Long-term cultures of chronic lymphocytic leukemia B cells generate B suppressor cells

B suppressor cells (Bs) were generated by stimulation with PHA-P or concanavalin A of B cells from peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients. They suppressed well allogeneic mixed lymphocyte reactions. Long-term colonies of B cells from B-CLL were established for up to 6 weeks in liquid media, with B cells acquiring properties of suppressor cells while being cultured. Two types of cultures were observed: cells either grew in compact multicellular colonies or formed diffuse monolayers. Cells that remained alive in cultures for a number of weeks, but did not multiply, did not develop suppressive characteristics. Bs cells retained their phenotypic markers in cultures. Mitosis and lymphoblasts, but no differentiation into plasma cells, were observed. PHA-P-generated Bs were cultured in long-term colonies, retaining their suppressor properties. The establishment of long-term cultures of B-CLL cells may facilitate a better understanding of the nature and characteristics of normal and leukemic B cells.     

B- and T-lymphocyte markers on transformed lymphocytes from mitogen-stimulated cultures of normal and CLL lymphocytes and on tonsil blasts

Mitogen-transformed human peripheral blood lymphocytes and tonsil blasts were examined by rosette formation to detect the presence of membrane-bound immunoglobulin (Ig) and surface receptors for fixed IgG and fixed C₃. In addition, the capacity of these cells to rosette with sheep erythrocytes was evaluated as a reaction characteristic of T lymphocytes. In order for clear morphological recognition of the rosetting transformed lymphocytes and the rosetting tonsil blasts a cytocentrifuge technique was developed and used in conjunction with autoradiography and/or with Romanowsky stains. Using these techniques and the culture methods described in this paper phytohaemagglutinin, pokeweed mitogen, streptococcal filtrates and purified protein derivative stimulated predominantly T cells in the peripheral blood of man. A minority of the transformed cells in these mitogen-stimulated cultures (<24%) did rosette with B lymphocyte markers and presumably represent a B-cell response. No significant differences were found between the T- or B-cell specificity of the mitogens investigated. Lymphoid preparations from tonsils excised from normal donors with recurrent tonsillitis were found to contain 6–15% lymphoblasts and the large majority of these cells formed rosettes with the B-cell markers, less than 20% of these lymphoblasts formed spontaneous sheep erythrocyte rosettes. Using a mixed rosetting technique a small proportion (<5%) of PHA-transformed cells and tonsil lymphoblasts were found to have combined sheep Fc or combined sheep C₃ receptors. The investigation of B- and T-lymphocyte surface markers on mitogentransformed lymphocytes was extended to neoplastic lymphocyte populations and it was found that the majority of transformed cells (> 70%) present in chronic lymphocytic leukaemia cultures stimulated with PHA after 6 days incubation were transformed T lymphocytes.     

Selective loss of B-cell phenotype in lymphocyte predominant Hodgkin lymphoma

The neoplastic Reed-Sternberg cells characteristic of classical Hodgkin's lymphoma (cHL) are of B-cell origin but they almost always show striking loss of a range of B-cell-associated molecules. In contrast, the neoplastic cells found in lymphocyte predominant Hodgkin's lymphoma (LPHL) (L&H cells) are traditionally thought of as possessing the full repertoire of features associated with germinal centre B cells (eg BCL-6 expression, 'ongoing' Ig gene mutation). In the present paper, we report an extensive phenotypic analysis of L&H cells which revealed down-regulation of a number of markers associated with the B-cell lineage (eg CD19, CD37) and with the germinal centre maturation stage (eg PAG, LCK). The promoter methylation status of three of these down-regulated genes (CD10, CD19, and LCK) was further studied in microdissected L&H cells, and this revealed that their promoters were unmethylated. In contrast, these genes showed promoter methylation in cell lines derived from CHL. Further investigation of the mechanisms responsible for the deregulation of these molecules in L&H cells may provide new insights into the genetic abnormalities underlying LPHL.     

Studies on the immune status of children with acute lymphocytic leukaemia. II. In remission with and without cytostatic treatment.

In eleven children with acute lymphocytic leukaemia (ALL), cytostatic treatment was stopped after sustained remission lasting for 9 months--6 1/2 years. Their immunological status was monitored every 6--12 weeks during the first year after cessation of therapy. Rebound of depressed parameters was observed for absolute lymphocyte, B-cell and T-cell counts and for immunoglobulins. A simple follow-up scheme for such patients is proposed.     

