RAB-1: a new monoclonal antibody to leukemic hairy cells

RAB-1, a new monoclonal antibody (McAb) to human leukemic hairy cell (HC) was produced. Using indirect immunofluorescence methods and microscopic or flow cytometric analysis, it was found that the RAB-1 antigen was expressed on few resting B cells and not on resting T lymphocytes, platelets, monocytes, erythroid and myeloid cells. RAB-1 expression on malignant cells was as follows: strongly positive in 15/15 hairy cell leukemia (HCL), negative with non-T and T-acute lymphoblastic leukemia and T-chronic lymphocytic leukemia (CLL); weakly expressed on myeloma and Waldenstrom cells; moderately on 10-25% of the cells in 4/10 B-CLL and 6/10 B lymphomas and in 7/7 B-prolymphocytic leukemia (PLL). Amongst human cell lines that were tested, RAB-1 reacted strongly with one HC line, moderately with the EBV-lymphoblastoid Daudi and Raji Burkitt's lines and was not expressed on Ramos, ALL, myeloid and erythroid cell lines. Normal B cells activated with PWM or anti-mu beads, and malignant B cells activated with anti-mu and TPA did not show an increase of expression of RAB-1 antigen. Interestingly, 30-40% of T4-Class II antigen positive cloned cells and T cells activated with PHA and Con.A expressed RAB-1, suggesting that this McAb recognizes surface molecule, newly induced during T-cell activation and constitutively expressed on HC and some B-cell malignancies.     

Expression of the T1 (CD5, p67) surface antigen in B-CLL and B-NHL and its correlation with other B-cell differentiation markers

The T1 surface antigen (CD5,p67) expression on blood lymphocytes (PBL) and lymphoid cells from lymph node biopsies (LN) from 31 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 79 with B non-Hodgkin lymphoma (B-NHL), was detected in 25 B-CLL (80 per cent) and in 11 B-NHL (13 per cent) belonging to the following histologic subtypes: lymphocytic of CLL type (DLWD) one case, lymphoplasmacytoid (DLWD) four cases, centrocytic (DLPD) five cases, immunoblastic (DH) one case. All B-CLL and the T1 + B-NHL were also tested with monoclonal antibodies against the Common Acute Lymphoblastic Leukemia Antigen, B cells (FMC7, FMC8, BA1, Y29-55), T cells (OKT11a), HLA-DR and HLA-DQ monomorphic determinants. All the B-CLL and the T1+ B-NHL were CALLA-, BA1+, Y29.55+. FMC7+ cells were detected in large numbers six B-CLL (three T1+ and three T1-) and in four centrocytic lymphomas. FMC8 reacted with 70 per cent of leukemias (where it stained 30 per cent of neoplastic cells) and with 8/9 T+ B-NHL. HLA-DR and HLA-DQ molecules were detected in 100 per cent and 90 per cent of cases respectively. In vitro treatment of HLA-DQ- or T1- B-CLL with phorbol ester TPA led to the expression of these antigens as well as of the receptors for Interleukin 2 and MLR3 activation antigen. Surface membrane Ig (SIg) was detected in 79 per cent of cases, its density measured by FACS analysis varied, even markedly, from case to case. Among the B-CLL, cells with high SIg content were either T1+ or T1- and more likely FMC7+. The SIg- cases were seven B-CLL (five T1+ and two T1-) and two B-NHL, in which, however, cytoplasmic IgM was detected. This study reveals the existence of four major B-CLL subgroups: T1- SIg-, T1+ SIg+, T1+ SIg+, T1- SIg+. It also indicates that the T1 antigen may be transitionally present during B-cell differentiation and that its expression may precede that of SIg as supported by the in vitro studies. In addition, the finding that some B-NHL are T1+ suggests that they derive similarly to the B-CLL from a common progenitor.     

