Proliferation of precursor B-lineage acute lymphoblastic leukaemia by activating the CD40 antigen

No reliable culture system exists for B-lineage acute lymphoblastic leukaemia (ALL). Recently we found that many different mature B-cell malignancies proliferate upon stimulation via the CD40 antigen, and this led us to investigate whether a similar CD40 activation on ALL cells could also induce proliferation. First, we measured CD40 expression in 21 ALL cases; all were CD40⁺, although mostly weak. Next, we triggered the CD40 antigen by anti-CD40 antibodies and by a CD40 ligand-expressing cell line. In addition, we measured the influence of IL-3, IL-4 and IL-7 with and without these stimuli.   In 8/10 cases proliferation, measured by ³H-thymidine incorporation, could be induced after CD40 crosslinking, especially in the presence of IL-3. Stimulation via the CD40 ligand was more successful than using crosslinked anti-CD40 antibodies. IL-4 inhibited the spontaneous proliferation found in three cases, but stimulated proliferation after CD40 crosslinking. IL-7 did not contribute to proliferation. Morphology, immunophenotyping and surface marker analysis, combined with DNA flow cytometry confirmed that the proliferation found could be ascribed to the ALL cells.   In conclusion, B-lineage ALL cases are CD40⁺, and many can be cultured using CD40 stimulation and IL-3.     

Responsiveness of chronic lymphocytic leukemia B cells activated via surface Igs or CD40 to B-cell tropic factors.

Recent studies performed in the laboratory have established that interleukin-4 (IL-4) used in combination with anti-CD40 monoclonal antibody (MoAb) 89 presented on Ltk- mouse fibroblasts stably expressing human Fc gamma RII/CDw32 (referred to as the CD40 system) sustains long-term proliferation of normal human B cells. In the present study, B-cell chronic lymphocytic leukemias (B-CLLs) activated through slgs or CD40 were examined for their capacity to proliferate and differentiate in response to various cytokines. Our results indicate that the outcome of IL-4 stimulation on the in vitro growth of B-CLL depends on the signalling pathway used for their activation. Whereas IL-4 did not display any growth-stimulatory effect on B-CLL activated by Ig cross-linking agents, it could stimulate DNA synthesis and enhance the viable cell recovery when leukemic B cells were cultured in the CD40 system. Most B-CLL samples were induced for IgM synthesis upon Staphylococcus aureus strain Cowan I stimulation. This Ig response was potentiated by IL-2 and antagonized by IL-4. Anti-CD40 MoAb used alone or in combination with cytokines (IL-1 alpha to IL-6, interferon gamma, tumor necrosis factor gamma, and transforming growth factor beta) failed to induce Ig secretion from B-CLL cells. No evidence for Ig isotype switching was obtained with the cytokines listed above, regardless of the mode of activation. Taken together, our results suggest that B-CLL cells can be partially released from their apparent maturation block by IL-2 and Ig cross-linking agents. In contrast, combinations of IL-4 and cross-linked anti-CD40 antibodies induced entry of B-CLL cell into cycle, but poorly stimulated their differentiation into Ig secreting cells.     

T-cell malignancies with mature phenotypes: altered cell cycle regulation by HLA class I molecules.

