Large granular lymphocytes from B-chronic lymphocytic leukemia patients inhibit normal B cell proliferation

Large granular lymphocytes (LGL) may exert regulatory influences on B cell immunoglobulin synthesis. We, therefore, investigated the influence of LGL from controls and B cell chronic lymphocytic leukemia patients (B-CLL) on control B cell proliferation to costimulation with the F(ab')2 fragment of goat antihuman mu and B cell growth factor (BCGF). Purified LGL (greater than 90% by morphology) from control and B-CLL peripheral blood were added in various concentrations to purified control B cells and incubated with anti-mu and BCGF for 3 days. [3H]-thymidine uptake of B cells was then measured. There was no proliferation of control or CLL LGL alone to the costimulatory signals of the F(ab')2 fragments of goat antihuman mu chain and BCGF. Addition of control LGL to equal numbers of control B cells did not blunt control B cell responsiveness to BCGF (with control LGL 8,649 +/- 298 cpm vs. control B cells alone 8,336 +/- 556 cpm, mean +/- SEM). When control LGL were increased to 10:1 LGL:B cell ratio, the maximal inhibition by control LGL of control B cell proliferative response to BCGF was 23%. In contrast, addition of CLL LGL at a 1:1 LGL:B cell ratio resulted in marked impairment of the control B cell proliferative response to BCGF (with CLL LGL 3,586 +/- 954 cpm vs. control B cells alone 8,649 +/- 298 cpm). Inhibition by CLL LGL occurred in a cell-concentration-dependent manner. No difference in CLL LGL's inhibitory effect on either resting or activated control B cell responsiveness to BCGF was noted. Inhibition of de novo protein synthesis (by cycloheximide inhibition) of CLL LGL did impair CLL LGL's inhibitory capacity for BCGF-induced B cell proliferation. A possible explanation for these findings includes the possibility that a subgroup of LGL with B cell suppressive activity may have expanded as a host response to the B cell leukemia or as part of the disordered cell regulation in B-CLL.     

Heterogeneous proliferative effect of tumor necrosis factor-α and lymphotoxin on mitogen-activated B cells from B-chronic lymphocytic leukemia

The proliferative effect of tumor necrosis factor-α (TNF-α) and lymphotoxin on B cells from patients with B-chronic lymphocytic leukemia (B-CLL) was studied. Fresh purified B-CLL lymphocytes showed no proliferative response to either recombinant (r) TNF-α or r-lymphotoxin. However, after 'in vitro' activation of B-CLL lymphocytes for 2 days with Staphylococcus aureus Cowan 1 (SAC), four of seven patients showed enhanced blastogenic response in the presence of either rTNF-α or r-Iymphotoxin. We also found that the proliferative response of SAC-activated B-CLL lymphocytes to the two cytokines was independent of that found in the presence of interleukin-2. These results demonstrate that TNF-α and lymphotoxin can heterogeneously support the proliferation of in vitro activated B cells from B-CLL patients and may reflect the biological heterogeneity of B-CLL disease.     

T lymphocyte subpopulations in chronic lymphocytic leukemia of B cell type in relation to immunoglobulin isotype(s) on the leukemic clone and to clinical features

Total blood T lymphocytes and subpopulations (OKT4+ and OKT8+ cells) were studied in 59 patients with B cell chronic lymphocytic leukemia (B-CLL). In 48 previously untreated patients, total T lymphocytes were higher as compared to healthy controls (p less than 0.001). T-cells and OKT8+ cells were significantly increased in patients in advanced clinical stage and with progressive disease in comparison to patients with low stage and indolent disease. High numbers of OKT8+ lymphocytes were also seen in patients with a dominance of mu heavy chains on the leukemic clone. Moreover, in this patient group the OKT8+ subpopulation correlated with total B cells (r = 0.68, p less than 0.001) while in patients with a mu delta phenotype no such correlation was seen. After successful cytostatic therapy there was a reduction in total numbers of both OKT4+ and OKT8+ cells, in particular, with a concomitant increase in OKT4/OKT8 ratios.     

