Immunophenotypic characterization of mononuclear phagocytes and dendritic cells in lymphoid organs of the rhesus monkey

Mononuclear phagocytes and dendritic cells are potent antigen-presenting cells that localize to distinct microenvironmental compartments in many different organs. These cells are particularly plentiful in spleen and lymph node. Recently, these cells have been identified and immunophenotypically characterized in human tissue sections using monoclonal antibodies. However, similar studies in animal species, particularly those representing models of human diseases, have yet to be completely performed. We have evaluated 18 monoclonal reagents raised against human determinants for their reactivity with macrophages and dendritic cells in lymphoid organs of rhesus monkeys. Six of the 18 (EBM11, 25F9, Mol, R4/23, To5, and SK9) produced labeling patterns in rhesus monkey lymphoid tissue that paralleled the staining patterns described for human tissues. Seven others (KB90, FMC17, Mo3, PHM3, PHM2, G16/1, and 27E10) stained varying subsets of specific cells types in these simian tissues. These reagents are requisite for the future study in an experimental animal of the afferent immune response in both normal and disease states.     

Detection of cell surface antigens in cryostat sections with immunogold-silver staining

Immunogold-silver staining was used for the detection of lymphocyte cell surface antigens in cryostat sections of lymphoid tissues. The sections were incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained, and mounted. In light microscopy, the tissue architecture was well preserved, and a dark labeling was seen on the positive cells. Optimal labeling conditions were determined. The distribution of the lymphocyte subsets, as defined by a panel of monoclonal antibodies in tonsil and reactive lymph nodes, was similar to that found with a biotin-avidin-horseradish peroxidase method. The monoclonality of the neoplastic cells in lymph nodes of B-cell non-Hodgkin's lymphomas clearly could be demonstrated. The sensitivity of the technic was comparable with that of the biotin-avidin-horseradish peroxidase labeling method. In addition, immunogold-silver labeling was combined with acid phosphatase cytochemistry.     

Heterogeneity of phenotypic expression in normal and neoplastic B-cell proliferations detected by monoclonal antibodies LN-1 and DLC-48

Recently developed monoclonal antibodies DLC-48 and LN-1 have been shown to be effective reagents for complement-mediated cell lysis of human B-cells. The authors have initially found that these reagents are useful in elimination of malignant lymphomas cells from human bone marrow for autologous bone marrow transplantation. In the current study, the authors have further characterized the reactivity of these antibodies in B-cell neoplasia and have analyzed the heterogeneity of antigenic expression among benign and neoplastic lymphoid cells for B-cell monoclonal antibodies. With the use of quantitative flow cytometric techniques to assess heterogeneity of antigen expression, the findings indicate that phenotypic heterogeneity within neoplasms from individual patients exists for a number of B-cell-related monoclonal antibodies, including B1, common acute lymphocyte leukemia antigen (CALLA), Ia, Ba-1, DLC-48, and LN-1. Within a given histologic class of lymphoid neoplasia, phenotypic heterogeneity was found to be a property of some individual cases and not others for all B-cell monoclonal antibodies examined. This heterogeneity was not consistently expressed in patients with similar tumors, nor was it a consistent property of a given antigen. Using cell lines derived from large cell lymphomas, the authors were able to find phenotypic heterogeneity within neoplastic cell lines that can be used to develop models for dealing with phenotypic heterogeneity in the context of complement-mediated cell lysis. They also established that for DLC-48 and LN-1, the heterogeneity was not related to the phase of the cell cycle of the proliferating cells. The results suggest that single monoclonal antibody treatment for bone marrow depletion of neoplastic cell may be insufficient to overcome phenotypic heterogeneity of B-cell neoplasms.     

Double immunoenzymatic labeling of lymphomatous tissues for both immunologic phenotype and a malignancy-associated nucleolar antigen

Defining cell lineage in the non-Hodgkin's lymphomas (NHL) is challenging for the immunopathologist. Cell surface marker techniques have made a major contribution to the understanding of the biology and classification of lymphoproliferative disorders by permitting the determination of the lymphoid (B- or T-cell) or monocytic lineage of the tumors. Because lymphoma cells often simulate the morphologic features and cell surface phenotype of their normal lymphocytic counterparts, it is difficult to discriminate normal from neoplastic lymphocytes. The authors have used representative monoclonal antibodies (MAb) to cell surface antigens to assess tumor cell surface antigens associated with various lymphoreticular cell lineages. Heteroantisera to the human malignancy-associated nucleolar antigen (HMNA) was utilized as a marker for neoplastic lymphoid cells as previously described. The use of double immunoenzymatic staining with both peroxidase and alkaline phosphatase allow us simultaneously to determine lymphoid lineage and malignancy on human lymphoma cells. In 101 cases of various cell types of NHL, the anti-HMNA antiserum reacted with nucleoli in the morphologically neoplastic lymphoma cells, but not with normal-appearing lymphoid and other cell types present in the lesions. Control specimens from normal and hyperplastic lymphoid tissue also failed to react with anti-HMNA antibodies.     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

