B-CLL Patients Research Articles

Clinical significance of CD38 expression in chronic lymphocytic leukemia

AbstractB-cell chronic lymphocytic leukemia (B-CLL) follows heterogeneous clinical courses, and several biological parameters need to be added to the current clinical staging systems to predict which patients will experience an indolent or an aggressive outcome. This study analyzed CD38 expression by flow cytometry and soluble APO1/Fas (sAPO1/Fas), Bcl-2 (sBcl-2), and CD23 (sCD23) proteins by immunoenzymatic methods to evaluate their effect on the clinical course of 168 unselected B-CLL patients. Intermediate/high risk modified Rai stages were characterized by a higher CD38+ B-cell number (P = .0002) and higher sCD23 levels (P < .0001). Moreover, CD38+ B-cell percentages were significantly and directly associated both with β2-microglobulin and sCD23 concentrations (P < .0001 and P = .002, respectively). Both a higher tumor burden (lymphadenopathy/splenomegaly) and a lymphocyte doubling time less than 12 months were significantly associated with higher CD38+ percentages (P < .0001 and P = .0001, respectively). With regard to clinical outcome, progression-free survival was significantly longer (75% versus 37% at 5 years; P = .00006) in patients with lower CD38+ B-cell percentages. Furthermore, the risk of partial or no response to fludarabine increased with increasing CD38 expression (P = .003), and a shorter overall survival (50% versus 92% at 8 years; P < .00001) characterized patients with more than 30% CD38+ B-cell number. The predictive value of CD38 expression was maintained among the patients within the Rai intermediate risk group and was confirmed in multivariate analysis. Thus, the percentage of CD38+ B cells appears to be an accurate predictor of clinical outcome and therefore could be used to indicate when more novel chemotherapeutic approaches are needed.

Influenza virus vaccination and booster in B-cell chronic lymphocytic leukaemia patients

Background: Influenza vaccination is recommended for patients with B-cell chronic lymphocytic leukaemia (CLL). Because response rates are often low, we decided to evaluate antibody response to single and booster vaccinations with influenza A and B virus vaccine in these patients. Methods: Twenty patients with B-CLL received two subunit virus vaccine injections 21 days apart. Antibody titres were determined before and 21 days after the single and booster vaccinations. The serological response was expressed using the following criteria: (1) response rate, i.e. the proportion of subjects with at least a 4-fold titre increase; (2) the protection rate, i.e. the proportion of subjects exceeding the threshold of 100 (influenza A) or 200 (influenza B); and (3) the mean fold increase (MFI), i.e. the difference between the log-adjusted geometric mean titres of pre- and post-vaccination sera. Results: Response rates were 5% for influenza A and 15% for B after the single vaccination and 15% for A and 30% for B after the booster vaccination. Protection rates were 0% for influenza A and 25% for B after the single vaccination; they were 5% (H1N1) and 10% (H3N2) for influenza A and 30% for B after the booster. The MFI±S.D. (range) after the booster vaccination was 0.26±0.33 (0–1.00), 0.17±0.34 (0–1.00) and 0.35±0.34 (0–1.20) for H1N1, H3N2 and influenza B, respectively. Conclusion: In this study with B-CLL patients, immune response to influenza vaccination was poor. Thus, single and booster vaccinations with influenza virus vaccine do not appear to be of great value to patients with B-cell CLL.

Interleukin 4 content in chronic lymphocytic leukaemia (CLL) B cells and blood CD8+ T cells from B‐CLL patients: impact on clonal B‐cell apoptosis

B-chronic lymphocytic leukaemia (CLL) clonal B cells are characterized by resistance to apoptosis. We evaluated clonal B cells and blood T cells for interleukin 4 (IL-4) content as IL-4 is able to increase CLL cell resistance to apoptosis. The content of IL-4 in CD8+ T cells of CLL patients (n = 9) ranged from 37% to 63% of the total CD8+ T cells (mean level of 49% +/- 3.4) compared with a range of 5-10% for control CD8+ T cells. Clonal B cells positive for cytoplasmic IL-4 ranged from 1% to 97% (mean value 57.8 +/- 6.9%). CD8+ T cells and clonal B cells secreted detectable levels of IL-4, but only clonal CLL B cells (n = 4) secreted IL-4 in association with increasing cell numbers. Fludarabine (F-ara-AMP, 0.1-100 micromol/ml) was able to downregulate the IL-4 content of CD8+ T cells, but not clonal B-cell IL-4. Culture supernatant from CLL CD8+ T cells decreased the spontaneous apoptotic rate of clonal B cells that was reversed with anti-IL-4 and soluble IL-4 receptor. These findings show that IL-4 is present in the microenvironment of B-CLL. In addition, use of agents that can interfere with IL-4 presentation to clonal B cells can be effective in increasing clonal B-cell apoptosis.

