Phorbol ester treated chronic B lymphocytic leukaemia cells induce autologous T cell proliferation without generation of cytotoxic T cells.

The ability of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to modify the stimulatory capacity of leukaemic B cells in autologous mixed leucocyte reactions (AMLR) was studied. T lymphocytes from patients with chronic lymphocytic leukaemia (CLL) did not proliferate when stimulated with untreated autologous non-T cells. In contrast, vigorous 3H-thymidine incorporation was seen in six of eight cases when CLL stimulator cells were pre-incubated for 3 days with TPA. No cytotoxic T cells against TPA treated or untreated autologous target cells were generated in such AMLR. These results indicate that enhancement of AMLR stimulatory capacity may be among the characteristic features of TPA driven in vitro differentiation of CLL cells.     

Interferon induces proliferation and differentiation in primary chronic lymphocytic leukaemia cells.

The influence of interferon-alpha (IFN-alpha) on the differentiation of malignant cells from six patients with chronic lymphocytic leukaemia was studied in vitro. IFN induced differentiation in the leukaemic cells from four of the patients. In cells from two of these patients, IFN also induced proliferation. When tested by immunofluorescence, the clonality of the differentiating cells was established by the presence of intracellular light chains of one type only.     

Activation of B chronic lymphocytic leukaemia cells by Branhamella catarrhalis.

Cells from the blood of patients with chronic lymphocytic leukaemia were cultured in the presence of two polyclonal activators of human B cells, the bacteria Branhamella catarrhalis (Bc) and Staphylococcus aureus Cowan 1 (SAC). Although the magnitude of the responses varied, cells from seven of the eight patients studied were induced to proliferate in response to Bc. In contrast, the response to SAC was low or negligible in seven of the eight patients, and only one patient responded well to this mitogen. Bc was also effective in inducing secretion of IgM in cells from seven of the eight patients, and this was unaffected by removal of T cells. Fractionation of CLL cells on density gradients showed that the highest level of IgM production was induced in cells with a low buoyant density, whilst cells with a high buoyant density secreted little or no immunoglobulin in response to Bc. Together, these results demonstrate that Bc is an effective, T-independent activator of both DNA synthesis and immunoglobulin production in CLL cells.     

Patterns of nuclear proto-oncogene expression during induced differentiation and proliferation of human B chronic lymphocytic leukaemia cells.

Phorbol ester-induced differentiation of human B-chronic lymphocytic leukaemic cells was found to be preceded by a rapid transient induction in expression of the c-jun proto-oncogene, which paralleled that of c-fos. Induced expression of c-myc but not of c-fos/c-jun proto-oncogenes was markedly higher in a proliferating variant leukaemic cell population compared with that seen in typical lymphocytic leukaemia cells. These data suggest that the c-fos/c-jun nuclear oncogenes play a role in induced differentiation, whilst c-myc is more important in the proliferative response of B lymphocytes.     

Phenotypic heterogeneity of B cell chronic lymphocytic leukaemia

The peripheral blood lymphocytes from 39 patients from the Latvian S.S.R.T., U.S.S.R. with chronic lymphocytic leukaemia (CLL) have been phenotyped with various monoclonal antibodies representing the major clusters of differentiation (CD) used for phenotyping B cells. A clear delineation of two groups of patients was evidenced. The major group (33/39) possessed leukaemic cells bearing surface immunoglobulins (SIg) at a low density, Class II HLA, and CD5, CD24 and CD37 molecules but not CD21, CD22 and CD35. CD23 antigen was seen only once under microscope examination, but could be visualized by flow cytometry. CD6 antibody reacted with cells from about 1/3 of this group of patients. In the six patients of the second group the leukaemic phenotype was SIg+, Class II HLA+, CD5+, 24+, 37+, 21+, 22+, 35+, 23+ and 6-. The main finding is the concomitant expression of CD22, CD21 (CR2) and CD35 (CR1) molecules, all involved in B cell activation. It is not yet known whether these observations correlate with different clinical evolutions of the disease.     

Effect of interleukins on the proliferation and survival of B cell chronic lymphocytic leukaemia cells.