Immunohistologic analysis of lymphoid cells by a rapid double staining method

The development of monoclonal antibodies has allowed characterization of subpopulations of lymphoid cells and of their in situ distribution in tissues. The feasibility of simultaneous localization of two surface antigens was studied by double staining with monoclonal antibodies to B cells, T cell subsets and follicular dendritic reticulum cells (DRC) using the avidin-biotin-complex (ABC) method in sections of human lymphoid tissues (tonsil, lymph node, spleen) and a non-lymphoid tissue, endometrium. Color mixture was avoided when an additional incubation with avidin-biotin-labeled peroxidase and subsequent development in the respective substrate of the first sequence preceded the second staining sequence using the primary antibodies at optimal concentrations. The antigenic profiles were portrayed by contrasting and distinct colors of the reaction products. It was observed that T lymphocytes of the cytotoxic/suppressor and helper/inducer phenotypes were topographically associated with each other and with B cells in B and T cell areas of lymphoid tissues as well as in lymphocytic aggregates in endometria. Subpopulations of these cells were mantled by processes of DRCs in lymphoid follicles. The findings indicate that the double ABC staining method can be used for simultaneous demonstration of two surface antigens in tissue sections.     

Lymphoma phenotyping in formalin-fixed and paraffin wax-embedded tissues. I. Range of antibodies and staining patterns

Recently, monoclonal antibodies capable of phenotyping malignant lymphomas in routinely fixed and processed tissue have become available. Some of these reagents identify lineage-restricted variants of the leucocyte common molecule, whereas others identify unique fixation-resistant epitopes on lymphoid cells, some of which are shared by non-lymphoid tissues. A new generation of antibodies recognizing 'classical' leucocyte antigens such as CD3 are also emerging. Refinements in antigen detection systems, especially for immunoglobulin recognition, combined with these new reagents promise to improve the accuracy of lymphoma diagnosis in routine histopathology. These new antibodies are reviewed, and their limitations, cross reactivities and profiles of staining in lymphoreticular disease are discussed. A strategy for their optimal use is proposed.     

Lymphoma phenotyping in formalin-fixed and paraffin wax-embedded tissues: II. Profiles of reactivity in the various tumour types

Recently, monoclonal antibodies capable of phenotyping malignant lymphomas in routinely fixed and processed tissue have become available. Some of these reagents identify lineage-restricted variants of the leucocyte common molecule, whereas others identify unique fixation-resistant epitopes on lymphoid cells, some of which are shared by non-lymphoid tissues. A new generation of antibodies recognizing 'classical' leucocyte antigens such as CD3 are also emerging. Refinements in antigen detection systems, especially for immunoglobulin recognition, combined with these new reagents promise to improve the accuracy of lymphoma diagnosis in routine histopathology. These new antibodies are reviewed, and their limitations, cross reactivities and profiles of staining in lymphoreticular disease are discussed. A strategy for their optimal use is proposed.     

Immunophenotyping of Nigerian cases of non-Hodgkin''s lymphomas on paraffin sections

One hundred cases of routinely fixed and processed non-Hodgkin's lymphoma from Nigeria were immunostained with a small panel of monoclonal antibodies against B-, T- and macrophage antigens. The aims of the study were to assess the suitability of stored material from a country like Nigeria for immunohistochemical examination and the ability of the antibody panel to evaluate the distribution of B- and T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. There were no tumours of macrophage lineage. Four cases gave satisfactory staining of reactive lymphoid cells but no reactivity with malignant cells and thus were not phenotyped. The remaining nine cases gave no staining of neoplastic or reactive cells, suggesting that they were unsuitable for immunohistochemical study, presumably because of inappropriate fixation and handling. We concluded that a panel of three monoclonal antibodies is suitable for routine immunostaining of conventionally fixed and processed blocks in Third World countries and will give diagnostically useful information in approximately 95% of cases.     

