Application of orthogonal light scattering for routine screening of lymphocyte samples

Orthogonal and forward light-scattering properties of lymphocytes were measured from patients with different lymphocytic diseases in order to determine the potential value of light scattering as a screening device. Monitoring of orthogonal light scattering of lymphocytes of a B-cell chronic lymphocytic leukemia patient during splenic irradiation (SI) revealed the selective decrease of malignant cells and the fact that the major part of the residual lymphocytes were cytotoxic lymphocytes. By combining forward and orthogonal light scattering it was shown that lymphocytes from a patient with T gamma lymphocytosis were abnormal. Orthogonal light scattering also showed an increase in cytotoxic lymphocytes in a patient with mononucleosis infectiosa and in a splenectomized patient. Orthogonal light scattering of lymphocyte subpopulations showed that the leu8+ population of a patient with mononucleosis infectiosa was bidisperse. For elderly donors the occurrence of CD3+, CD4+, CD8+, and HNK-1+ lymphocytes with a large orthogonal light scattering varied considerably. The CD8+ lymphocytes of these donors consisted mainly of cytotoxic lymphocytes. These results show that determination of light-scattering properties of lymphocytes may yield important diagnostic information and can indicate when further investigation of the lymphocytes by means of immunofluorescence is necessary.     

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia. A flow cytometric study of peripheral blood, lymph nodes and bone marrow.

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD 57+ and cytotoxic CD 8+) wa studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57+, CD 16+ and cytotoxic CD 8+ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3+ lymphocytes, which were 1.7-27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4+ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57+ receptors in comparison with their peripheral blood CD 57+ lymphocytes or the CD 57+ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.     

SC3 monoclonal antibody defines a novel specific human B-cell surface antigen differentially expressed on B-cell leukaemias and lymphomas and involved in the proliferation of normal and malignant B lymphocytes

The monoclonal antibody SC3 was raised against the NK leukaemia cell line YTindi. It detected a 98-kDa surface antigen with weak expression on a restricted number of leukaemia cell lines under reducing conditions. SC3 mAb labelled 5-10% of normal peripheral blood lymphocytes corresponding almost exclusively to B lymphocytes, and 60-70% of tonsillar B cells. It did not react with erythrocytes, platelets or monocytes whereas it stained granulocytes. The aim of the present study was to examine the expression and functional effects of SC3 mAb reactive epitope on normal and malignant B cells. Most SC3+ B cells from healthy donors were CD23+, some co-expressed CD5 and CD27 and a few were CD38+. SC3 epitope was expressed exclusively by B-lineage malignant proliferations, including B-lineage ALL. Practically, all B-CLL studied expressed SC3 mAb reactive epitope although with variable intensity, while MCL and PLL were negative. Other low grade and high grade B-NHL were variably stained. SC3 mAb alone triggered the proliferation of CD2-depleted PBL and significantly increased the proliferation induced by suboptimal concentrations of LPS. This effect was much weaker with B-CLL cells but was increased after cross-linking with an anti-IgM antibody. The restricted expression pattern combined with molecular weight and functional data indicate that SC3 mAb may detect a novel B-cell antigen mostly expressed by early and naive B cells. Although its expression in B-cell malignancies was not limited to a single differentiation stage, it might confer specific functional characteristics to the positive malignant cells.     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

Phenotype of chronic lymphocytic leukemia (CLL) B-cells. B-CLL cells express the Leu-8 antigen

In the present report we studied the phenotype of peripheral blood mononuclear cells (PBMC) from 25 patients with B-cell chronic lymphocytic leukemia (CLL). Cells from all the cases expressed monoclonal surface immunoglobulins (SmIg), formed rosettes with mouse erythrocytes (MRFC) and were positive with OKB 2 and OKIa monoclonal antibodies. In addition, CCB 1 monoclonal antibody was positive in 17 out of 20, Leu-1 in 18 out of 21 and Leu-8 in 23 out of 25 cases. Double labelling experiments confirmed that the Leu-8 antigen was co-expressed on Leu-1+, CCB2+, HLA-DR+ B-CLL cells. Thus, B-CLL cells generally express the SmIg+, MRFC+, Leu-1+, OKB2+, Leu-8+ phenotype. Since it is known that normal peripheral blood B cells may be divided into two subpopulations according to Leu-8 expression, our data indicate that B-CLL cells originate from the more immature Leu-8+ B-cell subset which will respond to anti-IgM, whereas it reacts poorly to pokeweed mitogen.     