Differences in constitutive and activation-induced expression of CD69 and CD95 between normal and chronic lymphocytic leukemia B cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a sustained accumulation of long-lived and well-differentiated B lymphocytes in lymphoid tissues, peripheral blood and bone marrow. Although the pathogenesis of this disease is not entirely understood, altered apoptosis is believed to play a relevant role in B-CLL. In this study, we compared the expression of CD95, the best characterized surface molecule involved in triggering the apoptotic machinery, on normal and CLL B cells before and after in vitro activation with polyclonal stimulators. Cell activation was monitored by verifying the induced expression of the early activation antigen CD69. Freshly analyzed CLL B cells showed significantly lower levels of CD95 than normal B cells. Moreover, following in vitro culture with phorbol 12-myristate 13-acetate (PMA) + ionomycin, phytohemagglutinin, or pokeweed mitogen, CLL B cells failed to upregulate CD95 expression as efficiently as normal B cells. Impairment of CD95 upregulation was mainly observed following PMA + ionomycin treatment. In contrast, CLL B cells were shown to express CD69 as well as normal B cells, regardless of the activator used, indicating that CLL B cells retain the ability to respond to activating stimuli but are unable to efficiently implement the CD95-mediated activation-induced cell death (AICD) program. In conclusion, these results suggest that prolonged survival of CLL B cells may be contributed to by alterations in AICD mechanisms.     

Proliferative response of highly purified B chronic lymphocytic leukemia cells in serum free culture to interleukin-2 and tumor necrosis factors alpha and beta

Recent studies have suggested that interleukin-2 (IL-2), tumor necrosis factor (TNF) and a crude preparation of B-cell growth factors (BCGF) have a regulatory role in the proliferation of B-CLL cells. However, interpretation of the experimental data has been complicated by potential methodological pitfalls. To establish the role of these factors in B-CLL growth, we investigated their stimulatory effects under accurately defined in vitro conditions. Purified B-CLL cells were obtained by fluorescence activated cell sorting on the basis of coexpression of CD5 and CD19/CD20/CD24 surface antigens in order to avoid interference of normal cell contamination. Subsequently, the cells were cultured free of serum. TNFs alpha and beta and BCGF induced DNA synthesis in the purified B-CLL cells in five out of six cases, as assessed by 3H-thymidine (TdR) uptake on days 3 and 6 of culture. The stimulating activity of BCGF could be suppressed by the addition of specific anti-TNF monoclonal antibodies, indicating that the BCGF activity had been, to a major extent, due to the presence of TNFs in this impure preparation. IL-2, when added as a single stimulus, induced DNA synthesis in the B-CLL cells in three out of six cases on day 3, but not on day 6 of culture. In some patients, cooperative effects of IL-2 and TNF were observed. Cytogenetic analysis was applied to confirm the CLL origin of the proliferating cells. The phorbol ester TPA or anti-IgM were not required for induction of DNA synthesis in the CLL cells, although TPA potentiated 3H-TdR uptake in three out of six cases, and anti-IgM in one case. Our data demonstrate that IL-2 and TNFs alpha and beta can act as growth factors for B-CLL cells under fully defined in vitro conditions, essentially without the need of TPA or anti-IgM as primary activation signals.     

Proliferation of leukemic B cells in response to SAC and anti-mu. Evidence for different modes of action and comparison to proliferation and differentiation induced by conditioned medium

We investigated the proliferative responses and immunoglobulin production of highly purified E-rosette negative largely leukemic B cells from patients with CLL to Staphylococcus aureus Cowan I (SAC) or to SAC in combination with anti-mu or conditioned medium (CM). The latter was derived by stimulating human peripheral blood mononuclear leukocytes with PHA. We observed: (1) that purified E-rosette negative largely leukemic B cells from 25% (five out of 20) of the patients exhibited proliferative responses to SAC; (2) inhibition of SAC-induced proliferation by anti-mu in certain patients, whereas synergism between SAC and anti-mu in inducing proliferative responses in others; (3) the lack of synergism between SAC and CM in inducing proliferative responses, which is in contrast to the strong synergism that was observed between anti-mu and CM in inducing proliferation; and (4) induction or enhancement by SAC alone of Ig production by largely leukemic B-cell populations from few patients with CLL and purified tonsillar B lymphocytes, but not peripheral blood B cells from normal donors. These results suggest that SAC and anti-mu induce proliferation of B cells by different mechanisms and that B-cell proliferation and differentiation is dependent not only on the mitogen but also on the activation state of the cells.     