The human T-cell leukemias: clinical, cytomorphologic, immunophenotypic, and genotypic characteristics

The distribution of the conventional lymphoid cell markers on T lymphocytes and the principal panels of monoclonal antibodies used to recognize distinctive T-lymphocyte-associated differentiation antigens are discussed. These reagents have been used to probe the early and late stages of T-cell differentiation, and a hypothetical schema of T-cell differentiation has been constructed. Application of these reagents to the investigation of neoplastic T cells has resulted in the determination of the subset of origin and the stage of differentiation of the neoplastic cells in T-cell-derived lymphoproliferative malignancies. Recent advances in molecular biology have made possible the Southern blot hybridization analysis of DNA extracted from neoplastic T cells for patterns of T-cell-receptor gene rearrangements. Examination of these patterns in benign and malignant T and non-T cell has provided the basis for the use of T-cell-receptor gene rearrangements as specific genetic markers of T-cell lineage, clonality, and differentiation. These and other advances have resulted in the delineation of a new category of T-cell neoplasia, the adult T-cell leukemia/lymphoma syndrome. They have also demonstrated that the majority of clinically indolent neoplasms composed of large granular lymphocytes in so-called T gamma-lymphoproliferative disease are monoclonal proliferations. Further phenotypic, functional, and genotypic analyses of the T-cell malignancies should provide better understanding of T-lymphocyte differentiation and heterogeneity. Such studies should also lead to better clinicopathologic correlations and greater understanding of the basis for the clinical diversity of the T-cell-derived lymphoproliferative malignancies.     

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).     

Molecule detected in formalin fixed tissue by antibodies MT1, DF-T1, and L60 (Leu-22) corresponds to CD43 antigen.

Three monoclonal antibodies MT1, L60 (Leu-22), and DF-T1, were reported independently as recognising human T cells in routinely processed, paraffin wax embedded tissue. The present study was performed to compare these three reagents in terms of their immunocytochemical reactions and target molecule(s). On Western blotting of white cell extracts the three antibodies reacted with antigens of the same molecular weight (range 110-160 kilodaltons). Furthermore, their immunocytochemical reactivity with normal human cells, as analysed by two-colour flow cytometry, was essentially identical (labelling of monocytes, most T lymphocytes, and weak reactions with some B cells), and the antibodies gave closely similar reactions on 54 white cell derived neoplasms. To identify the target antigen for these three reagents, antibodies from the Third International Workshop on Leucocyte Antigens were reviewed and it was shown that the Western blotting and immunocytochemical reactions of MT1, L60 (Leu-22), and DF-T1 were identical with those of the reagents which defined the CD43 antigen (also known as leucosialin or sialophorin). Furthermore, all these antibodies reacted with cells transfected with a cDNA clone encoding CD43. It is concluded that antibodies MT1, L60 (Leu-22), and DF-T1 all recognise the heavily glycosylated myeloid/lymphoid associated CD43 antigen.     

Neoplastic angioendotheliosis—further evidence supporting a lymphoid origin

Neoplastic angioendotheliosis is a rare condition, most commonly presenting with a bizarre neurological illness associated with multifocal cerebral infarction due to the occlusion of small blood vessels by neoplastic cells. The histogenesis of the malignant cells is controversial with previous suggestions of an endothelial, epithelial or lymphoid origin. We have studied a case immunohistologically using a panel of monoclonal and polyclonal antibodies recognizing a range of lymphoid, endothelial and epithelial antigens. The results indicate that the malignant cells were of B-lymphoid origin. We also report a second case in which a patient with a high grade B-cell non-Hodgkin's lymphoma developed multiple painful skin nodules which showed the histological features of neoplastic angioendotheliosis. These findings support the view that neoplastic angioendotheliosis is a lymphoma with a propensity to localize and proliferate in small blood vessels throughout the body.     

Role of antigen density in immune lysis of interferon-treated human lymphoid cells: analysis with monoclonal antibodies to the HLA-A,B antigenic molecular complex and to Ia-like antigens

Cultured human lymphoid WI-L2 cells incubated with human leucocyte interferon (final concentration 500 and 2000 U/ml) for 16 h at 37 degrees C acquire increased susceptibility to complement and cell-dependent lysis mediated by monoclonal antibodies to HLA-A,B antigens and to human beta 2-microglobulin but do not change in their susceptibility to immune lysis mediated by monoclonal antibodies to human Ia-like antigens. Changes in susceptibility to immune lysis of interferon-treated lymphoid cells are likely to reflect changes in antigen density, since binding assays with monoclonal antibodies and quantitative absorption assays with alloantisera have shown that the expression of HLA-A,B antigens and beta 2-microglobulin is significantly increased on interferon-treated lymphoid cells, whereas that of Ia-like antigens in not changed.     

Origin of human mast cells studied by dual immunofluorescence.

