Immunofluorescence analysis of normal and malignant lymphoid tissues with selected combination of antisera

Tissue sections stained with combinations of antisera labeled with different fluorochromes (i.e., conventional antisera to human immunoglobulin classes, T lymphocyte antigens, and Ia-like p28,33 antigens used in various double combinations with each other or with different mouse monoclonal antibodies) allow the identification of the different areas of lymph nodes in serial sections and provide great flexibility as well as precision in the analysis of the distribution and relationship of normal and malignant cells. Lymphoid microenvironments in the thymus and the paracortical areas of lymph nodes are described. The close association of T lymphocytes and nonlymphoid cells expressing large amounts of Ia-like antigens (such as interdigitating reticular cells and endothelium) may be relevant for the understanding of immunoregulatory disorders such as dermatopathic and angioimmunoblastic lymphadenopathies and some malignancies (e.g., mycosis fungoides) were the expression of Ia-like antigens on non-T cells seems to be abnormally abundant. The analysis of immunoglobulin and membrane marker expression of normal and malignant B cells and their relation to T cells can also be related to the histology of the disease. These studies are clinically useful for the classification of childhood lymphomas, the differential diagnosis of anaplastic carcinomas and lymphomas, and in the study of the early stages of lymphomas.     

Anomolous staining patterns in immunohistologic studies of malignant lymphoma.

A number of immunoperoxidase studies of malignant lymphomas have reported polytypic light chain staining of neoplastic cells, thus bringing into question the concept that the monoclonality of B cell lymphomas is reflected in their synthesis of monotypic light chain. In this study of a large number of Ig positive lymphomas, staining for a wide variety of antigens has identified clear differences between monotypic Ig synthesizing cells and cells staining polytypically which appear to be taking up Ig from the environment. Attention to the nature of Ig staining and staining for J chain were the two most useful criteria in differentiating Ig synthesis from uptake. The results confirm that malignant B cell lymphomas synthesise monotypic Ig.     

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.     

Differentiation between chronic lymphocytic leukaemia (B-CLL) and non-Hodgkin lymphomas in the leukaemic phase by the mouse red blood cell rosette assay

A distinction between B-CLL and other malignant B-cell lymphomas in the leukaemic phase may be difficult. Mouse red blood cell rosette formation of lymphocytes from 97 patients with B-CLL and 19 patients suffering from other B-cell lymphoproliferative disorders was examined together with lymphocyte rosette formation of healthy controls. The majority of circulating lymphocytes of B-CLL patients formed rosettes with mouse red cells, whereas there was no relationship between the number of peripheral neoplastic B-cells and that of rosette forming cells in other lymphoproliferative diseases. The relatively simple mouse red blood cell rosette assay proved to be of value in the differentiation of otherwise nearly related conditions.     

Cytologic and immunocytologic studies of peripheral T-cell lymphomas

Lymph node aspirates from 18 peripheral T-cell lymphomas (PTLs) were analyzed. Cytologic and immunocytologic studies were performed on Cytospin preparations using the alkaline phosphatase-antialkaline phosphatase method with a panel of monoclonal antibodies (CD3, CD4, CD8, CD19 and CD30). The cytologic diagnosis was confirmed by histologic investigation. Nine lymph node aspirates from patients with Lennert's lymphoma, angioimmunoblastic (AILD)-type PTL and pleomorphic small-cell-type PTL were composed predominantly of small-to-intermediate-sized lymphocytes. An admixture of plasma cells, eosinophils, neutrophils, lymphocytes with an irregular nucleus, granula in the cytoplasm or abundant cytoplasm was also seen. Nine lymph node aspirates from patients with T-immunoblastic lymphoma, pleomorphic large-cell-type PTL and large-cell anaplastic (Ki-1+) lymphoma showed marked cytologic heterogeneity. Immunocytologic investigation of the aspirates using the antibodies CD3, CD4, CD8, CD19 and CD30 was helpful for the differentiation of PTLs from reactive lymphadenopathy and other malignant lymphomas. A strong predominance of CD3+ cells was found in only seven cases. The aspirates expressed a helper/inducer phenotype in 11 cases and a suppressor/cytotoxic phenotype in 4 cases. A T-cell phenotype not corresponding to the normal T-cell phenotype was found in nine cases. In 15 of the 18 cases, the number of CD19+ cells was found to be less than 15%. The large cells of the large-cell anaplastic (Ki-1+) lymphoma expressed the antigens CD30 and CD45 and were negative for CD15. These findings indicate that immunocytologic studies can be used in improving the cytologic diagnosis of PTLs.     