AIMS--To investigate the effects of interleukin (IL) 1, 2, 4, and 5 on the proliferation and survival of peripheral blood B cells from patients with B chronic lymphocytic leukaemia (B-CLL) and compare them with the effects on normal peripheral blood B cells. METHODS--The proliferation and survival of pokeweed mitogen (PWM) activated B cells from B-CLL (n = 12) and normal peripheral blood (n = 5) were studied in vitro in response to IL-1, IL-2 IL-4, and IL-5. Survival of cells in cultures with or without added interleukins was studied by microscopic examination of cells and DNA agarose gel electrophoresis. RESULTS--Proliferation was observed in both B-CLL and normal peripheral blood cells on culture with IL-2 alone and also in some, but not all, B-CLL and normal peripheral blood cells with IL-1 and IL-4. However, there was greater variability in B-CLL cell responses than in normal peripheral blood cells. Il-5 did not affect normal peripheral blood cell proliferation but it increased proliferation in two B-CLL cases. Synergistic effects of these cytokines were not detected. IL-4 inhibited normal peripheral blood and B-CLL cell proliferation after the addition of IL-2. Inhibition of B-CLL cell responses to IL-2 was also observed with IL-5 and Il-1. Survival of B-CLL cells in cultures was enhanced with IL-4 not by an increase in proliferation but by reduced apoptosis. No such effect was seen in normal peripheral blood cells. IL-2 had a less noticeable antiapoptotic effect; IL-5 enhanced apoptosis in B-CLL cells. CONCLUSIONS--B-CLL and normal peripheral blood cells proliferated equally well in response to IL-2. IL-4 had a much lower effect on B-CLL cell proliferation, but had noticeable antiapoptotic activity. IL-5 enhanced cell death by apoptosis.     

Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.

BACKGROUND AND OBJECTIVES: Freezing is a practical approach for cell preservation for retrospective studies. The aim of this work was to check the cryopreservation impact on B cell chronic lymphocytic leukaemia phenotype. MATERIAL AND METHODS: Blood samples from 15 CLL patients were analyzed freshly and after freezing at -196 degrees C, without separation, and thawing. Results were compared by Student's paired t-test. RESULTS: The phenotype of fresh CLL cells was as follows: CD19+, CD5+, faint CD20, CD23+/-, weak CD22 and sIg, CD37+, HLA-DR+, FMC7-. After cryopreservation, the percentage of CD5 and CD23 positive cells decreased, whereas HLA-DR positive cells increased moderately. The CLL Matutes's score was modified in 6 cases out of 15 (40%). CONCLUSION: Cryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression.     

Autoimmunity in chronic lymphocytic leukaemia.

The prevalence of autoantibodies in B cell chronic lymphocytic leukaemia (B-CLL) was investigated. A lower prevalence of autoimmune haemolytic anaemia than that found in other series was found: large numbers of non-progressive stage A disease cases were included, in which the prevalence of autoimmune haemolytic anaemia is low. Non-haematological autoantibodies were no commoner than in age matched controls. Whatever explanation is offered for autoimmune phenomena in B-CLL it must take account of the fact that those phenomena are virtually confined to autoantibodies against the formed elements of the blood.     

J chain synthesis and ultrastructural changes in chronic lymphocytic leukaemia cells during in vitro differentiation

The synthesis of J chain and the ultrastructural changes in chronic lymphocytic leukaemia (CLL) cells during in vitro cultures stimulated with PWM and/or allogenic T cells were investigated by immunoelectron microscopy, radioimmunoassay and anti-idiotype antibodies, in an attempt to demonstrate changes in the maturation stage of CLL cells. The cells from four patients showed the induction of J chain synthesis in parallel with the increase of Ig synthesis. Two of revealed cytological transformations, in which CLL cells resembled immunoblasts or plasmacytoid cells. These findings suggest that some CLL cells are not 'frozen' within the original maturation stage and can be induced to differentiate to a more mature stage.     

Functional and phenotypic heterogeneity of electrophoretically separated leukaemic B cells from patients with chronic lymphocytic leukaemia.