Restoration of impaired natural killer cell activity of B-chronic lymphocytic leukemia patients by recombinant interleukin-2

Natural killer (NK) function in the majority of B-cell chronic lymphocytic leukemia patients is markedly deficient. This study was undertaken to determine if the biological response modifier interleukin-2 (IL-2), which is a potent augmenter of normal individuals' NK activity, could augment the low NK activity in these patients. Peripheral blood lymphocytes (PBL), depleted of B-cells, from most B-CLL patients exhibited low natural killer activity against NK-sensitive K562 cells and against herpes simplex virus (HSV)-infected lymphoblastoid cell lines (LCL). Incubation of patients' B-cell-depleted PBL with recombinant IL-2 resulted in augmentation of their NK activity against both K562 cells and HSV-infected cells. Furthermore, whereas large granular lymphocytes (LGL) isolated from CLL patients are deficient in cytoplasmic granules, which are thought to play a role in NK-cell-mediated lysis, treatment of patients' LGL resulted in increased granulation by 4 hr after treatment with IL-2 and showed a concomitant increase in lytic activity comparable to that of normal individuals.     

Interleukin 2 (IL 2) and interferon-gamma production by T lymphocytes from patients with B-chronic lymphocytic leukemia: evidence that normally released IL 2 is absorbed by the neoplastic B cell population

The capacity of T lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) to release interleukin 2 (IL 2) and interferon (IFN)-gamma was assessed following various stimuli. The spontaneous release of IL 2 and IFN-gamma was practically absent both with B-CLL and normal T lymphocytes. By contrast, after stimulation with phytohemagglutinin (PHA) or with PHA plus 12-O- tetradecanoylphorbol-13-acetate, the production of IL 2 and IFN-gamma by B-CLL T lymphocytes was similar to that of normal T lymphocytes, irrespective of the reversed T lymphocyte subset distribution (OKT4/OKT8 ratio) observed in B-CLL. However, the titer of IL 2 was greatly reduced when autologous leukemic B cells were added to the culture system. Unlike IL 2, the presence of leukemic B cells did not affect the titer of IFN-gamma in the culture supernatants. The indication that IL 2 may be adsorbed in vivo by the neoplastic B cells was further confirmed by the demonstration of the IL 2 receptor (revealed by anti-Tac monoclonal antibody) on the leukemic B cells, particularly following mitogenic stimulation, and by the evidence that exogenous IL 2 can be directly absorbed by untreated B-CLL T lymphocytes to release IFN-gamma and IL 2 is preserved, but that IL 2 may be rapidly removed by the neoplastic B-CLL cells, thus contributing to the well-documented T lymphocyte abnormalities present in this disease.     

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 γ (IFN-γ) and Tumor Necrosis Factor (TNF-α) by mitogen-activated CD2+ lymphocytes from B-CLL patients was higher than that found in healthy controls, while no differences were found in TNF-β production. IFN-γ and TNF-α 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-γ 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-α, and TNF-β 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. Am. J. Hematol. 57:93–100, 1998. © 1998 Wiley-Liss, Inc.     

Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients

Recent studies have shown that normal human T cells contain a high- molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.     

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL)

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.     

Phase-I/II study to evaluate dose limiting toxicity, maximum tolerated dose, and tolerability of bendamustine HCl in pre-treated patients with B-chronic lymphocytic leukaemia (Binet stages B and C) requiring therapy