Antigenic expression of B-cell chronic lymphocytic leukemic lymphocytes

A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.     

Blast Cells with Monoclonal Surface Immunoglobulin in Two Cases of Acute Blast Crisis Supervening on Chronic Lymphocytic Leukaemia

Acute blast crisis occurred in two patients with previously well-confirmed chronic lymphocytic leukaemia. The finding by direct immunofluorescence of membrane-bound monoclonal immunoglobulins on the surface of the blast cells showed that they were related to B cells in the same way as the proliferating lymphocytes in most patients with chronic lymphocytic leukaemia. In one patient the surface monoclonal IgM detected on both the lymphocytes and the blast cells had the same rheumatoid antibody activity, supporting the concept that the leukaemic cells found during the acute and chronic phases of the disease originated from the same clone.     

Quantification of the leukocyte common antigen (CD45) in mature B-cell malignancies.

CD45 is a glycoprotein expressed on all lymphohematopoietic cells. Its expression increases during normal B-cell differentiation and remains stable on mature cells. Although it is widely known that CD45 antigen expression is decreased in B-acute lymphocytic leukemia (ALL), only scarce and contradictory information is available on CD45 expression on mature B-cell malignancies. In healthy adults (n = 15), CD45 expression on B lymphocytes was lower than that on T cells. In patients with chronic lymphocytic leukemia (CLL; n = 22), CD45 expression on malignant cells was lower than that on the whole lymphocyte population of healthy adults (n = 28) and on normal B lymphocytes (n = 15). In 6 of the 22 CLL patients, the malignant cell population could be separated from the normal lymphocyte population on the CD45-side scatter (SSC) plot. In 16 CLL patients, there was some degree of overlap between the malignant and normal cells with respect to CD45 expression. For these patients, there was an inverse correlation between CD45 expression on the whole lymphocyte population and the percentage of malignant cells in this population. In two patients with mantle cell lymphoma (MCL), CD45 expression on the malignant cells appeared lower than that on normal B cells and on the whole lymphocyte population. In six patients with hairy cell leukemia (HCL), CD45 expression on hairy cells was comparable to that on the whole lymphocyte population of healthy adults, but slightly higher than that of normal B cells. Evaluation of CD45 expression may help to characterize mature B-cell malignancies.     

B cells in chronic lymphocytic leukaemia. Comparative analysis of blood and bone marrow

A study was performed on cell suspension from peripheral blood and bone marrow aspirates and on cryostat sections from bone marrow biopsies in order to investigate the membrane phenotype of neoplastic B cells in chronic lymphocytic leukaemia (B-CLL). The immunological analyses, performed on 43 patients, included rosetting ability with sheep and mouse erythrocytes, evaluation of surface immunoglobulins and reactivity with anti-HLA-DR, UCHT 1 (OKT-3 like) and RFA-1 (OKT-1 like) monoclonal antibodies. The results demonstrate that neoplastic B lymphocytes in B-CLL display an identical phenotype in peripheral blood and bone marrow. Possible interpretations on the origin of proliferating cells in B-CLL are discussed.     

EBV-gp350 confers B-cell tropism to tailored exosomes and is a neo-antigen in normal and malignant B cells--a new option for the treatment of B-CLL

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.     

Lymphoma immunophenotyping: "borderline" lymphomas

Immunophenotyping of B-cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(-) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as "borderline" CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.     