Soluble anti-HLA class I monoclonal antibodies (MoAbs) modulate normal T-lymphocyte proliferation induced via the CD3/Ti and the CD2 pathway, but do not induce proliferation of normal T lymphocytes in the absence of additional mitogenic stimuli. In this report, we show that anti-HLA class I MoAbs induce DNA synthesis in peripheral blood mononuclear cells from a patient with a CD4+CD8+T-prolymphocytic leukemia (T-PLL) and from a patient with a CD4-CD8+ T-chronic lymphocytic leukemia (T-CLL), in the absence of detectable additional mitogenic stimuli. Proliferation of leukemic T cells is induced by both whole Igs and Fab' fragments of anti-HLA class I MoAbs, arguing in favor of their direct interactions with the proliferating cells as the mechanism underlying the mitogenic effect. This interpretation is also supported by the ability of anti-HLA class I MoAbs to induce proliferation of leukemic T-cell preparations, depleted of accessory cells. DNA synthesis in T-CLL and T-PLL cells is preceded by expression of G1-specific messenger RNAs, ie. c-myc, 2F1, Tac, and interferon-gamma, in activated cells. Cell proliferation is inhibited by the protein kinase C inhibitor H7, indicating that activation of this enzyme is required for the mitogenic effect of anti-HLA class I MoAbs. The latter inhibit the proliferation of T-CLL cells as well as that of normal T cells stimulated with anti-CD3 MoAbs and enhance that of both types of cells stimulated with anti-CD2 MoAbs. In addition, anti-HLA class I MoAb Q6/64 in combination with anti-CD2 MoAb 9.6 or MoAb 9-1 induces proliferation of leukemic T cells to a greater extent than the individual MoAbs, but is not mitogenic for normal T cells. Anti-HLA class I MoAbs restore the cytolytic activity of T-CLL cells that is lost after 5 days of incubation of control medium, suggesting that HLA class I antigens may mediate a signal contributing to the activation state. The present results indicate that leukemic T-cell proliferation can be triggered via HLA class I molecules and suggest a potential role for these antigens in the in vivo growth of malignant clones.     

Activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes.

The activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes (LGL) were studied in vitro. Anti-CD3 monoclonal antibody (MoAb) alone (P less than .01) and recombinant interleukin-2 (IL-2) alone (P less than .01) caused significant stimulation of peripheral blood mononuclear cells (PBMC) from four CD3+ LGL leukemia patients, as measured in a 3H-thymidine incorporation assay. Recombinant interleukin-4 (IL-4) alone had no effect (P = .11). The combination signals of anti-CD3 MoAb and either IL-2 or IL-4 produced a proliferative response greater than anti-CD3 MoAb alone (P less than .01) or lymphokine alone (P less than .01). Leukemic LGL, purified by two-color sorting, were subsequently activated by anti-CD3 MoAb and IL-2 and assessed for DNA content by viable Hoechst No. 33342 (HO) staining. Results of these studies demonstrated that leukemic LGL were stimulated directly by anti-CD3 MoAb and IL-2, with the percentage of cells in cell cycle (S + G2/M) ranging from 16% to 72%. Normal CD3+ LGL were also stimulated to enter the cell cycle by anti-CD3 and IL-2. These results show that leukemic LGL proliferate in vitro after activation through the T-cell receptor and/or lymphokine.     

Activation via the CD3 and CD16 pathway mediates interleukin-2-dependent autocrine proliferation of granular lymphocytes in patients with granular lymphocyte proliferative disorders.

Granular lymphocytes (GLs) in patients with GL-proliferative disorders (GLPDs) are known to express the interleukin-2 receptor (IL-2R) beta chain (p70-75) constitutively and to proliferate in response to stimulation with IL-2 via the beta chain. In this report, we found that the anti-CD3 monoclonal antibody (MoAb) OKT3 could induce the proliferation of GLs from patients with T-cell lineage GLPDs (T-cell receptor-alpha beta+/CD3+16+), but not that of natural killer (NK) cell lineage GLs (T-cell receptor-alpha beta-/CD3-16+). In contrast, the anti-CD16 MoAb 3G8 that reacts with NK-lineage GLs could induce the proliferation of these GLs but not that of GLs with a T-cell phenotype. Furthermore, the anti-CD16 MoAbs CLB FcR gran1 (VD2) and OK-NK, which react with both T- and NK-lineage GLs, induced the proliferation of GLs with both T- and and NK-cell phenotypes. The proliferative response induced via the CD3 or IgG Fc receptor III (Fc gamma RIII: CD16) pathway was shown to be associated with the IL-2-dependent autocrine pathway by various findings, including the induction of endogenous IL-2 production, the coexpression of the IL-2R alpha chain (p55) and the IL-2R beta chain, and the inhibition of GL proliferation by anti-IL-2 or anti-IL-2R MoAb. These results suggest that GL proliferation is mediated at least partly through the IL-2-dependent autocrine pathway, and that the TCR/CD3 complex in T-cell phenotype GLs and the Fc gamma RIII in both T- and NK-cell phenotype GLs play a role in their activation in GLPDs.     