CD38 expression levels in chronic lymphocytic leukemia B cells are associated with activation marker expression and differential responses to interferon stimulation.

CD38, a surface protein whose expression increases upon normal B-cell activation, is a marker of disease aggression in B-cell chronic lymphocytic leukemia (B-CLL). Higher percentages of CD38-expressing CLL B cells may be found in lymphoid compartments compared to peripheral blood. Therefore, it is possible that although CLL B cells are resting, CD38 may be a marker of recent cell activation prior to entry into the periphery. To address this hypothesis, we examined the association of CD38 expression with other activation antigens identified in gene expression profiling experiments and include CD18, CD49d, CD20, and subunit 5 of the anaphase-promoting complex/cyclosome. We found that all these markers were more highly expressed in leukemic B cells from CD38-positive CLL patients. Lastly, because interferon is known to modulate CD38 expression, we used IFN-alpha to test the ability of CLL B cells to increase CD38 expression in vitro. Interestingly, IFN stimulation only modulated CD38 expression in CLL B cells that already expressed CD38. Taken together, these data suggest that CD38 is a marker of a more recently activated CLL B cell. This in turn may explain the biological and clinical differences between CD38-positive type B-CLL and CD38-negative type B-CLL.     

Effect of tetravalent bispecific CD19xCD3 recombinant antibody construct and CD28 costimulation on lysis of malignant B cells from patients with chronic lymphocytic leukemia by autologous T cells

To develop an effective antitumor immunotherapy for B-lineage non-Hodgkin's lymphoma, we constructed a tetravalent tandem diabody (tanDb) specific for both human CD19 (B-cell marker) and CD3 (T-cell antigen). Here, we report the effective killing of malignant primary B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) by autologous T cells induced by tanDb at very low E:T ratios. Mononuclear cells from patients with B-CLL were cultured with bispecific antibody fragments in either the presence or absence of monospecific anti-CD28 antibody. Use of tetravalent tanDbs caused almost quantitative elimination of malignant B cells from the blood samples of 19 patients and some cytotoxic activity in 3 of 23 analyzed cases. In contrast, the structurally similar but bivalent diabody and single-chain diabody demonstrated nearly no antitumor activity in an autologous system. tanDb-induced activation and proliferation of T cells occurred only in the presence of CD19+ target cells. Expression of the B7-1 (CD80) and B7-2 (CD86) molecules on the surface of leukemia cells made unnecessary the additional CD28-costimulation of T cells. When only a few tanDb molecules were present, the effect of CD28 costimulation on T-cell activation was more pronounced. Depending on the patient sample, we observed a 10- to 1,000-fold decrease of the half-maximal concentrations of tanDb for cell lysis. Upon CD28 crosslinking by agonistic MAb, specific tumor cell lysis was found at tanDb concentrations as low as 0.5 pM. These data demonstrate that the tetravalent CD19xCD3 tanDb might be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.     

Occurrence of particular isoenzymes in fresh and cultured leukemia-lymphoma cells. I. Tartrate-resistant acid phosphatase isoenzyme