Fine needle aspiration cytology and immunocytochemistry in the diagnosis of lymphoid lesions of the thyroid gland

Immunocytochemical analysis was used in conjunction with cytomorphology to evaluate fine needle aspirates from 18 patients with nodular lymphoid infiltrates of the thyroid. The smears from 13 patients had a cytomorphology that was diagnostic of chronic lymphocytic thyroiditis; immunocytochemistry showed the lymphoid population to be composed of T cells and polyclonal B cells. Three patients had high-grade malignant lymphomas by cytomorphology; immunologic evaluation showed the B-cell phenotype of the lymphoma cells and monoclonal light chain expression. Cytomorphology could not differentiate between an inflammatory and a neoplastic lymphoid infiltrate in two cases. Immunocytochemical analysis showed that the cells of B-cell phenotype were monoclonal (neoplastic) in one case and polyclonal (inflammatory) in one case.     

Modulation of apoptosis by cytokines in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the slow and progressive accumulation of monoclonal apparently mature, CD5(+) B lymphocytes. The majority of circulating cells appear to be nondividing, and it has been suggested that a prolonged life span is mainly responsible for the accumulation of the leukemic cells. However, spontaneous programmed cell death by apoptosis occurs when B chronic lymphocytic leukemia cells are cultured in vitro. This may be because of the lack of an unidentified essential cytokine present in vivo. Thus, we investigate interleukin-2 (IL-2), IL-4, IL-6 and IL-10 in vitro effects on apoptosis of B cells from 32 previously untreated patients with B-CLL in initial clinical stages. B cells were isolated from peripheral blood, and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with the different cytokines, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of cytokines. Our results indicate that both IL-2 and IL-4, but not IL-6, inhibit in vitro apoptosis in a large percentage of B-CLL patients. IL-10 increases in vitro apoptotic cell number in stage 0 patients, but not in stage I and II. These data support the hypothesis that IL-2 or IL-4, may be cell survival factors in vivo and that IL-10 might be a candidate for immune therapy of early B-CLL.     

The use of CD38 expression by monoclonal B lymphocytes as a prognostic factor in B-cell chronic lymphocytic leukemia

In B-cell chronic lymphocytic leukemia (B-CLL) the Rai and Binet staging criteria are not always able to accurately predict the prognosis of each patient. Rapidly evolving, violent disease is often seen in the so-called "good-prognosis" group, which highlights the need of additional and more refined prognostic markers. Several of these markers are described in the literature, with varying abilities to predict patient survival. Among the promising prognostic markers is flowcytometric analysis of CD38 on the monoclonal B cells in CLL. Several studies have shown that expression of CD38 is associated with a decreased overall-, or progression free survival. CD38 expression may be analyzed as percentage positive cells or as antibodies bound per cell. Addition of CD38 to the flow cytometry antibody panel for B-CLL analysis is a relatively easy way to obtain important prognostic information.     

Physical discrimination between human T-lymphocyte subpopulations by means of light scattering, revealing two populations of T8-positive cells

Light-scattering properties of human T-lymphocyte subpopulations selected by immunofluorescence were studied. Based on differences in orthogonal light scattering, two subpopulations of T8-positive cells can be distinguished. The first population (T8a) has the same orthogonal light-scattering properties as T4-positive cells, whereas the orthogonal light scattering of the second population (T8b) was about 70% larger. Orthogonal light scattering of Leu7-positive lymphocytes resembles that of the T8b population. We have studied the occurrence of the subpopulation in healthy individuals and we discuss their possible functional identification. Light-scattering properties of lymphocyte subpopulations in two patients with B-cell chronic lymphatic leukemia suggest that this observation is of clinical interest.     