Variability in B-cell antigen expression: implications for the treatment of B-cell lymphomas and leukemias with monoclonal antibodies.

Antigen expression intensity is becoming important for decision-making in relation to monoclonal antibody therapy. By quantifying CD20, CD22 and CD52 expression on chronic lymphocytic leukemia and normal (control) B cells, over time. The effect of Interleukin-4 therapy on CD20 antigen intensity on B-CLL cells in vivo was also determined. Lymphocytes were purified at weeks 0, 4 and 8 from five B-CLL patients, five healthy volunteers and seven B-CLL patients receiving IL-4 therapy. The number of antigen receptor sites was calculated in molecules of equivalent soluble fluorochrome using flow cytometry. The mean number of CD20 receptors at baseline was significantly lower on B-CLL cells compared to normal B cells (8160 vs 87 046; P<0.0001). Similar results were obtained for CD22 (8630 vs 27 647; P<0.01), but not for CD52 (371 303 vs 409 484; P = 0.54). When soluble fluorochrome values at weeks 4 and 8 were analysed as change in per cent from baseline (delta%), there was <10 delta% variability in CD20 expression on control B cells, but considerable variability (22.5-67.5 delta%) on B-CLL cells. Expression of CD22 in CLL and control B cells varied by <15 delta%. CD52 on CLL B cells showed slightly greater variability (+/-35 delta%) than that of CD22 (+/-15delta%), but less than that of CD20. IL-4 therapy did not consistently increase the CD20 expression on B-CLL cells in vivo. Our data confirm differences in intensity between different target antigens on B-CLL cells, and draws attention to the fact that a substantial variability may occur over time, which may influence clinical decision-making. Caution must be taken when interpreting in vitro results on cytokine-mediated receptor intensity up-regulation.

Múltiplas infecções oportunistas em um paciente com leucemia linfocítica crônica tratado com cladribina

Summary Hypogamaglobulinemia is frequently associatedwith B-CLL rendering the patients prone to bacterialinfections. The use of purine analogs that causedepletion of T lymphocytes in the treatment of thisdisease, increases the spectrum of susceptibility ofthese patients to oportunistic agents. We report acase of a young B-CLL patient treated with 2-CdA,who presented multiple infectious complicationsduring the course of his disease associated with asevere cellular immune deficiency.Rev.bras.hematol.hemoter.,2000, 22 (3):420-423 Key words : Chronic lymphocityc leukemia,cryptococcal meningitis, cladribine Referencias Bibliograficas 1. Lorand-Metze I. Leucemias linfoidescronicas. Diagnostico, estadiamento, fatoresprognosticos e tratamento. Ser. Monogr.Esc. Bras. Hematol. 1999; 6; 18-22.2. Keating M.J. Translational research inchronic lymphocytic leukemia. AmericanSociety of Hematology 1999; 249-54.3. Cheson B.D. Infectious and immunossupressivecomplications of purine analog therapy. J ClinOncol

Hydroxychloroquine (Hcq) Induces apoptosis in b-chronic lymphocytic leukemia cells (B-cll) Via activation of CASPASE-3 and down-regulation of BCL-2