CD44 (Pgp-1) inhibits CD3 and dexamethasone-induced apoptosis

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones (GCH) induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, indicating that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether signals activated by adhesion receptors have a similar activity, we analyzed the effect of CD44 (Pgp-1) adhesion molecule receptor stimulation on T-cell apoptosis induced by three stimuli (anti-CD3 MoAbs, dexamethasone [DEX] treatment, and exposure to ultraviolet irradiation [UV]) on a 3DO T-cell line. The results show that CD44 engagement, either by hyaluronic acid (HA) or anti-CD44 MoAbs, inhibits DNA fragmentation and apoptosis induced by DEX and anti-CD3 MoAbs, whereas that induced by UV, a p53-dependent phenomenon, was not inhibited. Furthermore, the antiapoptotic effect exerted through CD44 activation does not seem related to overexpression of bcl-2 or to have appreciable effects on cell proliferation. Our results indicate that adhesion molecules modulate T-cell survival by counteracting apoptosis induced by DEX or anti-CD3 MoAbs.     

Detailed studies on expression and function of CD19 surface determinant by using B43 monoclonal antibody and the clinical potential of anti-CD19 immunotoxins

Extensive immunologic surface marker analyses and binding competition assays demonstrated that B43 monoclonal antibody (MoAb) is a new member of the CD19 cluster that recognizes the same surface epitope as several other anti-CD19 MoAbs. We used B43 MoAb to test for CD19 expression on neoplastic cells from 340 leukemia and 151 malignant lymphoma patients and on nonneoplastic cells in normal lymphohematopoietic and nonlymphohematopoietic tissues. Our study more than doubles the total number of cases with classified hematologic malignancies that have been examined for CD19 antigen expression. The data presented confirm that CD19 is the most reliable B lineage surface marker and support our view that this B lineage-restricted surface determinant may be an important functional receptor. Our findings provide unique and direct evidence that (a) CD19 is expressed on leukemic B lineage lymphoid progenitor cells freshly obtained from B lineage acute lymphoblastic leukemia patients but not on normal myeloid, erythroid, megakaryocytic, or multilineage bone marrow progenitor cells; (b) ligation of CD19 with B43 MoAb induces sustained increases in [Ca2+]i when crosslinked and inhibits high-molecular weight B cell growth factor (HMW-BCGF)-induced proliferation of activated B cells without affecting their low-molecular weight B cell growth factor (LMW-BCGF) response; therefore CD19 may be a unique signal receptor; (c) HMW-BCGF and LMW-BCGF augment expression of CD19, which suggests that CD19 and BCGF receptors may be under coordinate regulatory control; (d) approximately two million B43 MoAb molecules per cell can be bound to target B lineage lymphoma cells with a Ka of 1.9 x 10(8)/mol/L; (e) CD19 can undergo B43 MoAb-induced internalization; and (f) the opportunity is thus provided for using anti-CD19 MoAb to deliver toxins to B lineage neoplastic cells for more effective treatment of high-risk leukemia/lymphoma patients.     