In Vitro studies on human B and T cell purified populations. Stimulation by mitogens and allogeneic cells, and quantitative binding of phytomitogens.

The property of T cells to form rosettes with sheep red blood cells has been used to separate peripheral blood lymphocytes into purified T- and B-cell suspensions after density gradient centrifugation. A study of lymphocyte markers has shown that 2-6 per cent of E rosettes only were recovered in the B cell-enriched population. Lymphocyte stimulation in vitro was obtained with PHA, con A and PWM in unseparated and T-cell populations, but never in B-cell suspensions. Experiments of recombination between the two purified fractions have demonstrated that 10% of T cells added to B cells were able to induce a response to PHA and PWM. Otherwise, only T cells responded to allogenic stimulation. Lastly, B and T cells seemed to bind qualitatively and quantitatively the same mitogens on their membranes.     

Polyclonal Lymphocyte Responses to Murine Trypanosoma cruzi Infection

Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi-infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results in rapid blast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and, to a lesser extent, of Lyt 2+ T cells are still detected. Similar results were obtained in C57BL/6 (resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.     

Flow cytometric analysis of Epstein-Barr virus receptor among the different B-cell subpopulations using simultaneous two-color immunofluorescence

The distribution of Epstein-Barr virus receptor (EBVR) among the different B-cell subpopulations was analyzed by flow cytometry, using simultaneous two-color immunofluorescence of EBVR and cell-surface markers. The expression of EBVR was established by the binding of fluorescein isothiocyanate (FITC)-labeled EBV to the cells, while surface markers were stained by phycoerythrin (PE)-indirect immunofluorescence, using monoclonal antibodies. All cells of both the resting B-cell subpopulation defined by L30 and the B-cell subpopulations expressing surface IgM, IgD, IgA, and IgG were EBVR-positive. In contrast, EBVR was absent from about 10% cells of the activated B-cell subpopulation recognized by OKT9, as well as from about 10% cells of the highly differentiated B-cell subpopulation which reacted with OKT10. These results suggest that the expression of EBVR on the B-cell lineage varies with the maturation stage and with the state of activation. This postulation was further supported by analyses based on in vitro B-cell activation.     

The kinetic study on cell surface antigens of a hybridoma between human B cells and a mouse B-cell line

Human B cells obtained from tonsils were fused with a mutant clone of a murine B-cell line in the presence of polyethylene glycol and dimethyl sulfoxide. THT12.58, a subclone of the human-mouse B-cell hybridoma, was shown to express human B-cell surface antigens on the cell membrane, namely B1, B2, I2, human IgM and IgD derived from human B cells by analysis of flow microfluorometry (FMF), as well as murine B-cell markers; the amount of these human B-cell markers on THT12.58 did not change for more than 1 year after establishment. Interestingly, the expression of the human B-cell antigens significantly decreased after treatment of the cells with B-cell stimulatory factors (BSF) obtained from the supernatant of the culture of PHA-P activated T cells; this was followed by significant enlargement in cell size compared with the control. A marked decrease in the amount of each antigen on the hybrid was observed when treated with Staphylococcus aureus Cowan I strain (SAC) in addition to BSF. In contrast, the expression of these markers on the cells increased after exposure of the cells to recombinant human interferon-gamma (rINF-gamma). Additionally, the effect of BSF on the generation of IgM-secreting cells by human B cells markedly decreased after absorption of BSF with the hybrid cells. These results suggest that THT12.58 may possess a receptor for BSF on the cell surface, and be capable of differentiating into much more mature stage of B-cell lineage after exposure to BSF. Thus, this kind of a human-mouse B-cell hybridoma with human B-cell differentiation antigens can be a good model to investigate the kinetics of cell surface antigens and characterization of a receptor for BSF on human B cells.     

Lymphocyte surface markers in acute lymphoblastic leukemia of adults.

Leukemic cells from eight adult patients with acute lymphoblastic leukemia (ALL) were examined for the T-lymphocyte (T-cell) marker of sheep erythrocyte (E) receptors and for B-lymphocyte (B-cell) markers of surface immunoglobulins (SIg), complement receptors and FC receptors. Six of these patients' leukemic cells were devoid of both T- and B-cell markers, and therefore were "null" cells. The leukemic lymphoblasts of a 79-year-old patient had all B-cell markers, including monoclonal IgG K and receptors for complement and Fc. In cells from another patient (21 years old), only complement receptors were found. A review of the literature revealed that, similar to childhood ALL, adult cases of ALL were mostly of "null" cell type. All of the T-cell type was found in approximately 20% of patients. ALL of the B-cell type occurred only rarely. The latter cell type appeared to occur mainly in the middle-aged and the elderly.     