Immunohistochemical detection of oncoprotein 18 (Op18) in malignant lymphomas

Summary Expression of oncoprotein 18 (Op18), an intracellular phosphoprotein up-regulated in many malignant cell types, was evaluated in a series of normal lymphoid tissue and malignant lymphomas. In normal tonsils and reactive lymph nodes, the majority of Op18-positive cells were present in the germinal centres, whereas cells in the mantle zone were essentially negative and the interfollicular areas showed occasional positive cells. Double staining for PCNA and Op18 revealed that Op18 expression only to some extent was correlated with cell proliferation, as determined by PCNA expression.Non-Hodgkin's lymphomas exhibited a variable Op18 expression, and in Hodgkin's disease, Reed-Sternberg and Hodgkin cells frequently expressed Op18 with a strong staining intensity. Using Op18-PCNA double staining in malignant lymphomas, Op18 expression could also be partially dissociated from cell proliferation. By using confocal microscopy, the intracellular localization of Op18 was studied, demonstrating diffuse reactivity in the cytoplasm in interphase cells and during mitosis, whereas nuclei and condensed chromosomes were negative. In conclusion, Op18 was expressed at variable levels in most, perhaps all, proliferating lymphocytes in benign lymphoid tissue as well as in malignant lymphomas. However, the Op18 protein was also detected in a significant fraction of apparently non-cycling normal and neoplastic lymphocytes.     

Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Circulating CD4+ T lymphocytes with intracellular but no surface CD3 antigen in five of seven patients consecutively diagnosed with angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) accounts for less than 1% of all lymphatic malignancies. Oligoclonality or monoclonality for any of the T-cell receptor (TCR) chain genes can be demonstrated in the majority of the cases. During systematic screening for the presence of circulating lymphocytes with atypical coexpression of differentiation antigens in patients with T-cell lymphomas, we have discovered a minor population (accounting for 0.2% to 10.% of all lymphocytes) of atypical lymphocytes in the blood of five of seven patients consecutively diagnosed in 1997/1998 by lymph node histology to have AITL. The major distinguishing feature of these cells consists of the lack of the surface expression of the CD3 antigen, but not of the intracellular expression. These cells express the T-cell antigens CD2 and CD5 on their surface, but not CD7, and they express CD4 and CD45 at numbers of molecules per cell typical for T lymphocytes. Gene scan analyses for the TCR gamma chain revealed oligoclonality of these flow-sorted cells in one patient and monoclonality in two patients, the same patterns of TCR gamma chain gene as determined processing the respective diagnostic lymph nodes. Circulating CD4-expressing T lymphocytes with exclusively cytoplasmic expression of CD3 appear to represent the malignant population in patients with histologically diagnosed AITL.     

Flow cytometric characterization of chronic lymphocyte leukaemias using orthogonal light scattering and quantitative immunofluorescence