Proliferative pathways in CD1- CD3+ CD4+ CD8+ T-prolymphocytic leukemic cells: analysis with monoclonal antibodies and cytokines

The antigenic profile and the proliferative pathways in leukemic cells from the patient TRT with T-prolymphocytic leukemia (T-PLL) were analyzed using monoclonal antibodies (MoAbs) and cytokines. T-PLL cells expressed the phenotype CD1- CD3+ CD4+ CD8+. Incubation with the differentiating agent phorbol-12-myristate-13-acetate markedly increased the percentage of cells with the CD4- CD8+ phenotype, suggesting that leukemic cells were already committed towards a differentiated element with the CD4- CD8+ phenotype. T-PLL cells were induced to proliferate by anti-CD2 MoAb 9-1 + 9.6 and by anti-CD3 MoAb OKT3. The two pathways exhibited normal functional interactions and were susceptible to modulation by anti-HLA class I MoAbs. These results indicate that regulation of cell proliferation was preserved to a significant extent in the T-PLL cells analyzed. At variance with normal resting T cells that require previous activation to proliferate when incubated with interleukin-1 (IL-1) or interleukin-2 (IL-2), T-PLL cells proliferated vigorously when incubated with either interleukin. Furthermore, T-PLL cells proliferated when incubated with immune interferon (IFN-gamma). The latter finding parallels the enhancement by IFN-gamma of the proliferative response of lectin-activated murine T lymphocytes. These results suggest that T-PLL cells, which express a high constitutive level of c-myc mRNA, may be in an activated state. The antigenic phenotype and the characteristics of the proliferative pathways of T-PLL cells from the patient TRT are compatible with the possibility that they may be derived from an intermediate thymocyte.     

Prognostic significance of karyotypic abnormalities in B cell chronic lymphocytic leukemia: an update

Cytogenetic analyses by G-banding and/or Q-banding techniques of leukemic B cells were performed in 102 patients with chronic lymphocytic leukemia (CLL), including six with prolymphocytic leukemia (PLL), one with hairy cell leukemia (HCL), and one with Waldenstrom's macroglobulinemia (WM) from 1979 through 1983. Follow-up after cytogenetic study ranged from 24 to 70 months. Seventeen patients had stage 0, 10 had stage I, 31 had stage II, and 44 had stage III or IV. Adequate metaphases were obtained for karyotypic analysis in 86 (84%) of 102 patients. Of these 86 patients with adequate metaphases, 43 had normal karyotypes (50%) and 43 had abnormal karyotypes (50%), of which trisomy 12 was the most frequent. Ten patients had trisomy 12 as the sole abnormality, 14 had trisomy 12 in combination with other abnormalities, and the remaining 19 had other abnormalities without trisomy 12. Abnormal karyotypes were more frequently associated with patients with advanced stages than those with early stages of the disease. Response rate to chemotherapy was significantly higher in patients with normal karyotypes than in those with abnormal karyotypes. Of eight patients who subsequently developed Richter's syndrome, seven initially had complex karyotypic changes with or without trisomy 12. These observations suggest that the chances of development of Richter's syndrome in CLL patients with multiple chromosome changes may be much higher than in those with either simple trisomy 12 or a normal karyotype. Mean frequency of abnormal metaphases was significantly higher in patients with complex trisomy 12 in combination with other changes than in those with trisomy 12 as the sole abnormality.(ABSTRACT TRUNCATED AT 250 WORDS)     

Successful treatment of hairy cell leukemia with pentostatin

A 45-year-old woman was admitted to our hospital in August, 1999. Laboratory data showed a white blood cell count of 5,050/microliter with 78% abnormal lymphocytes, hemoglobin 6.8 g/dl, platelets 4.8 x 10(4)/microliter, and soluble IL-2 receptor 97,600/ml. The abnormal cells were characterized by a hairy appearance under phase contrast microscopy, and showed strong tartrate-resistant acid phosphatase activity. Immunophenotype analysis revealed that these cells were positive for CD11c, CD19 and CD25, and negative for CD5. Bone marrow biopsy showed diffuse proliferation of hairy cells with moderate myelofibrosis. We diagnosed the patient as having European-American-type hairy cell leukemia. Pentostatin was administered at a dose of 5 mg/m2 weekly. After twelve doses, the peripheral blood data returned to the normal range with no hairy cells in the blood or bone marrow, although slight splenomegaly remained. The patient underwent splenectomy in December of the same year, and we were unable to find any hairy cells by histological and immunohistochemical examination. Although most patients with hairy cell leukemia in Japan have the Japanese variant, and the European-American type is rare, pentostatin is as effective as it is for European and American patients.     