Natural Killer Cell Cytotoxicity and Paternal Lymphocyte Immunization in Women with Recurrent Spontaneous Abortions

PROBLEM: Natural killer (NK)-cell cytotoxicity in women undergoing lymphocyte immunization prior to and following treatment was investigated. METHOD OF STUDY: A cohort of 33 women with a history of two or more recurrent spontaneous abortions was prospectively studied. NK-cell cytotoxicity was determined at effector-to-target ratios of 50:1 and 25:1. Peripheral blood CD56+ NK-cell, CD 19+ B-cell, CD19+/5+ B-l-cell, and CD3+ pan T-cell levels were studied by flow cytometry before and after lymphocyte immunization treatment. Maternal antipaternal T- and B-cell antibody levels were measured before and after lymphocyte immunization by flow cytometric analysis. Paternal lymphocyte immunizations were given two times with a 4-week interval. Post-lymphocyte immunization testing was done 4 weeks after the second lymphocyte immunization. The controls were 8 normal healthy women. NK assays were done twice with an interval of 8 weeks. RESULTS: NK-cell activity at effector-to-target ratios of 50:1 (P = 0.005) and 25:1 (P = 0.001) were significantly suppressed after lymphocyte immunization. CD3+ pan T-cell levels after lymphocyte immunization were significantly increased compared with levels before lymphocyte immunization (P = 0.008). CD56+ NK-cell levels were significantly suppressed after lymphocyte immunization (P = 0.016). There was no correlation between changes in NK cytotoxicity and differences in antipaternal lymphocyte antibody levels before or after lymphocyte immunization. CONCLUSION: Lymphocyte immunization suppresses NK-cell cytotoxicity and CD56+ NK-cell levels and increases the peripheral blood CD3+ T-cell population in women with recurrent spontaneous abortions.     

Production of leucocyte migration inhibitory factor (LIF) in infectious mononucleosis. Spontaneous release and lack of response to concanavalin A.

The spontaneous release of LIF from blood lymphocytes was studied in patients with infectious mononucleosis. Mononuclear cells were separated from the blood and cultured for 22 hr, and LIF activity in the supernatant was determined. Supernatants depleted of LIF activity by means of anti-LIF antibodies or by treatment at 80 degrees C for 30 min were employed as controls; these two methods gave essentially similar results. In nine out of eighteen patients, spontaneous LIF production was demonstrated during the acute stage of the illness; this was not seen in any of the normal persons studied. 6 weeks later, spontaneous LIF production had ceased in most patients. Concanavalin A stimulated all normal lymphocytes to LIF production, but in sixteen out of seventeen patients with infectious mononucleosis this response was absent or diminished. At the follow-up study 6 weeks later, the lymphocyte response to concanavalin A was still suppressed.     

Shifts in Lung Lymphocyte Profiles Correlate with the Sequential Development of Acute Allergic and Chronic Tolerant Stages in a Murine Asthma Model

T lymphocytes have a central regulatory role in the pathogenesis of asthma. We delineated the participation of lymphocytes in the acute allergic and chronic tolerant stages of a murine model of asthma by characterizing the various subsets of lymphocytes in bronchoalveolar lavage and lung tissue associated with these responses. Acute (10-day) aerosol challenge of immunized C57BL/6J mice with ovalbumin resulted in airway eosinophilia, histological evidence of peribronchial and perivascular airway inflammation, clusters of B cells and TCRgammadelta cells in lung tissue, increased serum IgE levels, and airway hyperresponsiveness to methacholine. In mice subjected to chronic (6-week) aerosol challenge with ovalbumin, airway inflammation and serum IgE levels were significantly attenuated and airway hyperresponsiveness was absent. The marked increases in lung B and T cell populations seen in the acute stage were also significantly reduced in the chronic stage of this model. Thus, acute ovalbumin challenge resulted in airway sensitization characteristic of asthma, whereas chronic ovalbumin challenge elicited a suppressed or tolerant state. The transition from antigenic sensitization to tolerance was accompanied by shifts in lymphocyte profiles in the lung and bronchoalveolar lavage fluid.     

Lymphocyte depletion in ileal Peyer's patch follicles in lambs infected with Eimeria ovinoidalis.

A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer's patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5'- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer's patch follicles.     