Light scattering properties and antigen distribution of lymphocytes labeled with the monoclonal antibodies CD 5 and CD 20 were determined for 19 patients with a chronic B-cell derived leukaemia. The density of the antigen detected by the monoclonal antibody CD 5 appeared to be considerably lower on malignant B-lymphocytes of the patients as compared with T lymphocytes. A large variation was observed in the amount of receptors for the monoclonal antibodies CD 5 and CD 20 on the malignant cells of the different patients. B-cell chronic lymphocytic leukaemia (B-CLL) patients were clearly distinguishable from leukaemic follicular non Hodgkin lymphoma patients (LF-NHL, formerly lymphosarcoma cell leukaemia) and from a patient with a prolymphocytoid transformation (PLT) of the B-CLL according to the amount of the antigens for CD 5 and CD 20. Within the B-CLL patient population, no relation of progression of the disease with distribution of these antigens could be observed. In one patient the extraordinary phenotype CD 20+, CD 11+, leu 8+, CD 5- of the malignant lymphocytes was observed. An experimentally simple method to differentiate between the various chronic lymphocytic leukaemias (CLL) appeared to be the determination of orthogonal light scattering properties of lymphocytes. In healthy donors one can always distinguish two populations of lymphocytes in the orthogonal light scatter histograms. Lymphocytes of B-CLL patients show one uniform population with a relatively small orthogonal light scattering signal, lymphocytes of our patients with PLT of B-CLL or with LF-NHL show one uniform population with a relatively large orthogonal light scattering signal.     

Quantitative estimation of HLA-A and HLA-B antigens carrying the Bw4 supertypic specificity in human peripheral blood lymphocytes

A monoclonal antibody specific for HLA-Bw4 was employed for the quantitative estimation of class I antigens on human peripheral blood lymphocytes and on the human macrophage cell line U 937. The epitopes reactive with the HLA-Bw4-specific antibody, which are present on different antigens coded by the HLA-A or the HLA-B locus, were characterized in terms of equilibrium and kinetic binding parameters. The level of expression of class I antigens on human lymphocytes was found to be in direct proportion to the gene dose. Variations between donors of the same phenotype were of minor importance. Estimation of the association constant, association rate constant, and half-life of dissociation for the interaction of the antibody with lymphocytes heterozygous for either HLA-A24, HLA-A32, HLA-A9, or HLA-Bw4 strongly suggested that the public antigenic determinant shared by these antigens is identical.     

Expression of L-selectin and efficient binding to high endothelial venules do not modulate the dissemination potential of murine B-cell lymphoma

The homing receptor L-selectin is essential for the migration of naive lymphocytes into peripheral lymph nodes. In contrast to naive lymphocytes, activated and memory cells down-regulate L-selectin and enter peripheral lymph nodes by an L-selectin-independent mechanism. In view of the concept that lymphomas present the malignant counterparts of normal lymphocytes at a defined stage of differentiation, it has been suggested that in contrast to lymphomas with a memory/activated cell phenotype, L-selectin is essential for dissemination of lymphomas that represent naive cells. 38C-13 is a murine B-cell lymphoma with an immature naive cell phenotype. 38C-13 cells express high levels of L-selectin and bind to lymph node high endothelial venules in an L-selectin-dependent manner. In this study we demonstrate that treatment of 38C-13 tumor-bearing mice with anti-L-selectin antibodies did not inhibit tumor dissemination to peripheral lymph nodes. Moreover, L-selectin-negative 38C-13 variant cells disseminated as efficiently as wild-type cells. Thus, in spite of its expression, L-selectin is not required and does not affect the metastatic potential of the tumor. L-selectin of the malignant cells and of normal lymphocytes appears to be functionally different. Thus, whereas antibody cross-linking of L-selectin resulted in down-modulation of the receptor in normal lymphocytes, cross-linking had no effect on L-selectin expression in 38C-13 cells, suggesting that, in spite of comparable levels of surface expression in normal and malignant cells, L-selectin may be functionally impaired in some malignant cells. Received: 9 November 2000 / Accepted: 11 January 2001     

Cytomorphologic characteristics of non-Hodgkin's lymphoma

The cytologic samples of 84 non-Hodgkin's lymphomas, classified according to the Rappaport system, were reviewed, with particular attention to the cell pattern, the size of the cells and other morphologic characteristics. The analysis revealed that: (1) the subgroups of malignant lymphoma in the Rappaport classification display a heterogeneous cytologic picture; (2) cytology cannot make the differentiation between nodular and diffuse non-Hodgkin's malignant lymphomas; and (3) reliable cytologic classification is possible only in certain cases of well-differentiated lymphocytic and diffuse histiocytic lymphomas. Well-differentiated lymphocytic lymphoma is characterized by a uniform cell picture consisting of mature lymphocytes, prolymphocytes or centrocytes up to 12 micron in size, by the presence of paraimmunoblasts and by the absence of reticulum cells. The diffuse histiocytic lymphoma contains over 48% centroblasts or over 8% immunoblasts or multinucleated giant blast cells.     