Demonstration of high-affinity interleukin-2 receptors on B-chronic lymphocytic leukemia cells: Functional and structural characterization

Summary Functional and structural characteristics of interleukin 2 (IL-2) receptors on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed by a proliferation assay, IL-2 binding assay and cross-linking study. In the³H-thymidine incorporation assay, purified B-CLL cells from four out of sixteen cases, in which the percentage of Tac antigen (Tac Ag) positive cells in peripheral blood lymphocytes ranged from 0 to 48.8%, responded to IL-2 (100 U/ml) after both 3- and 6-day incubation. No relationship was found between the responsiveness to IL-2 and the percentage of Tac Ag positive cells. In the radiolabeled IL-2 binding assay, however, B-CLL cells from all seven cases examined, including three cases with mitogenic response to IL-2 and four cases without mitogenic response, were shown to have both high- and low-affinity receptors. The number of high- and low-affinity receptors per cell ranged from 29–186 and from 420 to 1,800, respectively. Furthermore, with the affinity cross-linking method p55 (Tac Ag) and p70/75 were found even in cases without mitogenic response in their B-CLL cells. In conclusion, the B-CLL cells so far examined possessed high-affinity IL-2 receptors consisting of p55 and p70/75; nevertheless, this was not sufficient to respond to the mitogenic signal of IL-2.     

Hairy cell leukemia: B-lymphocyte and phagocytic properties.

The diagnosis of hairy cell leukemia was made in three patients by phase-contrast microscopy and histochemistry of the abnormal peripheral blood cells. Both IgM and IgD surface immunoglobulins were resynthesized after these cells were trypsinized and cultured. Aggregate or Fc receptors were demonstrated on hairy cells. The ability to phagocytose latex was also a property of hairy cells; however, these cells did not demonstrate nonspecific esterase activity. Stimulation by phytohemagglutinin resulted in very low incorporation of tritiated thymidine. Cytofluorographic analysis of the phytohemagglutinin-stimulated cell population revealed less than 9% of the cells in an interploid or tetraploid state. The abnormal mitogen response was largely restored when purified T lymphocytes obtained from the peripheral blood of the patients were cultured with phytohemagglutinin. Hairy cells cultured with normal allogeneic mononuclear cells did not undergo blast transformation. These data strongly suggest that the cells of at least some patients with hairy cell leukemia are B lymphocytes with phagocytic capabilities.     

Downregulation of the CD95 receptor and defect CD40-mediated signal transduction in B-chronic lymphocytic leukemia cells

Cross-linking of the CD40 receptors has been shown to induce protein tyrosine kinases (PTK) phosphorylation and prevent apoptosis in Bcl-2 negative germinal center B cells. The expression of CD40 on B chronic lymphocytic leukemia (B-CLL) cells was found to be similar to that of normal B cells. Activation of normal B cells with soluble anti-CD40 monoclonal antibody (mAb) induced tyrosine phosphorylation, prolonged survival and prevented apoptosis. However, activation of CD40 on B-CLL cells using soluble anti-CD40 mAb does not influence survival or apoptosis. Normal B cells entered apoptosis when cultured in the presence of soluble anti-CD95 mAb. This process was independent of PTK activity. On B-CLL cells, the CD95 molecules were downregulated and a transient PTK signal was observed when cross-linking of the receptor by soluble anti-CD95 mAb occurred. Interestingly, B-CLL cells did not enter apoptosis in the presence of anti-CD95 mAb. Our study indicates that survival signals mediated through the CD40 molecule and death signals mediated through the CD95 molecule used different intracellular pathways in control donor B cells. In contrast, B-CLL cells do not respond to these signals. The leukemic B cells showed a defective CD40-mediated signal transduction and downregulated CD95 receptor expression. As a consequence, no apoptosis could be induced in B-CLL cells by a soluble anti-CD95 mAb. The abnormalities of these receptors may contribute to the long-lived status of B-CLL cells.     