Abstract Recently, it has been observed that HCQ, an antirheumatic drug, induces apoptosis in peripheral blood T lymphocytes but the mechanism of this induction remains unclear. A fortuitous observation in a patient with B-CLL led us to study the in vitro activity of HCQ against B-CLL cells and potential mechanisms of action. HCQ induced a decrease in cell viability in a dose- and time-dependant manner. The mean IC50 (concentration of drug required to reduce the cell viability to 50%) for 20 B-CLL patients evaluated was 32±7 μg/ml (range, 10–70 μg/ml) for 24 hours of treatment. This cytotoxic effect was due to apoptosis as demonstrated by morphological and DNA fragmentation studies, propidium iodide labeling and also annexin/PI assay. HCQ (100 μg/ml) produced apoptosis as early as 5 hours post incubation (28±4% versus 8± 3% for untreated cells) which increased to 82±4% at 18 hours post-treatment. Caspase-3 protease activity, measured by the colorimetric assay, was increased in HCQ-treated cells and correlated with the level of apoptosis. Moreover, pretreatment of B-CLL cells with the caspase inhibitor Z-VAD.fmk prevented the activation of Caspase-3 and the cytotoxic effect of HCQ. The amount of Bcl-2 was significantly reduced during apoptosis as observed by western blot and flow cytometry. Compared with levels before treatment, the decrease of Bcl-2 was 22±2% (range, 15–32%, p

Bcl-2 and Bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients

Chlorambucil and other cytotoxic drugs kill cells, non-selectively, by inducing apoptosis. In this study, we measured the apoptotic response to chlorambucil in T- and B-cells from untreated B-CLL patients and T-cells, from normal control subjects. We found increased chemosensitivity in the T-cells of B-CLL patients compared to the controls ( P=0.0002). The chlorambucil ID 50 values for T-cells from B-CLL patients showed a direct correlation with Bcl-2 expression ( P=0.002) and an inverse correlation with CD3 cell count ( P<0.0001), suggesting a trend of increasing chemosensitivity and decreasing Bcl-2 expression with an elevated T-cell count. There was no differential expression of Bcl-2 or Bax between the CD4 + and CD8 + cells of B-CLL patients, isolated by immunomagnetic separation. We found correlations in the leukaemic B-cells between chlorambucil ID 50 values and both Bcl-2 expression ( P=0.006), and Bcl-2/Bax ratios ( P=0.002), suggesting a role for the Bcl-2/Bax ratio in predicting the response of untreated CLL patients to cytotoxic treatment. Chlorambucil produced almost identical changes in Bcl-2 and Bax expression in normal T-cells and leukaemic B-cells triggered to die by apoptosis, which together with the correlation between Bcl-2 and chemosensitivity confirms a pivotal role for Bcl-2 in regulating a distal step in the apoptotic pathway following cytotoxic cellular damage.

Influence of dexamethasone, fludarabine, and their synergism on apoptosis and BCL-2 expression in b-cll cells in vitro according to differentiation stage of cells

Abstract The purpose of this work was to investigate apoptosis and bcl-2 expression in cells of classical CLL, CLL/PL and PLL, to examine the influence of dexamethason and fludarabine separately and in combination on these two phenomena. There is evidence in the literature that morphological types B-CLL can be regarded as stages of different cell maturation. The hypothesis was that different maturation stages differ in their tendency to spontaneous and drug-induced apoptosis. Another hypothesis concerning additive or synergistic effect of mentioned drug combination was tested. Cells collected from 15 B-CLL patients (5 typical CLL, 5-CLL/PL and 5-PLL) were divided into control cultures, cultures treated with single drug and cultures treated with drug combination. The percentage of apoptotic cell nuclei was calculated in the subdiploid DNA peak of the DNA fluorescent histogram at days 3 and 7 using a flow cytometric analysis of propidium iodide staining of DNA. In parallel, morphology was examined on MGG stained slides using light microscopy. Bcl-2 protein expression was estimated in 9 patients using semiquantitative analysis of antibody by flow cytometry. Results of this study do not show significant differences between spontaneous and drug induced apoptosis in all 3 different stages. On estimating of drug induced apoptosis, drug influence was corrected according to spontaneous apoptosis measured in cell population of the same source. Additive cytotoxic effect of dexamethasone and fludarabin combination was observed in all different stages with no significant differences between influence of each drug. Analysis of bcl-2-expression demonstrated bimodal pattern as described earlier, which disappears after drug addition. Percentage of cells in control and drug treated cultures positive for bcl-2 protein was constant and high in all stages for an entire period of incubation. Dexamethasone, fludarabine and their combination induced upregulation, downregulation or did not change bcl-2 expression in different cases. No specific pattern was shown to characterize individual differentiation stage; but each drug alone and their combination tended to influence bcl-2 expression in a similar way in every given case, suggesting existence of a common pathway of bcl-2 modulation for these drugs. In conclusion, this study of in vitro apoptosis of B-CLL cells demonstrated: 1) no significant differences between maturation stages of B-CLL according to their susceptibility to spontaneous or drug-induced apoptosis, 2) a cumulative effect of fludarabine and dexamethasone combination on apoptosis in comparison to a single drug, 3) bimodal pattern of bcl-2 expression evolving into different unimodal patterns after drug addition with no specific pattern characterizing each stage and with similar direction of change for each drug, suggesting a common pathway for bcl-2 modulation.

Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: Costimulation with IL-2 results in a highly immunogenic phenotype

Objective CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Methods Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-α), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-γ Elispot assays. Results The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor α chain in malignant as compared with normal B cells ( p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN–stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-α and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Conclusion Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B-CLL patients.

Deregulated expression of promyelocytic leukemia zinc finger protein in B-cell chronic lymphocytic leukemias does not affect cyclin A expression.

The promyelocytic leukemia zinc finger (PLZF) gene encodes a transcription factor expressed in myeloid, lymphoid and CD34(+) progenitor cells. Structurally related to BCL-6, which is involved in human lymphoma, PLZF may have a role in proliferation, differentiation and survival of hematopoietic cells, that could be mediated by transcriptional repression of the cyclin A gene. Quantitative competitive reverse transcription-polymerase chain reaction was used to measure the levels of expression of PLZF and cyclin A in normal leukocyte subsets (including CD19(+) lymphocytes, n=21) and malignant B lymphocytes (including B-chronic lymphocytic leukemias [B-CLL], n=63). Results obtained with this method were confirmed by Western and Northern blot analysis. Transactivation assays were performed using an expression construct for PLZF and two cyclin A promoter luciferase reporters in an Epstein-Barr virus (EBV)-transformed B-cell line. Cyclin A expression, cell growth kinetics, and cell cycle were analysed in stable clones of the Burkitt lymphoma (BL) B-cell line DG75 with inducible expression of PLZF, generated using the tetracycline-regulated expression system. Expression of PLZF was 100-fold downregulated in 90% B-CLL (56/63) compared to normal B lymphocytes (P<0.001). B-CLL patients with the highest levels of PLZF had a poorer survival (P<0.013). In transactivation assays, PLZF inhibited the activity of the cyclin A reporters by 50%, demonstrating that PLZF can repress cyclin A expression in non-malignant B lymphocytes. However, in B-CLL patients, the level of cyclin A expression was found to be within the normal range. Altered PLZF function in B lymphoid malignancies was further corroborated in the PLZF-regulatable DG75 clones, where induction of PLZF expression did not significantly alter the levels of cyclin A expression, the cell growth kinetics, or the cell cycle phase distribution. The lower survival of patients with the highest levels of PLZF suggests that this protein may be a marker of progression in B-CLL. The absence of co-ordinated regulation of PLZF and cyclin A genes in B-CLL and in a malignant B-cell line may indicate a loss of cyclin A control by PLZF in B-CLL and other B-cell disorders. Deregulation of PLZF could thus play a role in B-cell malignancy.

Attempted Reconstruction of the Immune System using Low Doses of Interleukin 2 in Chronic Lymphocytic Leukemia Patients Treated with 2-Chlorodeoxyadenosine: Results of a Pilot Study

This study was designed to investigate the immunostimulatory effect of low dose 11–2 treatment in B-CLL patients previously treated with 2-chlorodeoxyadenosine (2CdA) in whom severe depletion of T lymphocyte subsets was observed. Four patients enrolled into the study had previously been treated with 3–6 courses of 2 CdA. All patients suffered from recurrent infections and showed CD4+ and CD8+ immunosuppression. Recombinant 11–2 was given subcutaneously at a dose of 100 μg (1.8 × 1061U) daily for 6 weeks. The drug was administered between 2CdA courses. These preliminary studies showed a marked increase in T cell subsets after Il-2 treatment. All patients displayed an increase of NK cells and there was increased expression of Il-2 receptors (CD 25 and CD 122) on lymphocytes.It is possible that the combination of cytotoxic therapy with 2CdA and low dose rII-2 could stimulate the T-cell immune system and may be a promising regimen in patients with B-CLL with severe depletion in T-cell subsets.