Classification of non-Hodgkin's lymphomas: facts and perspectives]

Previous classifications of malignant non-Hodgkin lymphomas (nHL) are based on morphological and histological-cytological findings. The rapid development of immunology, the application of immunocytological, cytochemical and electromicroscopic methods led to new classifications of nHL based on immunology. They are founded on the idea that the prevailing cells of nHL correspond to a cell type from the differentiation line of lymphocytes. Thus, a distinction can be made between malignant lymphomas (ml) of stem, B and T-cells; the more frequently occurring lymphomas originating from B-cells are represented by chronic lymphatic leukaemia originating from B1-cells, by ml of follicular genesis (centrocytes, centroblastic-centrocytic ml, centroblasts) and by ml synthesizing and releasing immunoglobulins (immunocytoma, immunoblastoma). The type formerly called "reticulosarcoma" (histocytic ml) could be identified to be an immature, Ig synthesizing ml (immunoblastloma) in most cases. The new classification enables a more extensive differentiation and a better prognostic statement to be made than previous ones. The new units of disease can be diagnosed in most cases with light microscopic routine methods, although borderline cases and unclassifiable ml may occur.     

Cross-reactivity between human and murine lymphocyte antigens: III. Reactivity of H-2 allo- and xenoantisera with human lymphoid cells

H-2 alloantisera and antimouse lymphocyte xenoantisera react with 14%–100% of human lymphocytes from a panel of at least 80 unrelated people. Population and family studies did not reveal HL-A specificity of such lymphocytotoxic antibodies but indicated that the antibodies are directed against polymorphic antigenic determinants inherited in association with HL-A antigens. H-2 allo- and xenoantisera absorbed with human lymphoid cells and a panel of platelets bearing all the known HL-A specificities were still cytolytic when tested against murine lymphocytes, suggesting that only a small proportion of the heterogeneous population of H-2 antibodies react with human lymphocytes. On the other hand, HL-A alloantisera could be absorbed by lymphocytes from certain murine strains. These results suggest that the crossreactivity between human and murine lymphocytes is caused by antigens common to several HL-A (or H-2) types or by antigens linked to HL-A but not identical with them.     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Rabbit antiserum against Daudi-cell membrane fractions detects cell surface antigens present on subpopulations of human lymphocytes.

An antiserum was produced by immunization of rabbits with membrane preparations of a human lymphoblastoid B cell line, Daudi. After absorption with a human endometrial carcinoma cell line, this antiserum appeared to be specific for antigen(s) present on adult and fetal thymocytes as well as on tonsillar lymphocytes but absent, or present in very small amounts, on normal or phytohemagglutinin- (PHA) stimulated peripheral blood lymphocytes (PBL). When T and B cell-enriched fractions from tonsillar lymphocytes were tested with the anti-Daudi serum, the reactivity was equally distributed in each population. Among 13 human lymphoblastoid cell lines tested, reactivity was demonstrated on three out of four T cell lines, and on four out of nine B cell lines. The positive reacting B cell lines were derived from two African and two American Burkitt lymphomas. The antigen(s) described does not seem to be related either to human Ia-type antigens or to Epstein-Barr virus-associated antigens because these antigens are not present on fetal or adult thymocytes. Reciprocal absorption experiments indicate that this anti-Daudi serum detects the same antigenic structures present on certain subpopulations of T and B lymphocytes.     