Purpose: Bendamustine hydrochloride, an anti-neoplastic agent with unique mechanism of action, is known to cause impressive remissions in relapsed nonHodgkin’s lymphoma and chronic lymphocytic leukaemia (CLL). Optimal bendamustine dosing for CLL patients had not been finally established and a phase I/II study was conducted to determine the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) of bendamustine. Methods: The open-label, single-centre phase I/II study was conducted from March 2001 to September 2002 in Sofia, Bulgaria. The 15 study patients were extendedly pre-treated, but fludarabine-naive (3 female, 12 male, 47–72 years of age, 61 years on average). Bendamustine was given at a starting dose of 100 mg/m² on days 1 and 2 every 3 weeks based on the previous results in lymphoma. Results: Bendamustine was well tolerated in spite of heavy pre-treatment of the study participants. Toxicity corresponded to the known safety profile of bendamustine, with the exception of bilirubin elevation. The level of 110 mg/m² was established as MTD. A bendamustine dose of 100 mg/m² is the recommended dose for further clinical investigations. A 4-week interval is recommended to allow for sufficient recovery. Efficacy results confirmed powerful anti-neoplastic activity of bendamustine even in extendedly pre-treated CLL patients. Based on the remission criteria, nine patients were defined as responders (four CRs, two PR, three NC) and two patients as nonresponders to therapy. Four patients were not evaluable for response, because they had received less than three courses bendamustine. After a follow-up period of 15 months, the four patients with CR were still in remission. One patient with PR had relapsed, the other had ongoing response. Conclusions: Bendamustine is a very active and well-tolerated drug in patients with pre-treated and refractory CLL. Fludarabine-naivity of patients appears to markedly improve their bendamustine tolerability. First-line use of bendamustine is a safe option for CLL-patients requiring treatment, because bendamustine—owing to its unique pharmacodynamics—(1) is highly effective, (2) reasonably safe, and (3) does hardly produce cross-resistance against other anti-neoplastic drugs effective in this indication.     

Identification of leukemia cell-derived inhibitory activity (LIA) in conditioned media from human myeloid leukemic cell line ML-2

Summary Conditioned media from the human myeloid leukemic cell line ML-2 contain a factor that inhibits the entry of normal CFU-GM into S phase of mitotic cycle as measured by the ³H-TdR suicide technique. This factor was detected in conditioned media prepared by incubating 5×10⁶ ML-2 cells/ml or 1×10⁶ ML-2 cells/ml in serum-free RPMI for 5 or 24 hours respectively, and was isolated by ultrafiltration through an XM300 Diaflo membrane followed by chromatography on Sepharose 6B. Ferritin, prepared from human placenta, had the same inhibitory effect on CFU-GM. Antibodies against human placental ferritin completely inactivated the inhibitory effect of both human placental ferritin and the factor released from ML-2 cells. The inhibitory activity produced by the cell-line ML-2 was considered as LIA (leukemia cell-derived inhibitory activity) earlier found in HL-60 cell line and AML and CML cells.     

Mitogenic stimulation of human tumor-infiltrating lymphocytes by secreted factor(s) from human tumor cell lines.

Tumor-infiltrating lymphocytes (TILs) have shown in vivo antitumor efficacy in both animal and human studies. Functions thought necessary for antitumor activity include cytolysis, homing, and proliferation at tumor sites. TILs, which are T lymphocytes grown ex vivo directly from tumors, bear interleukin 2 (IL-2) receptors capable of transducing the IL-2 mitogenic signal. However, IL-2 is not normally synthesized by solid tumor cells. This study was aimed at exploring the possible presence of T-cell mitogens of tumor origin. To this end four TIL lines derived from four melanoma patients were studied for their ability to use the environments of cultured tumor cell lines as mitogenic sources. The presence of four irradiated cultured human tumor cell lines, three of which were derived from the same melanoma patients as the TILs, were found to stimulate proliferation of human TILs in the absence of IL-2. Further investigation showed that the observed proliferative stimulation by the fourth tumor line was due to secreted factor(s) as mitogenic activity was present in the serum-free tumor cell supernatant. Both immunologic analyses of this medium and proliferative assays in which TILs were stimulated with recombinant lymphokine standards suggest the presence of a yet uncharacterized T-cell mitogen.     

Unmasking of cryptic natural killer (NK) cell recognition sites on chronic lymphocytic leukemia lymphocytes