Prediction of imminent complications in HIV-1-infected patients by markers of lymphocyte apoptosis

OBJECTIVE: The aim of the study was to compare accepted surrogate markers of HIV disease progression with markers of lymphocyte apoptosis in their ability to predict short-term disease progression. METHODS: In all, 40 HIV-positive patients were studied prospectively and observed during follow-up for HIV-related adverse clinical events. Ex vivo apoptosis was measured with the markers CD95 expression, annexin V binding, and Apostain dye uptake by flow cytometry at baseline. Established markers of disease progression (CD4 count, HIV-RNA level, and CD8/38 count), CD8, B-cell, and natural killer (NK) cell counts were determined by standard procedures at baseline and after 6 months. RESULTS: In HIV-infected patients, CD95 expression and annexin V binding showed significantly elevated apoptosis in peripheral blood lymphocytes and all lymphocyte subsets at baseline compared with HIV-negative, healthy controls. Apostain failed to differentiate between HIV-infected patients and healthy controls. HIV-related complications could be predicted by CD4 and CD8/38 counts, but not HIV viral load as assessed by relative operating characteristic (ROC) analysis (CD4, p = .003; CD8/38, p = .031). A similar or even better diagnostic accuracy was found for CD95 expression in total lymphocytes (p<.001), the CD4+ (p = .003) and CD8+ (p = .005) T-cell subsets and for annexin V binding in CD4+ T cells (p = .005). When patients with CD4 counts <200 cells/microl were analyzed separately, only annexin V binding in CD4+ T cells, but none of the other prognostic markers could predict complications (p = .001). CONCLUSION: Determination of annexin V binding on CD4+ T cells may be a useful tool to monitor HIV-infected patients with low (<200 cells/microl) CD4 counts, as it can reliably assess the risk for imminent complications in such patients.     

Pre-B-cell leukemia. A new phenotype of childhood lymphoblastic leukemia.

Large lymphoid cells containing small amounts of cytoplasmic IgM (clgM) but undetectable surface immunoglobulin (slg) have recently been recognized as precursors of B lymphocytes. They are a small, rapidly dividing pool of normal marrow lymphoblasts. Since lymphoblasts in most childhood acute lymphoblastic leukemias lack slg and other conventional B-lymphocyte and T-lymphocyte markers, we examined the possibility that some leukemias represent "pre-B"-cell neoplasms. In four of 22 consecutive patients, leukemic cells had the clgM+.slg- phenotype of pre-B cells. These patients' cells shared "common acute-lymphoblastic-leukemia" antigens and "B-cell" alloantigens, but differed in expression of several developmental features characteristic of the B-cell line. Pre-B-cell leukemias were readily responsive to chemotherapy. We conclude that a distinct subpopulation of previously unclassified leukemias reflects oncogenic transformation at the earliest recognizable stage in differentiation along the B-cell axis.     

Malignant lymphomas involving the central nervous system—a morphological and immunohistochemical study of 32 cases

The morphological features of 32 cases of malignant lymphomas involving the central nervous system presenting over a 32 year period were reviewed and the lymphomas redefined using current classifications. Twenty-four cases (75%) of non-Hodgkin's lymphomas were reclassified using the Kiel classification. There were 18 low grade non-Hodgkin's lymphomas (comprising 11 lymphoplasmacytoid, five lymphocytic and two centroblastic-centrocytic) and six high grade tumours (comprising two centroblastic, two immunoblastic, one unclassifiable and one lymphoblastic lymphoma). Cytologically the great majority of non-Hodgkin's lymphomas were B-cell lymphomas. The eight cases (25%) of Hodgkin's disease were classified by the Rye subtype and consisted of three mixed cellularity, two lymphocyte depletion, two lymphocyte predominant and one nodular sclerosis. The presence of intracytoplasmic immunoglobulins as well as markers for histiocytic cells were studied by the immunoperoxidase technique using polyclonal antisera. A monoclonal staining pattern, as revealed by light chain restriction, was found in nine cases (38%) of the non-Hodgkin's lymphomas confirming their B-cell origin. With the Marshall's metalophil method and the other histiocytic markers, scattered reactive microglial cells and histiocytic reticulum cells were found throughout the tumours in most cases. No histiocytic lymphomas were present in the series.     

