly established mAb that CD79 beta was expressed on the surface of pro-B cells which had not undergone the productive Ig gene rearrangement. Biochemical analysis showed that CD79 beta on pro-B cells existed either as monomers or as disulfide-linked heterodimers with CD79 alpha, non-covalently associated with four unidentified membrane molecules. Our finding that CD79 beta is expressed on earlier B-lineage cells than previously expected coincides with the recent study in which CD79 beta-deficient mice exhibit a blockade of B cell differentiation at the pro-B cell stage. Thus, it is speculated that the CD79 beta-containing complexes on pro-B cell surfaces may function to induce early B cell differentiation.

Distribution of the CD68 macrophage/myeloid associated antigen.

The distribution of the pan-macrophage CD68 antigen, recognized by six different monoclonal antibodies, was examined in human blood, tissue, and cell lines using APAAP staining and Western blotting. All antibodies stained monocytes and macrophages, but labelling of neutrophils, basophils, and lymphocytes was seen with some of the reagents. In addition, the CD68 antibodies demonstrated a variety of staining patterns on some non-haemopoietic cells. The subtle differences between the reactions of the different antibodies suggested that the CD68 antigen may be heterogeneous, possibly due to differences in glycosylation. While CD68 antibodies are very useful markers of the macrophage/myeloid series, the presence of small amounts of the antigen on some lymphoid and non-haemopoietic cells means that care should be taken when using them for the diagnosis of tumours of unknown origin.

The immunohistological detection of platelets, megakaryocytes and thrombi in routinely processed specimens.

The production and characterization of a new monoclonal antibody, Y2/51, against platelet glycoprotein IIIa is described. A useful feature of this antibody is its ability to recognize platelets and megakaryocytes in formalin-fixed routinely processed material. It could also be used to reveal platelets both in thrombi in large vessels and in microthrombi too small to be readily apparent on conventional microscopic examination. For this purpose it was helpful to use the antibody in conjunction with a new monoclonal reagent (Ret40f) against red cell sialoglycoprotein beta which detects red cells and their precursors in routinely processed tissue. The use of these antibodies should be valuable for the detection of thrombi in a variety of situations such as renal transplant rejection, coronary artery disease and vasculitis.

CD5 is associated with the human B cell antigen receptor complex.

On human B cells the antigen receptor complex is composed of the membrane form of the immunoglobulin molecule and the non-covalently associated Ig alpha/beta heterodimer. A small subpopulation of normal B cells and chronic lymphocytic leukemia B cells express (analogous to T cells) the transmembrane molecule CD5, a counterstructure of B cell-specific CD72. Numbers of CD5+ B cells are increased in several physiological and pathological conditions. Moreover, CD5+ B cells are being held responsible for the production of autoreactive antibodies and seem to have signaling characteristics distinct from conventional B cells. On T cells, CD5 associates with the T cell receptor CD3 complex and ligation of CD5 leads to the generation of co-stimulatory signals, that act on T cell activation. We here demonstrate that CD5 is associated with the B cell receptor (BCR) complex and serves as substrate for BCR-induced tyrosine kinase activity. Hence, CD5+ B cells have a unique potential to modulate BCR signals.

Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation.

It has been reported previously that the bcl-2 protooncogene protein is detectable in neoplastic cells from cases of human lymphoma in which the 14;18 chromosomal translocation is present, but not in lymphomas that lack this chromosomal rearrangement or in normal lymphoid tissue. In the present study we confirmed, by immunohistologic labeling with polyclonal and monoclonal antibodies, that bcl-2 protein is strongly expressed in many cases of follicular lymphoma and that these neoplastic follicles differ clearly from their nonmalignant counterpart (reactive germinal centres) in which bcl-2 protein is undetectable. However we also found bcl-2 protein in normal T- and B-lymphoid cells and in a variety of lymphoproliferative disorders in which the 14;18 translocation is not present. It is therefore concluded that expression of bcl-2 protein is not a specific marker for lymphomas bearing the 14;18 chromosomal translocation and that the observations of other investigators may have reflected the inadequate sensitivity of their staining procedure.