E-rosette negative largely leukaemic B cells from patients with chronic lymphocytic leukaemia (CLL) were separated by density gradient electrophoresis, on the basis of their surface charge. The separated cells were pooled in seven fractions, according to their relative position in the electrophoretic distribution, and analysed by functional tests and cell-surface phenotypes. The ability of electrophoretically separated B cells from patients with CLL to differentiate into plasma cells in the presence of T cells from normal donors and in the pokeweed mitogen induced differentiation system was investigated. Lymphocytes able to differentiate into plasma cells under these conditions were highly enriched in the low-mobility fractions V, VI and VII. These plasma cells were of leukaemic origin, because they expressed only the light chain present on the cell surface of the leukaemic B cells before stimulation. Lymphocytes with Fc (IgG) receptors were relatively enriched in the high and intermediate mobility fractions I-IV, but they were present in the remaining of the fractions in smaller proportions. Lymphocytes with Fc (IgM) receptors were present in all fractions, but only in very small proportions in the very high mobility fraction I. Cells with complement receptors I and II were present in all fractions. Analysis of the density of cell surface immunoglobulin expression using the fluorescence-activated cell sorter, revealed that fractions of high and intermediate electrophoretic mobility (I-V) contained cells with both low and intermediate density of surface immunoglobulin, whereas the low mobility fractions VI and VII contained predominantly cells with low density of surface immunoglobulin. These results revealed significant phenotypic and functional heterogeneity of B cells from patients with CLL, suggesting the presence of subpopulations of leukaemic B cells in different differentiation or maturation stages.     

Non-cytotoxic antibodies in chronic lymphocytic leukaemia.

Non-cytotoxic Fc receptor blocking antibodies against autologous B lymphocytes were sought in sera from patients with chronic lymphocytic leukaemia (CLL), using a rosette inhibition assay. They were found in 11 of 52 (21%) of patients with CLL, but were not associated with previous blood transfusion or pregnancy, suggesting that they were unlikely to have resulted from allogeneic stimulation. Fc receptor blockade was more commonly detected in sera from patients with stage B rather than stage A CLL (Binet classification), though this did not achieve significance beyond the 90% level, and it was noted in 62.5% of those with lymphocyte doubling times of less than one year, compared with 36.3% of those whose lymphocyte doubling time was more than one year. The results indicate that autologous Fc receptor blocking antibody activity occurs in sera from patients with CLL, is more likely to be generated by the tumour itself than by allogeneic stimulation, and is associated with increased tumour load. Such antibodies may permit tolerance of tumour by the host.     

Serum paraproteins in chronic lymphocytic leukaemia.

The presence of paraproteins in the sera of 10 patients with chronic lymphocytic leukaemia (CLL) was investigated using immunoisoelectric focusing. Monoclonal immunoglobulins were found in nine of these 10 sera. Five sera contained a single monoclonal IgM paraprotein, one serum contained a single monoclonal IgG paraprotein, while three sera contained more than one monoclonal paraprotein--namely, IgM + IgD, IgM + IgG, and IgM + IgD + IgG. The results indicate that the malignant B cells of CLL may be at a later stage of differentiation than previously assumed and serum monoclonal immunoglobulin could be of value as a tumour marker.     

Density separation of chronic lymphocytic leukemia cells: Low-density non-T cells are efficient stimulator cells in allogeneic and autologous mixed leukocyte reaction

Unseparated mononuclear cells and E rosette-depleted non-T cells from a majority of patients with chronic lymphocytic leukemia (CLL) were found to be weak stimulators in the allogeneic mixed-leukocyte reaction (MLR). However, a minor population (1–5%) of highly active stimulator cells could be isolated from all patients studied by buoyant density centrifugation using discontinuous gradients of Percoll. The same low-density non-T-cell fraction also stimulated autologous T-cell proliferation in the autologous mixed-leukocyte reaction (AMLR). In contrast, Percoll-separated high-density non-T cells, including the leukemic B-cell pool, were completely inactive as stimulators in AMLR. These results suggest that the presence of efficient stimulator cells among CLL mononuclear cells may be masked by the large number of nonstimulating leukemic B cells.     