The sensitivity of chronic lymphocytic leukemia (CLL) lymphocytes to attack by natural killer (NK) cells has remained questionable. To clarify this issue, freshly isolated lymphocytes of 37 patients with B-CLL, five with WDLL and two with HCL, were tested with a standard cytotoxicity assay with NK cells from normal donors. All these targets were resistant to cytolysis by the effectors. Freeze-fracture analysis of CLL cell plasma membranes revealed that they have a larger number of intramembranous particles (IMP) associated with the external leaflet (E-face) than have normal lymphocytes. Unlike other neoplastic cells, exposure of CLL lymphocytes to phorbol esters or treatment with neuraminidase did not render them vulnerable to attack by NK cells, nor did 5 days of culture have an effect. Incubation of CLL lymphocytes with anti-Ig-mu (24-72 hr) or with 0.1% pepsin (15 min) resulted in 15% and 27% cytolysis, respectively. B-lymphocytes from the blood of healthy donors were not killed when treated similarly: These data establish that freshly isolated B-CLL lymphocytes are resistant to NK cytolysis but that in contrast to normal B-cells, they possess cryptic NK-recognition structures, which may be uncovered by surface modulation.     

Relationship of fluorescence polarization to cell lineage in lymphocytes from normal subjects and patients with chronic lymphocytic leukemia

Previous investigations have shown differences in fluorescence polarization between normal and chronic lymphocytic leukemia lymphocytes following incubation with the probe 1,6-diphenyl-1,3,5-hexatriene. In the present study, we determined the fluorescence polarization of unseparated or enriched subpopulations of T and B lymphocytes from normal subjects and patients with chronic lymphocytic leukemia. As had been observed by others, the mean polarization (P) value at 25 degrees C for unseparated chronic lymphocytic leukemia lymphocytes, .240 +/- .007 (N = 22), was lower than that of unseparated normal lymphocytes, .248 +/- .005 (N = 18), P less than .001 (Student's t-test). The difference was greater when B-enriched populations were compared. The mean P value of B-cell-enriched chronic lymphocytic leukemia lymphocytes, .240 +/- .007 (N = 5), was significantly lower than that of B-cell-enriched normal preparations, .256 +/- .004 (N = 5), P less than .001. In contrast, no significant difference was found between normal and chronic lymphocytic leukemia T cells. The anomalous fluorescence polarization manifested by chronic lymphocytic leukemia lymphocytes of B-cell origin serves to distinguish this lineage from its normal counterpart and from T cells of either source.     

Proteolytic cleavage of human von Willebrand factor induced by enzyme(s) released from polymorphonuclear cells

In vivo fragmentation of the von Willebrand factor antigen (vWF:Ag) molecule has been demonstrated on radiocrossed immunoelectrophoresis (CIE) in the plasma from patients with disseminated intravascular coagulation, in factor VIII concentrates, and in normal serum. Experiments reported here show that polymorphonuclear (PMN) cells contain a non-calcium-dependent protease(s) that when released and incubated with vWF:Ag results in an additional vWF:Ag peak on radio- CIE. Production of fragments of vWF:Ag by incubation with PMN cells occurred in a time-dependent manner. The protease(s) responsible was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin, but not by benzamidine, azide, epicron, or hirudin. Citrate, EDTA, and leupeptin also had no effect on the PMN cell enzyme's activity, indicating that the enzyme(s) is not calcium dependent. The PMN cell enzyme responsible for vWF:Ag fragmentation is located intracellularly and released by freezethaw lysis or cell activation by calcium or the calcium ionophore A23187.     

Production of B-cell growth factor interleukin 2 and gamma interferon by peripheral blood lymphocytes from patients with chronic lymphocytic leukemia of B-cell type

Chronic lymphocytic leukemia of B-cell type (B-CLL) is a malignant disease characterized by monoclonal proliferation of small lymphocytes of B-cell origin, usually associated with suppression of polyclonal B-cell activation (i.e., proliferation and differentiation). Normal human B-cell proliferation is controlled by different T-cell-derived lymphokines, including interleukin 2 (IL2) and gamma interferon (gamma-IFN), that account for the majority of the B-cell growth factor (BCGF) activity produced by mitogen-activated peripheral blood mononuclear cells (PBMCs). We have previously shown an increased and dysregulated secretion of IL2 in peripheral blood from patients with B-CLL. BCGF, IL2, and gamma-IFN productions by phytohemagglutinin (PHA)-stimulated PBMCs were investigated in 13 patients with active untreated B-CLL and 11 healthy donors. B-CLL PBMCs produced a significant amount of BCGF (6 U/ml) despite the low percentage of T cells (10%) associated with this disease as compared with that found in healthy donors (61%). BCGF production in normal controls and B-CLL patients was tripled after irradiation of PBMCs or addition of indomethacin. gamma-IFN secretion in B-CLL patients was decreased when compared with normal controls. Therefore, when gamma-IFN was calculated per fixed number of T cells, production was significantly higher in B-CLL patients than in normal controls, showing a dilution of the productive cells. This study suggests that T cells from B-CLL patients are functional in terms of BCGF production despite their decreased percentage and abnormalities in surface markers.     