Previous reports have suggested that mast cells derive either directly from basophils or mononuclear phagocytes, or from a common myeloid precursor. We have used monoclonal antibodies to investigate expression by normal human tissue mast cells of antigens characteristic of other haemopoietic lineages. We find that mast cells express panhaemopoietic markers, but not antigens typical of either myeloid or lymphoid cells. We propose, therefore, that mast cells form a distinct lineage, only distantly-related to other haemopoietic cells.     

Multiparameter immunofluorescence on paraffin-embedded tissue sections.

Immunohistochemical techniques have gained increasing importance in diagnostics and research. While formalin-fixed, paraffin-embedded human tissue retains excellent morphology, the detection of antigens by immunofluorescence in its sections and especially the demonstration of multiple simultaneous antibodies have limitations. Double immunofluorescence labeling of routinely processed paraffin sections has been described previously. The signal intensity observed after triple labeling has been reported to be significantly inferior to that obtained by application of double fluorochromes. The authors show multicolor labeling of three and four primary antibodies in routinely processed paraffin-embedded tissue sections using a standardized immunofluorescence technique. In addition, procedures to reduce background staining and to avoid nonspecific double staining are described.     

The preservation and loss of various non-haematopoietic antigens in human post-mortem tissues as demonstrated by monoclonal antibody immunohistological staining

Immunohistological methods using monoclonal antibodies have proved to be valuable in the differentiation between cells of various origins. We have previously shown most leucocyte differentiation antigens to be very resistant to post-mortem disintegration (Pallesen & Knudsen 1985). In the present study we have examined the preservation of several non-haematopoietic antigens in tissue samples of human skin, kidney, liver, pancreas, lung, thyroid gland, uterine tissue, female breast and brain from 30 autopsies performed at specific intervals after death. Frozen tissue sections were stained using monoclonal antibodies and an immunoperoxidase method. A total of 17 monoclonal antibodies against various intermediate filament proteins, epithelial antigens, various hormones and factor VIII related antigen were tested. We found surprisingly good preservation and staining of tissue antigens--even 3 d after death--in all organs except pancreas. It is concluded that many tissue antigens are fairly resistant to post-mortem disintegration and that immunohistology may be applied to diagnostic problems in human autopsy material.     

The heterogeneity of the reticulum of rat peripheral lymphoid organs identified by monoclonal antibodies

We have developed a panel of six monoclonal antibodies, ED10-ED15, directed against reticular cells in peripheral lymphoid organs. Immunohistochemistry revealed prominent differences between these antibodies with regard to their tissue distribution in lymphoid and non-lymphoid organs. Furthermore, the determinants recognized by ED10-ED13 were found to be differentially expressed by reticular cells occupying the various specialized compartments present in peripheral lymphoid organs. The reactivity patterns of these antibodies observed during the ontogenetic development of the spleen suggest that they recognize differentiation antigens expressed by reticular cells. In contrast, ED14 and ED15 were found to have a relatively ubiquitous tissue distribution recognizing reticular cells in each compartment, with a constitutive reactivity during splenic ontogeny. The present results indicate that reticular cells form a heterogeneous population within the lymphoid organs.     

An immunohistologic study of the epithelial and lymphoid components of six thymomas

Six thymomas were classified histologically and studied immunohistochemically with a panel of mouse and rat monoclonal antibodies directed against thymic epithelial and lymphoid components. The antibodies included monoclonal antibodies directed against cytokeratin, medullary epithelial cells (ER-TR5), and HLA-DR and HLA-ABC antigens, as well as antibodies with specificity for thymocytes. Histologically, one thymoma was characterized by epithelial predominance (EP type), two showed lymphoid predominance (LP type), and two showed mixed lymphoid/epithelial composition (MLE type); one thymoma was a malignant pure epithelial thymoma (PE type). In the thymomas of the MLE and EP types the major populations of cells consisted of HLA-DR-negative, cytokeratin-positive epithelial cells with large ER-TR5-positive subpopulations (i.e., the phenotype of medullary epithelium). In the thymomas of the LP type, the neoplastic population was composed of cytokeratin-positive, ER-TR5-negative cells that expressed the HLA-DR antigen (i.e., the phenotype of cortical epithelium). The thymoma of the PE type consisted of cytokeratin-positive cells, some of which were ER-TR5- and HLA-DR-positive. Double immunofluorescence studies revealed the presence of varying numbers of additional nonepithelial (nonlymphoid) HLA-DR-positive cells in thymomas of the LP, MLE, and EP types. The intervening lymphoid population in the thymomas of the LP, MLE, and EP types consisted largely of cortical thymocytes, as defined by immunologic characterization. These results suggest that thymomas can be classified as medullary or cortical epithelial neoplasms on the basis of their immunologic phenotypes.