Blood dendritic cells in patients with chronic lymphocytic leukaemia

Myeloid and plasmacytoid dendritic cells (MDC, PDC) play a key role in the initiation of immune responses. We found a reduction of both DC subsets in 42 patients with chronic lymphocytic leukaemia (CLL) at diagnosis (P<0.0001 and 0.0001 vs. controls, respectively), likely related to the high secretion of CCL22 and CXCL12 (P=0.04 and 0.008 vs. controls, respectively) by leukaemic cells. However, CD14+ monocytes from CLL patients could give rise to functional IL-12p70-secreting monocyte-derived DCs, capable of inducing a type 1 polarization immunostimulatory profile. These monocyte-derived DCs from CLL patients efficiently migrate in response to CCL19/MIP-3beta chemokine, suggesting that functional autologous DCs can be generated for immunotherapeutic purposes to circumvent DC defects in CLL.     

T cells in B cell chronic lymphocytic leukaemia. I. Decreased frequency of T lymphocytes secreting suppressor factor.

A reverse T cell plaque assay was employed to study the ability of purified T cells isolated from the blood of seven patients with B cell chronic lymphocytic leukaemia (B-CLL) to secrete antigen-specific helper or suppressor factors (ThF and TsF respectively) after activation in vitro. It was found that in spite of the phenotypical presence of CD8+ cells, the frequency of TsF-secreting cells was strongly decreased as compared to normal values. T cells secreting ThF could be generated in all B-CLL patients tested in about normal frequencies. These results may indicate a tumour induced change in the distribution of cellular subsets within the CD8+ cell compartment.     

B-cell maturation in chronic lymphocytic leukaemia. IV. T-cell-dependent activation of leukaemic B cells by staphylococcal enterotoxin 'superantigens'.

Staphylococcal enterotoxins (SE) are potent T-lymphocyte activators that stimulate T cells by directly cross-linking HLA-DR molecules on antigen-presenting cells with the V beta gene products of the T-cell receptor. The different SE activate all T cells expressing a given V beta, and, therefore, have been termed 'superantigens'. Here we show that SE are potent activators of leukaemic B cells from patients with chronic lymphocytic leukaemia (CLL). Purified B cells from seven of eight CLL patients with high WBC counts (greater than 80,000/microliters) responded to one or several of the tested SE (SEA, SEB, SEC1, SED, SEE) by proliferation ([3H]TdR incorporation) and/or Ig secretion. In several instances, the response of leukaemic B cells to SE was much stronger than was the response to other known B-cell activators including EBV, pokeweed mitogen (PWM), phorbolester (TPA), and Staphylococcus aureus Cowan I (SAC). The activation of leukaemic B cells by SE was strictly dependent on the addition of irradiated T cells isolated from healthy donors. FACS analysis of cultured cells ensured that the proliferating cells were indeed B cells. Taken together, these results demonstrate that SE are strong T-cell-dependent B-cell activators that, in some cases, can stimulate maturation of leukaemic B cells which are refractory to other activation signals.     

IL-13 has only a subset of IL-4-like activities on B chronic lymphocytic leukaemia cells.

Transgenic mice carrying a modified human tumour necrosis factor (huTNF)/beta-globin gene construct linked to the T-cell-specific locus control region of the human CD2 gene express huTNF in their T cells which is released into the circulation and causes the development of a wasting syndrome. We now report that the mice develop anaemia, probably through enhanced erythrophagocytosis rather than inhibition of reticulocyte production. Thus autologous erythrocytes, as well as sheep erythrocytes, were cleared more rapidly from the circulation of transgenic mice than from littermate controls. By contrast, peritoneal macrophages from transgenic mice were less phagocytic in vitro than cells from controls. They also secreted less murine (mu)TNF when stimulated by either bacterial lipopolysaccharide or toxic malarial antigens. The yields of muTNF approached normal levels, however, when these refractory cells from the transgenic mice were stimulated in the presence of a high concentration of indomethacin, suggesting that the production of muTNF was inhibited by enhanced synthesis of prostaglandins. The parasitaemia of transgenic mice infected with Plasmodium yoelii was about 10-fold less at its peak than in controls, although it followed the same time-course, and the multiplication of P. chabaudi was inhibited to an even greater degree. This control of parasitaemia may also be explained by enhancement of macrophage activity, mediated by huTNF acting on the murine p55 receptor, presumably by increasing the removal of parasites by phagocytosis or their killing by toxic products released by the activated macrophages. These observations suggest that a factor in the anaemia of human malaria may be macrophage activation caused by the secretion of TNF that occurs in this disease.