FcIgG receptor-bearing lymphocytes and monoclonal antibody-defined T cell subsets in atopic dermatitis: effect of treatment with thymopoietin pentapeptide (TP-5)

Lymphocyte subpopulations were determined in the blood of patients with atopic dermatitis (AD) before and after treatment with the thymopoietin pentapeptide TP-5. The relative and absolute numbers of lymphocytes bearing suppressor/cytotoxic cell markers (FcIgG+E+ and T8+ cells) were significantly decreased in the untreated patients and the T4+/T8+ cell ratio was increased, indicating an imbalance between lymphocyte subpopulations in AD. Patients who had been treated for 6 weeks with TP-5 displayed no significant abnormality of any of the lymphocyte subsets studied and comparison of pre- and posttreatment values revealed that there was a statistically significant increase in T8+ cell numbers, that by contrast did not take place in placebo-treated AD patients. The treatment had no demonstrable effect on IgE serum levels or on the spontaneous in vitro production of IgE by cultured lymphocytes from the patients.     

CD58 expression decreases as nonmalignant B cells mature in bone marrow and is frequently overexpressed in adult and pediatric precursor B-cell acute lymphoblastic leukemia

We used flow cytometry to determine the CD58 expression on nonmalignant B cells at different stages of maturation in the bone marrow and compared it with that of blasts in adult and pediatric precursor B-cell acute lymphoblastic leukemia (B-ALL). The mean fluorescence intensity (MFI) of CD58 expression decreased significantly as nonmalignant B cells differentiated in the bone marrow from an early to a mature stage. Few nonneoplastic B cells at a mid or mature stage of development expressed CD58 MFI values comparable to those seen in leukemic cases. Early-stage nonneoplastic B-cell precursors expressed relatively higher CD58 levels, which frequently overlapped with the variable level of CD58 expression observed among leukemic blasts. As a group, however, the malignant precursor B-ALL cells showed significantly higher expression of CD58 than nonmalignant B-cell populations at any maturational stage. These findings support the potential usefulness of CD58 expression in the diagnosis and monitoring of precursor B-ALL, but only when blasts express high levels of CD58.     

Frozen-Thawed Mononuclear Cells as a Source of B Lymphocytes for Antibody Screening

B lymphocytes were isolated from fresh blood and frozen-thawed lymphocyte preparations from the same donors. A panel of 55 human antisera, previously found to react with human peripheral blood B lymphocytes in cytotoxicity assays, was screened against these B-cell preparations. There was essentially no difference in the reactions of B-cell antisera with fresh B lymphocytes when compared with frozen-thawed cells. The percentage recovery of B cells from the fresh and frozen preparations was not significantly different.     

Selective suppressive effects of Trypanosoma cruzi on activated human lymphocytes.

The acute phase of Chagas' disease is accompanied by immunosuppression. To explore the underlying mechanism(s), we used an in vitro culture system in which the capacities of activated human peripheral blood mononuclear cells to express interleukin-2 receptors (IL-2R) and proliferate are markedly inhibited in the presence of Trypanosoma cruzi, the etiologic agent. The present work was designed to define the earliest time at which T. cruzi-induced suppression is manifested in terms of IL-2R expression on the cell surface and establish whether expression of other lymphocyte activation markers is also suppressed by the parasite. We found that expression of IL-2R by human peripheral blood mononuclear cells cocultured with T. cruzi and stimulated with either phytohemagglutinin or anti-CD3 (a monoclonal antibody specific for an epitope of the T cell receptor complex T3-Ti) was significantly suppressed as early as 12 h after culture initiation. Both the percentage of IL-2R+ cells and the surface density of IL-2R, measured by flow cytometry, were affected. However, expression of EA1, a human lymphocyte activation antigen known to be expressed 4 to 6 h after stimulation, was not altered by T. cruzi whether phytohemagglutinin or anti-CD3 was used. On the other hand, expression of transferrin receptors (TfR), which first occurs between 20 and 24 h after lymphocyte activation, was markedly suppressed by T. cruzi. This effect was denoted by significant reductions in both the percentage of TfR+ cells and the cell surface density of TfR whether phytohemagglutinin or anti-CD3 was used as the mitogen and was observed at all test times, i.e., at 24, 48, 72, and 96 h. Because expression of IL-2R and TfR is required for lymphoproliferation but that of the EA1 lymphocyte activation marker is apparently not, these results are consistent with the possibility that T. cruzi, at a relatively early stage during lymphocyte activation, selectively affects certain key events on which clonal expansion is dependent. Inhibition of IL-2R and TfR expression by the parasite might play a role in causing the suppressive effects associated with acute Chagas' disease.