Different isoforms of human FcRII distinguished by CDw32 antibodies.

The Third and Fourth International Workshops on Leucocyte Differentiation Antigens identified six mAb, designated CDw32, reacting with human Ig FcR type II (FcRII). We have examined the immunohistochemical and immunocytologic reactivities of these antibodies and find that the antibodies could be divided into three classes of reactivity: 1) antibodies IV.3, CIKM3, and CIKM5 reacted with monocytes, macrophages and neutrophils; 2) antibodies KB61 and 41H.16 gave strong reactions with B lymphocytes, placental and hepatic endothelium, and weaker reactions with monocytes, macrophages, and neutrophils; 3) antibody 2E1 gave an intermediate reaction pattern. Immunoprecipitation from U937 cell lysates showed that antibodies KB61 and 41H.16 recognized Mr 41,000 and Mr 37,000 molecules whereas the other antibodies detected a Mr 42,000 molecule. Preclearing with antibody KB61 removed the Ag recognized by the other five antibodies confirming the identity of the Ag and demonstrating reactivity of KB61 with the Mr 42,000 molecule. Antibodies KB61 and 41H.16 precipitated a Mr 41,000 molecule from B lymphocytes. Flow cytometry and immunoprecipitation studies of cells transfected with cDNA clones coding for two isoforms of FcRII showed that all six of the antibodies react with both transfectants but the only immunoprecipitations were obtained using KB61 and 41H.16 and one of the transfectants. The protein sequence of KB61 Ag isolated from leukemic B cells showed close homology with the proteins encoded by the cDNA clones but diverged in the intracytoplasmic carboxyl-terminal region. It was concluded that preferential recognition of one or more of the numerous isoforms of FcRII underlies the differing reaction patterns of CDw32 antibodies.

Antibody L26 recognizes an intracellular epitope on the B-cell-associated CD20 antigen.

Monoclonal antibody L26 is a highly selective marker of B cells and B-cell neoplasms in paraffin-embedded tissues, but it suffers from the drawback that the target molecule has not been identified. In this paper we provide evidence by two independent techniques that antibody L26 recognizes an intracellular epitope on the CD20 antigen (a pan B-cell marker). When this antigen was redistributed on the surface of unfixed viable B cells by incubation with monoclonal anti-CD20 followed by anti-mouse Ig, the diffuse cytoplasmic staining of L26 was abolished and replaced by coincident dotlike labeling for antibody L26 and the CD20 antigen. None of the other antibodies tested (covering 10 different B-cell-associated antigens) had this effect on the L26 staining pattern. Furthermore, COS-1 cells transfected with cDNA encoding the CD20 molecule gave positive staining with antibody L26 and with two other CD20 reagents, but not with antibodies to other pan B-cell markers (eg, CD19 and CD22).

A new monoclonal antibody to epithelial membrane antigen (EMA)-E29. A comparison of its immunocytochemical reactivity with polyclonal anti-EMA antibodies and with another monoclonal antibody, HMFG-2.

Two polyclonal rabbit antibodies to epithelial membrane antigen (EMA), two mouse monoclonal antibodies (E29 and HMFG-2), and a "cocktail" of these two monoclonals have been compared using an indirect immunoperoxidase technique. Sections from 25 tissues (17 malignant and 8 benign), were examined. The distribution of staining with each of these reagents was similar, but the polyclonal antibodies produced stronger staining in colorectal carcinomas and lactating breast, whereas staining with the monoclonal antibodies was stronger in non-neoplastic pleural mesothelium and in pulmonary alveolar cells. When the two monoclonals were mixed there was no increase in staining intensity. E29 gave a "cleaner" result than HMFG-2, with better discrimination between cells and stroma, and is highly suitable for routine diagnostic histopathology.