Cell lineage assignment of cytogenetic findings in acute lymphoblastic leukemia using combined immunomagnetic cell separation and chromosome preparation

In acute lymphoblastic leukemia (ALL) abnormal karyotypes frequently constitute a minor part of the dividing cells, and the origin of metaphases in normal diploid cases remains obscure. We used a combination of immunomagnetic cell separation and chromosome preparation (ICSCP) to focus on the metaphases of interest and to assign the chromosome findings to CD19+ or CD7+ leukemia cells.     

Establishment of a karyotypically normal cytotoxic leukemic T-cell line from a T-ALL sample engrafted in SCID mice

Bone marrow (BM) cells from a child with an immature (CD3-) acute T lymphoblastic leukemia (T-ALL) bearing no chromosomal abnormalities failed to grow in long-term culture in the presence or absence of recombinant human (rh) growth factors but could be engrafted in severe combined immunodeficient (SCID) mice and induced leukemia. The leukemic cells recovered from the animal tissues could be adapted to grow in vitro in the presence of rh interleukin-2 (IL-2) and give rise to a growth factor-dependent cell line designated TALL-107. This cell line expresses T-cell-specific mature markers (CD2, CD3/T-cell receptor [TCR] alpha beta, CD8, CD56), and its growth can be inhibited by IL-4 of all the cytokines tested. Similar to the original leukemic blasts, TALL-107 cells are clonal, have rearranged TCR-beta, gamma, and delta loci, and a normal 46 XY karyotype. However, unlike the patient's BM cells, the TALL-107 cell line displays potent tumoricidal activity that is not major histocompatibility complex restricted. The magnitude of mRNA expression of perforin and serine esterases and of lytic activity depends on the doses of IL-2 added. TALL-107 cells can also be triggered by CD3- and CD2-specific monoclonal antibodies (MoAbs) to mediate reverse tumor cell lysis. In addition, this cell line produces high levels of interferon gamma and tumor necrosis factor alpha on stimulation with anti-CD3 and/or anti-CD2 MoAb both in the presence or absence of IL-2. The overall data indicate that the SCID mouse is able to support the functional maturation and expansion of a cytotoxic T- cell subset from some types of T-ALL.     

Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell- induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3- mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP- sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be- identified target cell surface molecules.     

Hairy cell leukemia is characterized by clonal chromosome abnormalities clustered to specific regions

Cytogenetic analysis was performed on B-cell mitogen-stimulated cells from 36 patients with symptomatic hairy cell leukemia. Evaluable metaphases were achieved from 30 patients, and (67%) showed clonal abnormalities. Recurrent chromosomal aberrations involving chromosomes 1, 2, 5, 6, 11, 19, and 20 were found. The abnormalities were mostly deletions and inversions, whereas translocations and numerical abnormalities, except trisomy 5, were rare. Fourteen patients showed multiple clones, which mostly were unrelated and found in different combinations in individual cells. Cells with non-clonal abnormalities identical to those found in clonal changes in other patients were common. Chromosome 5 was involved in clonal aberrations in 12 of 30 (40%) patients, most commonly as trisomy 5 (n = 4), or pericentric inversions (n = 6) and interstitial deletions (n = 4) involving band 5q13. Three patients showed two and 1 patient three different clones that involved chromosome 5. In addition, 1 patient had a rare constitutional inversion of chromosome 5 with breakpoints at p13.1 and q13.3. Pericentric inversions and interstitial deletions of chromosome 2 occurred clonally in 4 patients (13%) and in single cells of another 6 patients. Deletions of chromosome 1 at band q42 was found in 5 patients, and 1 patient had a translocation between 1q42 and a supernumerary chromosome 5. Deletions of 6q and 11q were similar to those commonly found in other lymphoproliferative disorders. Trisomy 5, structural abnormalities involving the pericentromeric regions of chromosomes 5 and 2, and 1q42 abnormalities were findings distinguishing the karyotypes in hairy cell leukemia from those of other hematologic malignancies.     