The cells from 87 leukemia-lymphoma cell lines, 14 B-lymphoblastoid cell lines, 459 cases of leukemia-lymphoma, normal specimens, 22 leukemia-lymphoma cell lines treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) and 14 cases of chronic lymphocytic leukemia (CLL) and chronic myelocytic leukemia (CML) treated with TPA were analyzed for the expression of tartrate-resistant acid phosphatase (TracP) isoenzyme separated by isoelectric focusing. The TracP isoenzyme was seen in the following leukemia-lymphoma cell lines: 4 of 30 T-cell, 2 of 35 B-cell, 1 of 6 non-T/non-B-cell, 1 of 8 myelomonocytic, 3 of 4 erythroleukemia, and 3 of 4 Hodgkin's disease-derived cell lines. The expression of the TracP band could be induced by treatment with TPA in 3 myelomonocytic leukemia cell lines. Among the different types of leukemia-lymphoma cells freshly obtained from patients, the TracP isoenzyme was detected at a high incidence in cases of B-CLL, hairy cell leukemia (HCL), and B-lymphoma. Of the myeloid leukemias, 10% to 20% displayed the TracP isoenzyme. TracP positivity was detected in the peripheral blood, tonsil, bone marrow, spleen, and liver obtained from healthy donors, but not in the thymus. The expression of the TracP band could be newly induced by TPA in cases of CLL and in cases of CML. It is concluded that TracP activity is not specific for HCL, but is found at high incidences in cases of HCL, B-CLL and B-lymphoma. The TracP isoenzyme is not expressed by very immature lymphoid leukemia cells, but by cells arrested at later stages of differentiation of the T- or B-cell lineage, and by some myeloid cells.     

B8.7 antigen expression on B-CLL cells and its relationship to the LMW-BCGF responsiveness

In this work, we studied the expression of B8.7 antigen on B lymphocytes from patients suffering from B type chronic lymphocytic leukemia (B-CLL) as well as on non Hodgkin lymphoma cells (NHL). B8.7 is an activation marker, which has been reported to be associated with the capacity of activated B cells to respond to LMW-BCGF. B lymphocytes of 11 out of 22 patients tested were B8.7 positive. With the exception of one case, LMW-BCGF is able to induce DNA synthesis by these cells in the absence of costimulation by anti-mu antibodies (anti-mu Ab). The LMW-BCGF dependent proliferation of these malignant cells is inhibited by the anti-B8.7 monoclonal antibody (anti-B8.7 MoAb), in the same line as that of normal B cells. These results obtained with monoclonal B cells confirm that the B8.7 molecule is involved in the signalling pathway of the LMW-BCGF.     

Effects of preactivated autologous T lymphocytes on CD80, CD86 and CD95 expression by chronic lymphocytic leukemia B cells

Profound immune dysfunction is a constant feature in B-cell chronic lymphocytic leukemia (B-CLL) patients. Immunological abnormalities include hypogammaglobulinemia, impaired immunoglobulin class switching and diminished germinal center formation. This state of immune suppression renders B-CLL patients highly susceptible to infections, which contribute greatly to morbidity and mortality in this disease. Impaired T cell function in B-CLL is well-documented and has been suggested to result from inhibitory effects exerted by malignant B lymphocytes. Because the presence of leukemic cells may represent a major obstacle to efficient T cell activation, T lymphocytes were separated from CLL B cells, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4h, and then cocultured with autologous leukemic B cells both at a 1:1 ratio or at the same ratio as in vivo for 24-40 h. CLL B cell expression of CD86 and CD95 was markedly upregulated using this approach, whereas CD80 expression was augmented only in a minority of patients; these effects were partially preserved even when preactivated T cells were rechallenged with CLL B cells at the same low T/B cell ratio as that observed in vivo. Finally, CD80 upregulation on CLL B cells appeared to be mainly dependent on CD40L-mediated stimulation, whereas CD86 and CD95 expression was efficiently augmented by soluble factors released by preactivated T lymphocytes. In conclusion, efficient activation of T lymphocytes in B-CLL may be achieved which, in turn, may result in enhanced antigen-presenting capacity and susceptibility to apoptosis of leukemic cells via CD86 and CD95 upregulation, respectively.     