Monoclonal antibody FMC7 detects a conformational epitope on the CD20 molecule: evidence from phenotyping after rituxan therapy and transfectant cell analyses

Numerous studies have reported that monoclonal antibody (mAb) FMC7 detects an antigen present on only a subset of circulating B lymphocytes. In particular, this mAb may distinguish typical B-cell chronic lymphocytic leukemia (FMC7 negative) from other types of B-cell non-Hodgkin lymphoma (B-NHL; FMC7 positive). We treated patients with B-NHL with Rituxan, a chimeric CD20 mAb, and observed abrogation of staining not only with prototype CD20 mAb B-1 but also with mAb FMC7. To investigate the relation between antigens CD20 and FMC7, we performed mutual blocking studies that showed mutual inhibition of FMC7 and CD20. In addition, FMC7 modulated CD23 expression and confirmed the presence of mAb B-1 in B-lymphoblastoid cell lines CESS and JVM. Transient transfection of myeloid cell line K562 with plasmid containing CD20-encoding cDNA produced de novo expressions of CD20 and FMC7. Our data indicate that FMC7 binds to a particular conformation of the CD20 antigen, probably to a multimeric CD20 complex. We assume that FMC7 stains positively only when CD20 antigen is present in high densities and in the postulated multimeric complex formation.     

Determination of the number and relative position of tryptophan residues in various albumins.

Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis and from normal donor DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high a high content of Cr2+, Sb2+, Fe2+, and Zn2+, whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co2+ content was 20-fold higher than in DNA or normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn2+, which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr2+, Sb2+ and Co2+, whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones.     

IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.     

Modification of immunocytochemical ZAP-70 assay for potential clinical application in B-cell chronic lymphocytic leukemia

The ZAP-70 protein is a member of the Syk/ZAP protein tyrosine kinase family, normally expressed in T cells and NK cells but not found in normal, mature B cells. The protein plays a critical role in the initiation of T-cell signaling. Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) that expressed nonmutated immunoglobulin V genes were found to express levels of ZAP-70 protein that were comparable to those detected in T cells of healthy adults. The ZAP-70 protein expression can be evaluated by flow cytometry and may be used as a prognostic marker in B-CLL patients. We modified the method of immunocytochemical assessment of ZAP-70 expression. The traditional two-step method with monoclonal anti-ZAP-70 antibody in the first step followed by FITC-conjugated goat anti-mouse IgG was changed for one-step method with monoclonal anti-ZAP-70 antibody labeled by Zenon Alexa Fluor 488. The method is simple and fast. The major advantage of Zenon labeling technique is its compatibility with simultaneous staining of surface antigens. The cells may be earlier immunostained for CD3, CD19 and/or CD5 to compare of the ZAP-70 kinase expression in B and T cells.     

Human leukaemia-associated antigens expressed by acute lymphocytic leukaemias and their detection with heterologous antisera to T, B-, and non-T-non-B subtype AL blasts.

Antisera from rabbits and goats against subtypes of acute lymphocytic leukaemia (ALL with T-cell markers, ALL with B-cell markers, Non-T-non-B ALL) were tested for their specificity in complement-dependent in-vitro cytotoxicity testing. After absorption of the fivefold diluted antisera with erythrocytes and spleen cells of allogenous donors they reacted with ALL cells, but not with leukaemias of other types (AML, CLL, CML), lymphocytes of healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-stimulated lymphocytes, cord lymphocytes and bone marrow lymphocytes of patients in remission. In the reactions of the antisera against ALL cells the subtype of ALL is of major importance: Six rabbit antisera and one goat antiserum against T-subtype ALL reacted in all 19 tests with the leukaemia cells of 5 patients with T-cell ALL and in all 9 tests with thymocytes of 3 donors, but only in 14 out of 41 tests with the leukaemia cells of 14 Non-T-non-B ALL patients. One antiserum against a B-subtype ALL lysed B-cell ALL (1/1), but not T-cell ALL (0/3), Non-T-non-B-cell ALL (1/5) and thymocytes (0/2). Four antisera against Non-T-non-B-subtype ALL reacted in 22 out of 46 tests with the Non-T-non-B cells of 17 ALL patients, but did not react with the leukaemia cells of 4 children with T-cell ALL (0/16), one child with B-cell ALL (0/1) thymocytes of 2 donors (0/4). The reactions of the anti-ALL sera with fetal liver cells, complete absorbability of the antileukaemic activity of the antisera with fetal tissue and the reactions of an anti-fetal serum with ALL cells point to the existence of fetal antigen components as leukaemia-associated antigens.