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.

Detection of normal and chimeric nucleophosmin in human cells.

In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RARalpha and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.

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.

CD79a: a novel marker for B-cell neoplasms in routinely processed tissue samples.

The CD79 molecule, comprising two polypeptide chains, mb-1 (CD79a) and B29 (CD79b), is physically associated in the B-cell membrane with immunoglobulin. It transmits a signal after antigen binding and may, therefore, be considered the B cell equivalent of CD3. It appears before the pre-B-cell stage, and the mb-1 (CD79a) chain can still be present at the plasma cell stage. In this report, we describe a new anti-CD79a monoclonal antibody, JCB117, which reacts with human B cells in paraffin embedded tissue sections, including decalcified bone marrow trephines. When tested on a total of 454 paraffin embedded tissue biopsies, gathered from a number of different institutions, it reacted with the great majority (97%) of B-cell neoplasms, covering the full range of B-cell maturation, including 10 of 20 cases of myeloma/plasmacytoma. It is of interest that the antibody labels precursor B-cell acute lymphoblastic leukemia samples, making it the most reliable B-cell marker detectable in paraffin-embedded specimens in this disorder. All neoplasms of T cell or nonlymphoid origin were negative, indicating that antibody JCB117 may be of value to diagnostic histopathologists for the identification of B-cell neoplasms of all maturation stages.

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.

Evidence for a direct physical interaction of membrane IgM, IgD, and IgG with the B29 gene product.

The B cell Ag-receptor complex is composed of membrane immunoglobulin (mIg) and the mb-1/B29 heterodimer. In order to obtain insight into the architecture of the B cell receptor complex, we have looked for conditions that disrupt all disulfide bridges in the complex without affecting the noncovalent interaction between the mIg heavy chain and one or both members of the associated heterodimer. We show that in the presence of the reducing agent beta-mercaptoethanol the m mu, m delta, and m gamma heavy chains remain selectively associated with the B29 members. Our findings implied that if isotype-related differences exist between the mIg-associated dimers, they may reside in B29 and not, as initially suggested, in mb-1. However, sequence analyses of B29 gene transcripts from B cells expressing mIgM, mIgD, or mIgG only revealed no differences in their nucleotide composition. Thus, in spite of their close physical interaction with mIg heavy chain classes, which are significantly distinct in the C-terminal regions, no isotype-specific forms of B29 seem to exist.

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.

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 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.

The B29 and mb-1 polypeptides are differentially expressed during human B cell differentiation.

Surface immunoglobulin on mouse B cells is associated with a heterodimer comprising the products of the mb-1 and B29 genes. Here we report that antibodies raised against a peptide sequence from the intracytoplasmic C terminus of the B29 murine gene product detect the 37-kDa component of the human heterodimer, indicating that this component in man is also encoded by the B29 gene. The immunocytochemical reactivity of these anti-B29 antibodies was compared with those of antibodies to the mb-1 protein. Of 25 cases of acute lymphoblastic leukaemia (ALL), 24 were positive for mb-1 whereas B29 was expressed in only 13 cases. Most of these B29-positive ALL expressed immunoglobulin mu heavy chain in their cytoplasm (pre-B ALL). In lymphoid tissue sections, anti-B29-labeled B cell follicles in a similar fashion to anti-mb-1, with the striking exception that plasma cells were unreactive for B29, but positive for mb-1. These results suggest that the synthesis of B29 begins later in precursor B cells than that of mb-1, and ceases before the terminal plasmacyte phase.

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.

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).

The membrane IgM-associated heterodimer on human B cells is a newly defined B cell antigen that contains the protein product of the mb-1 gene.

7/8embrane IgM (mIgM) on human B lymphocytes is noncovalently associated with a disulfide-linked dimer that contains phosphoproteins of 47 and 37 kDa. In this study, the biochemical properties and the identity of these Ag receptor-associated components have been addressed. Both subunits carry N-linked carbohydrate groups. After deglycosylation, the 47-kDa and 37-kDa proteins have similar molecular masses, of about 23 kDa, and relatively acidic but different isoelectric points. The accumulated data, together with a previously performed comparison of tryptic peptides, suggest that the two components are structurally distinct and possibly encoded by different genes. Indeed, a mAb, raised against a synthetic peptide that was made on the basis of the published carboxyl-terminal amino acid sequence of the human mb-1 gene product, specifically reacted with the 47-kDa but not the 37-kDa subunit. None of the established B cell-specific mAb characterized in the Fourth International Workshop on Leukocyte Antigens, including CD24, CD37, and CD72, detect the mIgM-linked heterodimer, which makes it a newly defined human B cell Ag.

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.

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.

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).

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.

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.