Variations in proteasome subunit composition and enzymatic activity in B-lymphoma lines and normal B cells

We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymatic activity of proteasomes purified from tumor-derived and normal B lymphocytes representing different stages of B-cell activation/differentiation. The catalytic beta subunits (Lmp2 and Lmp7) and the regulatory subunits (PA28alpha and PA28beta) were expressed at equally high levels in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells and normal CD23(-) B lymphocytes. Lmp2 and Lmp7 were selectively down-regulated in germinal center cell-derived Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) cell lines. There was a direct correlation between the expression of Lmp2/7 and the chymotrypsin and trypsin-like activities in proteasomes purified from LCLs, BLs and CLL cells, whereas 5 HD cell lines expressing B or T-cell markers exhibited a variable pattern of subunit expression and enzymatic activity. Poor hydrolysis of the fluorogenic substrates by proteasomes from BL cells correlated with a distinct pattern of cleavage of a reference 50mer peptide, production of different sets of degradation products and significantly reduced recovery of a known cytotoxic T-lymphocyte (CTL) target epitope. The enzymatic activity of proteasomes from normal CD23(-) "resting" B lymphocytes resembled that of BL cells in spite of high Lmp2/7 expression. This pattern was not reversed by treatment with the B-cell mitogen, lipopolysaccharide (LPS). The results suggest that different stages of B-cell activation/differentiation are associated with distinct profiles of IFN-gamma-regulated subunit composition and enzymatic activity of the proteasome. This may have important implications for the analysis and manipulation of tumor-specific immune responses.     

Differential expression of TRAP Isoenzyme in B-CLL Cells Treated with Different Inducers

Purified B-cells from normal tonsils and from the peripheral blood of eight patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with the protein kinase C (PKC) activators TPA, Bryostatin 1 (Bryo), mezerein, with the calcium ionophore A23187, and with the cytokines interleukin-1/2, interferon-alpha/gamma, tumor necrosis factor. The induction of the lysosomal enzyme tartrate-resistant acid phosphatase (TRAP) was examined at the RNA (by Northern blotting analysis) and the protein level (by isoelectric focusing). TRAP mRNA and protein were induced by treatment with PKC activators while the combination of PKC stimulator and calcium ionophore was not effective, TRAP mRNA was detected as early as 2 hours after initiation of the culture. This induction of positivity for TRAP which is the enzymatic hallmark of hairy cell leukemia (HCL) by TPA or Bryo is consistent with the previously reported acquisition of HCL-type cellular features in TPA-driven CLL cells; CLL cells exposed to the double stimulus of TPA or Bryo + A23187 have previously been described to differentiate to plasmacytoid cells which is in accord with their remaining TRAP negative. The present data provide evidence that 1) B-CLL cells can be induced by direct stimulation of PKC to convert to HCL-type cells; 2) TPA/Bryo-exposed B-CLL cells display phenotypes different from those of cells treated with combinations containing calcium ionophore; 3) the phosphoinositol signal transduction pathway distal to PKC can be activated effectively in B-CLL cells; 4) Bryo, a new natural PKC activator, induces responses in CLL similar to those caused by TPA; 5) TRAP is an inducible indicator of cellular activation. These observations provide support for the idea that HCL and HCL-like cells might differentiate along a separate branch of B-cell lineage and might represent mature “activation end stage” cells.     

CD5 is A potential selecting ligand for B-cell surface immunoglobulin: a possible role in maintenance and selective expansion of normal and malignant B cells

Although the function of CD5 on B cells is unknown, previous studies suggested that CD5 interaction with V(H) framework regions of surface immunoglobulins (Igs) may contribute to survival and expansion of B cells. Here we used B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from normal and B-CLL patients to study CD5-Ig interactions. Immobilized Ig binds and permits isolation of CD5 from lysates of CD5-expressing cell lines. Immunoglobulins or Fab fragments of different V(H) families varied in their effectiveness as inhibitors of anti-CD5 staining of CLL cells, appendix and tonsil tissue sections. Human Ig also binds to purified recombinant CD5. We show here for the first time that the unconventional Ig-CD5 interaction maps to the extracellular CD5-D2 domain whereas conventional epitopes recognized by anti-CD5 antibodies are localized in the D1 domain of CD5. We propose that interactions of VH framework regions with CD5 as a ligand may maintain, select or expand normal, autoimmune or transformed B cells and also contribute to skewing of the normal V(H) repertoire.     

Release of interleukin-2-like material by B-chronic lymphocytic leukemia cells. An autocrine or paracrine model of production and utilization?