Spontaneous release of leukocyte migration inhibition factor (LMIF) by lymphocytes in chronic lymphatic leukemia patients

Leukocyte migration inhibition factor (LMIF) activity expressed as a migration index was studied in sera of 13 chronic lymphatic leukemia (CLL) patients and 14 healthy subjects. LMIF activity was present in 92.8% of CLL patients and 14% of controls. The LMIF activity of lymphocyte culture supernatants in the same patients and 7 controls was also studied. A LMIF-like activity was found both, in the sera and phytohemagglutinin (PHA) unstimulated cell supernatants in 9 of 13 patients (69.2%). These results demonstrate the ability of malignant B cells to release LMIF or LMIF-like factors spontaneously.     

Evaluation of apoptosis induced in vitro by cladribine (2-CdA) combined with anthracyclines in lymphocytes from patients with B-cell chronic lymphocytic leukemia

The aim of the study was to evaluate the effect of three anthracyclines [doxorubicin (DOX), mitoxantrone (MIT), and idarubicin (IDA)] on the rate of apoptosis triggered by 2-chlorodeoxyadenosine (2-CdA) in peripheral blood mononuclear cells isolated from 52 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL). The cells were cultured up to 48 h in the presence of drugs alone and in the following combinations: 2-CdA+DOX, 2-CdA+MIT, and 2-CdA+IDA. Apoptosis was assessed after 24 h and 48 h of incubation using annexin V/propidium iodide assay by flow cytometry. The apoptotic index (AI) was defined as a percentage of annexin V-positive B-CLL cells. Additionally, in some patients other hallmarks of apoptosis (activation of caspases, DNA fragmentation) were assessed in parallel for confirmation of apoptotic mode of induced cell death. All of the cytostatics induced apoptosis of B-CLL cells at a rate significantly higher than the index of spontaneous apoptosis occurring during 24 h and 48 h of cell culture. 2-CdA in combination with DOX significantly increased the percentage of annexin V-positive cells, particularly after 48 h of incubation, as compared with DOX used in monotherapy (median AI for 2-CdA+DOX=37.9%, median AI for DOX =13.8%, P=0.0011, and median AI for 2-CdA=22.1%, P=0.013). Combination of 2-CdA with MIT induced a similar effect, also more distinct after 48 h (median AI for 2-CdA+MIT=41.05%, median AI for MIT=16.3%, p=0.0012, and median AI for 2-CdA=22.1%, p=0.017). For both combinations median AI were similar to the sum of median AI for each drug when used alone. IDA in a concentration ten times lower (0.1 micro g/ml) than used before in acute leukemia cells produced high cytotoxic effects, masking the additive effect of combination with 2-CdA. Only at a dose of 25 ng/ml of IDA, significant differences in AI after 24 h and 48 h were detected between samples treated with 2-CdA+IDA (median 27.5% and 65.0%, respectively) and those incubated with IDA alone (median 10.5% and 33.4%; P=0.0004 and 0.0274, respectively). Similarly, there were significant differences between AI of cells treated with 2-CdA+IDA and 2-CdA alone (median 9.5% at 24 h and 23.5% at 48 h; P=0.0013 and 0.0207, respectively). In conclusion; these data indicate an additive cytotoxic effect on B-CLL cells of DOX, MIT, and IDA applied in vitro with 2-CdA; all of them induced apoptosis with similar efficacy. We suggest that further preclinical and clinical studies concerning combined use of 2-CdA with anthracyclines are desirable. High sensitivity of B-CLL cells to IDA suggests the possibility of lowering its dose in patients, especially when combined with 2-CdA.