Response of human platelets to activating monoclonal antibodies: importance of Fc gamma RII (CD32) phenotype and level of expression.

Certain monoclonal antibodies (MoAbs) specific for platelet membrane glycoproteins are known to be capable of activating platelets, and it is generally thought that platelets from normal subjects are equally susceptible to stimulation by such MoAbs. We found that platelets from 20 normal donors varied significantly in their sensitivity to three IgG1 murine MoAbs specific for membrane glycoproteins CD9, GPIV (CD36), and the GPIIb/IIIa complex (CD41), respectively. The response of platelets to these MoAbs was blocked by prior addition of MoAb IV.3 specific for the Fc gamma RII receptor, indicating that activation was Fc receptor mediated. Platelets that responded poorly to these MoAbs failed to bind the MoAb 41H.16, specific for the "responder" form of Fc gamma RII, but platelets that responded well reacted with this MoAb. The average number of Fc gamma RII receptors on platelets from "responders" and "non-responders" was approximately the same. However, the number of Fc gamma RII receptors expressed influenced sensitivity of a subgroup of "responder" platelets to the anti-CD41 MoAb. These platelets were judged on the basis of MoAb binding studies to be heterozygous for the two alleles of Fc gamma RIIA. In contrast to their varying sensitivity to IgG1 MoAbs, members of the platelet panel responded equally well to 50H.19, an IgG2a MoAb specific for CD9, and these responses could not be blocked by MoAb IV.3 in the presence of plasma. This appears to be because of dual actions of 50H.19 on platelets: one FcR-dependent and the other complement-dependent. Our findings confirm previous reports that certain IgG1 MoAbs activate platelets through binding of their Fc domains to Fc gamma RII receptors and demonstrate that this response is influenced both by Fc gamma RII phenotype and (in the case of the anti-CD41 MoAb) by the number of Fc gamma RII receptors expressed. The failure of nonresponding platelets to bind detectable amounts of MoAb 41H.16, which is thought to recognize all Fc gamma RII receptors except for one allele of the Fc gamma RIIA gene, is consistent with the possibility that Fc gamma RIIA gene products, but not Fc gamma RIIB or Fc gamma RIIC gene products, are expressed on platelets.     

C5a-mediated neutrophil dysfunction is RhoA-dependent and predicts infection in critically ill patients

Sensitivity of chronic lymphocytic leukemia (CLL) cells to anti-CD20 mAbs is low and, therefore, the efficacy of monotherapy with current anti-CD20 mAbs is limited. At present, it is not known whether sensitivity of CLL cells to CD20 mAbs is modulated by microenvironmental stimuli. We have shown previously that in vitro CD40 stimulation of peripheral blood-derived CLL cells results in resistance to cytotoxic drugs. In the present study, we show that, in contrast, CD40 stimulation sensitizes CLL cells to the recently described novel type II anti-CD20 mAb GA101. Cell death occurred without cross-linking of GA101 and involved a lysosome-dependent mechanism. Combining GA101 with various cytotoxic drugs resulted in additive cell death, not only in CD40-stimulated CLL cells, but also in p53-dysfunctional CLL cells. Our findings indicate that GA101 has efficacy against chemoresistant CLL, and provide a rationale for combining cytotoxic drugs with anti-CD20 mAbs.