In a series of untreated patients with B-cell chronic lymphocytic leukemia (B-CLL), the capacity of the neoplastic B-cell population to release an interleukin-2 like factor (IL-2lf) was assessed. While unstimulated purified leukemic B-cells showed no IL-2lf production, in 16 of the 27 cases tested (59.2%) significant amounts of IL-2lf (4.3-125 U) were released following activation with phytohemagglutinin (PHA) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In seven further cases (25.9%), small quantities of IL-2lf (0.2-1.7 U) were detected, while only in four (14.8%) no release was found. In 11 of the 20 cases (55%), PHA alone was also capable of inducing the production of limited amounts of IL-2lf (0.4-8 U). Only small amounts were released from B lymphocytes isolated from normal tonsils both with PHA and PHA plus TPA. Further purification using a fluorescence activated cell sorter suggests that the IL-2lf is truly produced by leukemic B cells and blocking experiments with the PC-61 monoclonal antibody indicate that IL-2lf and IL-2 use the same cell membrane receptor. However, co-cultures of leukemic B cells with small amounts of autologous or allogeneic T lymphocytes enhanced the amount of IL-2lf released into the supernatant to values markedly higher than those released by T- or B cells alone. Unlike normal B lymphocytes, unstimulated purified leukemic B cells from 17 out of 23 B-CLL cases (73.9%) were capable of absorbing variable amounts of exogenous IL-2. In addition, in six of the 11 cases tested (54.5%) IL-2 alone was capable of producing a 2-4 fold increase of thymidine uptake. In six out of eight cases (75%), a 2-5 fold enhancement of the proliferative response was observed when the leukemic B cells were co-stimulated with Staphylococcus aureus Cowan 1 (SAC) and IL-2. Moreover, when the cells were pre-activated with SAC or with PHA plus TPA and then further stimulated with IL-2, a 2-20 fold increase in proliferative response was found in the majority of cases studied. These findings indicate that elevated quantities of IL-2lf may be released in B-CLL particularly due to the B- and T cell interconnections, and that the leukemic B cells appear capable of absorbing IL-2 and of proliferating after costimulation with IL-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunophenotypic similarities of mediastinal clear-cell lymphoma and sinusoidal (monocytoid) B cells

Using 14 well-defined (clustered) monoclonal antibodies (MAbs) against B-cell restricted/associated differentiation and activation (CD) antigens and 12 mediastinal clear-cell lymphomas (MCCL), 46 follicular-center-cell lymphomas (FCCL), and 20 non-neoplastic lymph nodes--including toxoplasmic and HIV-associated lymphadenitis--were immunohistochemically examined to determine the histogenesis of MCCL. Antigenically, MCCL was characterized as CD5-, CD10-, CD19+, CD20+, CD21-, CD22+, CD30-, CD37+, CDw40+, and by a frequent expression of CD11c and CD23, while other antigens were inconsistently expressed. The antigenic profiles of MCCL and FCCL showed statistically significant differences in 4/14 distinct antigens. When the neoplastic cells of both tumor groups were compared with morphologically defined normal B-cell types, the overall resemblance of their immunophenotypes was even closer between MCCL and sinusoidal (monocytoid) B cells than between FCCL and follicular-center B cells. We conclude that MCCL is a lymphoma type distinct from FCCL, most probably representing a highly malignant neoplasm corresponding to sinusoidal B-cell reaction.     