Cytomorphology and immunocytochemistry of fine needle aspirates from blastic non-Hodgkin's lymphomas

Fine needle aspirates from 54 consecutive patients with primary or recurrent blastic (high-grade malignant) non-Hodgkin's lymphomas (NHLs) were analyzed by cytomorphology and immunocytochemistry. The cytologic diagnoses induced follicular center-cell-derived (centroblastic or anaplastic centrocytic) lymphoma (31 cases), immunoblastic lymphoma (11 cases), lymphoblastic lymphoma (9 cases) and histiocytic lymphoma (3 cases). Immunocytochemistry showed a B-cell phenotype of the neoplastic lymphocytes in all lymphoblastic lymphomas, 29 follicle center-cell lymphomas and 4 immunoblastic lymphomas. Four of the immunoblastic lymphomas were of T-cell origin while one case was not evaluable due to necrosis. A histiocytic origin was confirmed in two of the three cases that had a cytologic diagnosis of histiocytic lymphoma; the third case was shown by immunocytochemistry to be a true Ki-1-positive large cell lymphoma. Histologic and immunohistochemical analysis were performed on surgical biopsies from 18 patients. The results were in agreement with those on the fine needle aspiration (FNA) material in 14 cases. Three lymphomas could be phenotyped on aspirated material while marker studies on excised material were inconclusive. One lymph node aspirate contained mostly necrotic cells, which were unsatisfactory for adequate immunocytochemistry. However, sections from a removed tonsil from the same patient could be used for conclusive histology and phenotyping. In conclusion, the high diagnostic accuracy of combined cytomorphologic and immunocytochemical assessment of FNA samples validates the use of the technique in the diagnostic work-up of blastic (high-grade malignant) NHLs. In fact, the diagnostic accuracy seems so high that the technique can safely be used in the final diagnosis of blastic NHLs.     

Inverse relationship between constitutive gamma interferon production and human T-cell lymphoma/leukemia virus expression in cultured T lymphocytes.

Particular interest in human T lymphocyte lymphoma/leukemia virus (HTLV) derives from the close association of HTLV with several types of human mature T lymphocyte malignancies and the strong possibility that HTLV is the causative agent of this group of leukemias and lymphomas. This is the first report to show that HTLV expression in T lymphocytes cultured in vitro is inversely proportional to constitutive gamma interferon production. Of 16 fresh T lymphocyte cultures established from patients with mature T lymphocyte neoplasias, 3 were grown continuously for over 3 years and 13 were grown for 2 to 8 months in culture. Of the 16 cultures, 9 were HTLVp19 positive and interferon negative, whereas the remaining 7 were HTLVp19 negative or weakly positive and also interferon positive (12 to 105 U/ml). The prototype HTLV-positive T-cell line (HUT102) was examined over a long-term culture and after selective cell cloning for high virus yield. Results indicate that early-passage, low-HTLV-producing HUT102 cells constitutively produced significant levels of gamma-immune interferon. In late-passage and cloned HUT102 cells, an increase in HTLV production was concordant with a decrease in constitutive interferon production and the loss of mature T lymphocyte antigens. Transformation of human umbilical cord blood lymphocytes by HTLV was possible only after cocultivation with the non-interferon, high virus-producing, cloned HUT102 T lymphocytes. The inverse relationship between interferon and HTLV production was also observed when normal human umbilical cord blood and adult T lymphocytes were transformed by HTLV and maintained in culture.     

A Specific Nuclear DNA Background Is Required for High Frequency Lymphoma Development in Transmitochondrial Mice with G13997A mtDNA

We previously found that mouse mitochondrial DNA (mtDNA) with a G13997A mutation (G13997A mtDNA) controls not only the transformation of cultured lung carcinoma cells from poorly metastatic into highly metastatic cells, but also the transformation of lymphocytes into lymphomas in living C57BL/6 (B6) mice. Because the nuclear genetic background of the B6 strain makes the strain prone to develop lymphomas, here we examined whether G13997A mtDNA independently induces lymphoma development even in mice with the nuclear genetic background of the A/J strain, which is not prone to develop lymphomas. Our results showed that the B6 nuclear genetic background is required for frequent lymphoma development in mice with G13997A mtDNA. Moreover, G13997A mtDNA in mice did not enhance the malignant transformation of lung adenomas into adenocarcinomas or that of hepatocellular carcinomas from poorly metastatic into highly metastatic carcinomas. Therefore, G13997A mtDNA enhances the frequency of lymphoma development under the abnormalities in the B6 nuclear genome, and does not independently control tumor development and tumor progression.