Preliminary immunocytochemical studies of MDR-1 and MDR-3 Pgp expression in B-cell leukaemias.

P-glycoproteins (Pgps) belong to the family of ATP binding cassette (ABC) transporter proteins. In humans two Pgp genes have been identified; mdr-1 and mdr-3. Classical Multiple Drug Resistance (MDR) is associated with over expression of the mdr-1 gene product, P-170. No role for mdr-3 in MDR has yet been proven. However there is evidence that mdr-3 overexpression may be associated with drug resistance in certain B-cell lymphocytic leukaemias. In an immunocytochemical study we have looked at a selection of B-cell leukaemias for mdr-1 and mdr-3 encoded Pgp expression using monoclonal antibodies specific for the mdr-1 and mdr-3 encoded gene products. In B-CLL patients a differential pattern of MDR-3 positive staining was observed; suggesting that MDR-3 positivity may be associated with a more malignant phenotype in B-CLL. This pattern was not observed with MDR-1 positivity. We also observed MDR-3 positivity in an AML stage M5a patient which is the first report of MDR-3 Pgp expression being detected in AML; suggesting that MDR-3 Pgp expression may be limited to particular subtypes of this disease. Results from B-NHL cases were inconclusive with varying expression of MDR-1 and MDR-3 Pgps observed. Work is currently underway to further explain the significance of these findings.

Functional interaction of the gp80 and gp130 IL-6 receptors in human B cell malignancies.

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.

CD57+/CD28- T cells in untreated hemato-oncological patients are expanded and display a Th1-type cytokine secretion profile, ex vivo cytolytic activity and enhanced tendency to apoptosis.

Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28- cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n = 54) as compared to healthy donors (n = 55), with CD57 and CD28 expression on the patients' T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor's major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients' CD57+/CD28- T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-gamma, and TNF-alpha than their CD57-/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28- subset. Moreover, a marked cytotoxic activity was detected within CD4+CD57+ T cells from some B-CLL patients. Finally, the patients' CD57+/CD28- T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28- T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.

Differential Sensitivity of CD4+ and CD8+T Lymphocytes to the Killing Efficacy of Fas (Apo-1/CD95) Ligand+ Tumor Cells in B Chronic Lymphocytic Leukemia

AbstractB-chronic lymphocytic leukemia (B-CLL) is characterized by cellular and humoral immune defects resulting in increased rates of infection and disturbed immune surveillance against cancer cells as well as by the expansion of slowly proliferating tumor cells. We found increased Fas receptor (FasR) expression in peripheral blood CD4+and CD8+ cells of B-CLL patients compared with the equivalent cells of healthy donors. Although increased Fas receptor expression was significant in both T-lymphocytic subsets, only CD4+ cells from B-CLL patients underwent apoptosis after treatment with the agonistic Fas antibody CH11. In CD4+cells of B-CLL patients, the Fas-sensitivity also correlated with a CD4+/CD8+ ratio below the lower threshold of healthy individuals (<1.0). By contrast, FasR expression in the CD19+ fraction of B-CLL patients was downregulated compared with normal controls, and this was associated with an insensitivity to CH11-induced apoptosis. The B-CLL cell line EHEB as well as CD19+ cells from B-CLL patients constitutively expressed Fas ligand (FasL). The FasL was functionally active, as the B-CLL cell line as well as T-cell–depleted CD19+ B-CLL fractions were able to kill target T-acute lymphatic leukemia (T-ALL) cells in vitro. This effect was inhibited by the antagonistic FasR-antibody ZB4, the neutralizing anti-FasL monoclonal antibody (MoAb) NOK-2 or by transfection of the caspase inhibitor crmA. These data point to the fact that expression of FasL on CD19+B-CLL cells, together with enhanced susceptibility of CD4+ T cells toward FasL-bearing effector cells, are causally linked to the relative reduction of CD4+ cells occurring during B-CLL progression. These findings could explain the inversion of the ratio of CD4+/CD8+ cell numbers, which may be causally linked to the immune deficiency observed in these patients and to the expansion of the neoplastic clone in B-CLL.

Altered pattern of cytokine production by peripheral blood CD2+ cells from B chronic lymphocytic leukemia patients.

To determine if activation-induced cytokine production is altered in CD2+ lymphocytes from B-CLL patients, cytokine levels were determined by ELISA in supernatants of PHA-stimulated cultures of CD2+ cells from 33 B-CLL patients and 22 healthy controls. The production of Interferon gamma (IFN-gamma) and Tumor Necrosis Factor (TNF-alpha) by mitogen-activated CD2+ lymphocytes from B-CLL patients was higher than that found in healthy controls, while no differences were found in TNF-beta production. IFN-gamma and TNF-alpha levels determined at 72 h in PHA-stimulated CD2+ cell cultures from B-CLL patients statistically correlated with the percentages of CD3+CD45RO+ and CD3-CD56+ lymphocytes, respectively. Although there were differences in the production kinetics of interleukins (ILs) 2 and 4 between B-CLL patients and the healthy controls, no differences were found at the time when the levels of both interleukins peak. The production of both IFN-gamma and IL-4 by PHA-stimulated CD2+ lymphocytes from non-smouldering B-CLL patients was significantly higher than that from smouldering B-CLL patients while no significant differences were found in the production of IL-2, TNF-alpha, and TNF-beta between the two B-CLL patient groups. These data suggest that functional alterations in the production of cytokines by CD2+ cells from B-CLL patients could help to explain the expansion of leukemic cells in B-CLL patients.

Peripheral blood lymphocyte subset shifts in patients with untreated hematological tumors: Evidence for systemic activation of the T cell compartment

Flow cytometry immunophenotyping of peripheral blood lymphocyte subsets and multivariate data-analytical techniques revealed that among untreated hemato-oncological patients ( n = 48) with lymphomas, acute and chronic myeloid and lymphocytic leukemias, monoclonal gammopathy of undetermined significance, and multiple myeloma, 42% had (nonmalignant) lymphocyte profiles clearly distinct from healthy donors. Notably, a similar pattern of increased CD3 +CD57 +, CD3 +HLA-DR +, CD3 +CD(16 + 56) +, CD4 −CD8 +, CD8 +CD57 +, CD8 +CD28 −, and CD8 +CD62L − subsets was detected. More extensive three-color immunophenotyping on an additional group of 49 untreated patients revealed that both CD4 + and CD8 + T cells displayed significant increases of activation markers: CD69, CD(16 + 56), HLA-DR, CD71, and CD57, and a loss of CD62L and CD28, which is also interpreted as a sign of activation. Consistent with the phenotypical signs of in vivo immune activation, polyclonal cytolytic activity, measured ex vivo in an anti-CD3-redirected assay, was detected within immunomagnetically purified CD4 + T cells of three out of six B-CLL patients investigated, but not within purified CD4 + T cells of five healthy donors. The purified CD8 + T cells of patients ( n = 28) and donors ( n = 5) on the other hand displayed similar polyclonal cytotoxic activities at the various effector:target ratios investigated. Tumor-directed cytotoxic activity of purified CD4 + ( n = 6) and/or CD8 + T cells ( n = 15) against freshly isolated autologous tumor cells was not detected in any of the experiments. Collectively, our results demonstrate systemic T cell activation as a common feature in hematological neoplasia, and a markedly enhanced cytolytic activity of the CD4 + subset in CLL patients. The reason(s) for this expansion of activated T cells and its pathophysiologic significance, however, remain unclear.

Elevated Plasma Levels of Interleukin-1 Receptor Antagonist are Associated with Decreased Cellular BCL-2 Oncoprotein Expression in B-Chronic Lymphocytic Leukemia

Plasma IL-1Ra levels and cellular BCL-2 oncoprotein expression were measured in a total of forty blood samples obtained from twenty-eight B-CLL patients and four healthy subjects. High IL-1Ra plasma levels (as defined by mean + three times standard deviation of normal controls) were observed in eleven samples (ten patients) which showed a significantly decreased cellular expression of BCL-2 protein (14.7 +/- 16.3% of cells as determined by immunofluorescence) when compared to B-CLL samples with no elevated IL-1Ra (BCL-2, 31.0 +/- 18.6%; p < or = 0.0115). Albeit correlational only, our results may encourage further experiments to elucidate potential regulatory effects of IL-1Ra for cellular BCL-2 oncoprotein expression.

Clinico-prognostic relevance of quantitative immunophenotyping in B-cell chronic lymphocytic leukemia with emphasis on the expression of CD20 antigen and surface immunoglobulins.

Expression of CD20, evaluated as antibody binding capacity (ABC) (i.e. absolute number of molecules of antibody per cell), was analyzed using flow cytometry on leukemic cells of 93 previously untreated patients, all fulfilling strict criteria of "immunologically typical" (i.e. CD5+, CD23+) B-cell chronic lymphocytic leukemia (CLL). Although changes of CD20 antigen density did not correlate with clinical parameters representative of either tumor mass (i.e. clinical stage, histological pattern of bone marrow involvement, absolute peripheral blood lymphocytosis) or disease progression (i.e. lymphocyte doubling time), a trend toward a better life-expectancy was observed in the low CD20 expression group compared with the high CD20 expression group (p = 0.05; relative risk of death, 0.51, 95% confidence interval, 0.24-1.04). Given the correlation between CD20 ABC and mean fluorescence intensity (MFI) of light chain (LC) surface immunoglobulins (Sm Ig) (r = 0.481, p < 0.0001), as well as the impact of MFI of Sm Ig LC on overall survival (p = 0.01; relative risk of death 0.44; 95% confidence interval, 0.10 to 0.76), we tried to verify whether a combination of B-cell markers, evaluated in a quantitative manner, could have additive prognostic properties. To this purpose we gave a value of 1 or 0 to each B-cell marker according to whether it was expressed at a low (i.e. CD20 ABC < 17.9 x 10(3) molecules/cell, MFI of LC Sm Ig < 100) or high (i.e. CD20 ABC > or = 17.9 x 10(3) molecules/cell, MFI of LC Sm Ig > or = 100) level thus allowing patient stratification into two groups with scores of 2 and 0-1, respectively. Survival of patients who scored 2 was significantly longer respectively. Survival of patients who scored 2 was significantly longer than that of patients who scored 0-1 (p = 0.02; relative risk of death, 0.44; 95% confidence interval, 0.22-0.72). However, when quantitative changes of CD20 antigen and LC Sm Ig expression, either alone or in combination, were simultaneously analyzed in a Cox model which included usual clinico-hematological features, only absolute peripheral blood lymphocytosis (p = 0.0001) and Binet clinical stages (p = 0.0001) maintained their prognostic power unmodified. Although variability of CD20 and Sm Ig expression make it possible to appreciate biological heterogeneity of B-cell CLL better, however, they cannot substitute well-established clinico-hematological features in the prognostic assessment of B-CLL patients.

Relationship between autoimmunity and immunodeficiency in CLL.

B-CLL patients have a high prevalence of autoimmune phenomena due to polyclonal autoantibodies (Abs) restricted to blood cell self-antigens (Ag) and progressively develop a severe hypogammaglobulinemia. Autoimmunity and immunodeficiency have been related to B-CLL biological properties and cellular origin. Three major issues have emerged: 1) the relevance of B cell receptor abnormalities. More specifically, the importance of the defective expression of CD79b which may explain the faint to virtually undetectable amounts of monoclonal sig, the anomalous signal transduction and the failure to present antigens (Ag). 2) the possibility that the relentless accumulation of B-CLL cells is also under microenvironmental influences. These are brought about by the interplay of cytokines produced through cell/cell contacts among malignant B cells, macrophages and T cells. 3) the view that the very properties of accumulating anergic self-reactive CD5+ B lymphocytes may provide a bridge between autoimmunity and immune incompetence. CD5+ malignant B cells influence the cytokine production by T cells, but are inefficient Ag presenting cells (APC). As such they are a hurdle to the production of normal Ab. However, by stimulating in vitro the CD40 molecule on the membrane of CLL cell with the CD40 ligand the inefficient APC is turned into an efficient APC. In vivo, such an activation may conceivably occur in specific environments like the spleen and favour the production by normal residual B-cells of polyclonal autoAb against red cell or platelet self Ag.