Effects of recombinant interferon-alpha and -gamma on B-CLL cells in serum-free medium: expression of activation, differentiation, and CALLA antigens

Chronic B-lymphocytic leukemia (B-CLL) cells from 10 patients were cultured serum-free with recombinant interferon (rIFN)-alpha 2, rIFN-gamma, or phorbol ester (TPA) for 5 days. All three agents induced functional differentiation, as evidenced by IgM secretion, without concomitant proliferation. A panel of monoclonal antibodies was used to detect changes in cell surface antigens defining pre-B cells (CALLA), resting B cells (HH1), early (4F2, MHM6) and late (anti-Tac, OKT9) B cell activation, and terminally differentiated B cells (OKT10). The activation markers 4F2, MHM6, and anti-Tac and the plasma cell marker T10 were all significantly induced with TPA, rIFN-alpha 2, an rIFN-gamma, whereas the expression of HH1 decreased. CALLA was detected on substantial proportions of differentiated (4-38%) but not resting (0-4%) B-CLL cells. The CALLA-positive B-CLL cells were negative for nuclear terminal deoxynucleotidyl transferase (TdT). The T9 antigen was expressed on TPA-treated cells (1-16%) only. The present findings indicate novel properties of IFN-alpha and IFN-gamma in inducing terminal differentiation of human monoclonal B cells without prior activation.     

Cellular distribution of a B-cell specific surface antigen (gp54) detected by a monoclonal antibody (anti-BL4)

A monoclonal antibody (anti-BL4) recognizing a previously characterized Mr 54,000 glycoprotein (gp54) was developed by immunizing BALB/c mice with cells from a precursor B-cell line (Josh-7). In normal individuals, this antigenic molecule was present on tonsillar B-cells (60-80%) and on a fraction of peripheral blood B-cells (5-25%). BL4 (gp54) expression was investigated in 186 patients with a variety of hematological malignancies using indirect immunofluorescence and flow cytometric analysis. Twenty-six of 37 cases of B-cell chronic lymphocytic leukemia (CLL) and 18 of 33 cases of B-cell non-Hodgkin's lymphoma were BL4 positive. Surface expression of BL4 on reactive cases of CLL and non-Hodgkin's lymphoma was brighter than those of B1, B2, and B4, BL4 positive CLL cases expressed a higher proportion of mouse rosette forming cells and Leu-1 positive cells than the BL4 negative subgroup and were not associated with elevated serum immunoglobulin levels. Four of 7 BL4 negative CLL cases were associated with increased serum levels of immunoglobulin M. Lymphoblasts from 14 of 14 cases of non-T acute lymphoblastic leukemia and 3 of 3 pre-B lymphoid blast crisis of chronic myeloid leukemia were BL4 negative. Neoplastic cells from 2 of 3 cases of Waldenstrom's macroglobulinemia and 4 of 7 cases of hairy cell leukemia were BL4 reactive. None of 7 cases of multiple myeloma and plasma cell leukemia were BL4 positive. All 11 T acute lymphoblastic leukemia cases, 6 other T-cell malignancies, 5 cases of Hodgkin's disease, 51 cases of acute nonlymphocytic leukemia, and 9 cases of chronic myeloid leukemia in chronic phase thus far studied were BL4 negative. An in vitro induction experiment using phorbol ester on a case of B-CLL demonstrated disappearance of BL4 accompanied with further B-cell differentiation. Our study further substantiates the previous finding that gp54 is a differentiation antigen restricted to the B-cell lineage and expressed during the intermediate stage of B-cell ontogeny.     

Production of chemokinetic inhibitory factor (CIF) by normal blood and spleen B lymphocytes

We have recently reported the partial purification and characterization of of a new lymphokine, the heat-labile chemokinetic inhibitory factor (CIF) which inhibits neutrophil movement. We have also shown that this lymphokine is produced and secreted by cultured B-chronic lymphocytic leukaemia (CLL) cells in vitro. The present study shows that highly purified resting normal B lymphocytes from blood and spleen have the capacity to produce CIF spontaneously. After activation with anti-IgM or EBV-infection the lymphocytes produced a number of other factors, heat-stable chemokinetic inhibitory factors and heat-labile chemokinetic enhancing factors. Supernatants from a collection of human B-cell lines representing different stages of B-cell differentiation were also investigated. None of these cell lines produced CIF. The present results show that the production of CIF is not restricted to the malignant B-CLL cell but is also produced by a subset of normal blood and spleen B cells.     

JAK2 tyrosine kinase mediates integrin activation induced by CXCL12 in B-cell chronic lymphocytic leukemia

Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment.