Antileukemia activity of a natural killer cell line against human leukemias.

We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lymphoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG1, HL60, Raji, NALM6, TALL-104, CEM-S, and CEM-T) and normal bone marrow cells was measured in 51Cr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (< or =19% lysis), sensitive (20-49% lysis), and highly sensitive (> or =50% lysis) at an E:T ratio of 9:1. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-mediated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileukemia activity against a patient-derived T-ALL as well as K562 and HL60 cells, both in in vitro CRA and in a xenografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA31) that was insensitive to the in vitro cytolysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhIL2 (2 x 10(7) NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In contrast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control.

Growth pattern and clinical correlation of subcutaneously inoculated human primary acute leukemias in severe combined immunodeficiency mice.

We examined the ability of patient-derived human leukemic blasts to generate leukemic growth and dissemination in severe combined immunodeficiency (SCID) mice by subcutaneous inoculation without conditioning treatment or administration of growth-promoting cytokines. Additionally, we correlated the growth pattern with the clinical outcome of patients from whom the leukemic cells were derived. The leukemias displayed three distinct growth patterns, ie, either aggressive, indolent, or no tumor growth. Leukemic cells from 6 of 13 patients with acute myeloid leukemia (AML), 4 of 7 T-cell acute lymphoblastic leukemia (T-ALL), and 11 of 16 patients with B-lineage ALL grew as subcutaneous tumors, with a significant number subsequently disseminating into distant organs in SCID mice. Patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SCID mice had a relatively poor clinical outcome, whereas patients with AML and T- or B-lineage ALL whose leukemic blasts grew indolently or whose cells failed to induce growth had a more favorable clinical course. Our study has shown that the subcutaneous inoculation of patient-derived human leukemic cells in SCID mice can engraft and grow as subcutaneous tumors with subsequent dissemination to distant organs in a manner analogous to their pattern of growth in humans. Additionally, these data suggest a clinical correlation to the growth and dissemination of some leukemic subtypes that may represent not only an additional prognosticator for patient outcome, but also a vehicle for the study of the biologic behavior of human leukemias and the development of novel therapeutic strategies.

A novel HLA class II-independent TCR-mediated T cell activation mechanism is distinguished by the V beta specificity of the proliferating oligoclones and their capacity to generate interleukin-2.

Superantigens can induce proliferative T cell responses after complex formation with major histocompatibility complex (MHC) class II antigens. In this study, highly purified variant T cells from a histocompatibility leukocyte antigen (HLA) (human major histocompatibility complex) class II-deficient patient were stimulated with toxic shock syndrome toxin 1 (TSST-1) in the presence of either HLA class II-negative or normal antigen-presenting cells (APC). The proliferative responses were similar to those of normal T cells even when HLA class II structures were absent on both responding T cells and costimulatory APC. However, the V beta specificity of responding T cells differed depending on the presence or absence of HLA class II antigens on the APC. In the presence of HLA class II-negative costimulatory APC, TSST-1 induced primarily proliferation of V beta 2-expressing T cells, whereas in the presence of normal APC virtually all responding T cells expressed V beta elements different from V beta 2. In addition, in the presence of normal APC, T cells responding to TSST-1 produced much higher amounts of interleukin-2 than T cells proliferating in the presence of HLA class II-negative APC. These findings suggest that T cells bearing selected V beta elements can directly interact with and respond to superantigen without involvement of HLA class II structures. Thus, our findings introduce a novel and distinct T cell receptor-mediated activation mechanism by which specific subpopulations of T cells respond to superantigen in the absence of HLA class II structures on APC.

Selective treatment of SCID mice bearing methotrexate-transport-resistant human acute lymphoblastic leukemia tumors with trimetrexate and leucovorin protection.

Impaired transport of methotrexate (MTX) is a common resistance mechanism of tumor cells to this drug. Trimetrexate (TMTX), a second-generation folate antagonist, is still active against MTX-transport-resistant cells because it enters cells by passive diffusion and does not use the reduced folate transport system for cell entry. Therefore, although leucovorin (LV) protects MTX-sensitive cells from TMTX toxicity, MTX-transport defective cells are poorly rescued by LV. Severe combined immunodeficiency mice bearing MTX-transport-resistant CCRF-CEM acute lymphoblastic leukemia tumors were treated with TMTX alone or with the combination of TMTX and LV, with tumor regressions in both groups (P < .001) and without significant toxicity. These results indicate that TMTX with LV protection may be a useful therapeutic regimen for patients with MTX-transport-defective acute lymphoblastic leukemia. Furthermore, resistance to TMTX plus LV may result in reversion to MTX sensitivity.

ICAM-1 (CD54) expression on B lymphocytes is associated with their costimulatory function and can be increased by coactivation with IL-1 and IL-7.

Recent studies have demonstrated that acute lymphoblastic leukemia-derived pre-B cell lines are deficient in their costimulatory function for T cell proliferation in response to the mitogen Con A and the superantigens TSST-1 and SEB. Stimulation of these pre-B cells with IL-7 increased their costimulatory function which involved the B7/CD28 pathway. In the present study, we stimulated T cells with Con A, TSST-1, and SEB in the presence of peripheral blood B lineage cells that do not constitutively express B7/BB1 on their surface and investigated whether their costimulatory function could also be enhanced by IL-7. We found that, in the presence of IL-1, stimulation with IL-7 increased the costimulatory function of B cells and their surface expression level of ICAM-1 (CD54). We then investigated whether costimulatory B cell function could be inhibited by blocking the ICAM-1/LFA-1 pathway. Addition of anti-ICAM-1 mAb to the coculture of T and B cells inhibited T cell proliferation by approximately 20%. In contrast, addition of anti-LFA-1 beta (CD18) mAb, directed against the T cell ligand of ICAM-1, inhibited T cell proliferation almost completely. To determine the role of ICAM-1 in costimulatory B cell function, we sorted B cells into ICAM-1low-and ICAM-1high-expressing populations. We found that B cells expressing high levels of surface ICAM-1 elicited significantly higher T cell responses than those with low levels, suggesting that the expression level of ICAM-1 on peripheral blood B cells correlates with their costimulatory function.

IL-7 induces surface expression of B7/BB1 on pre-B cells and an associated increase in their costimulatory effects on T cell proliferation.

Treatment with IL-7 of NALM-6 pre-B cells, but not of EBV-LCL, Daudi, HeLa, or K562 cells resulted in enhanced costimulatory activity for TSST-1-induced T cell proliferation. The effect of IL-7 on the costimulatory function of NALM-6 cells was dose-dependent, could be inhibited by a neutralizing anti-IL-7 mAb, and resulted in the need of less costimulatory cells, or less superantigen, to obtain proper T cell proliferation. Addition of anti-IL-7 mAb to the coculture of superantigen-stimulated T cells with IL-7-pretreated NALM-6 cells did not alter the T cell responses obtained without addition of this antibody. These findings suggest that a possible costimulation of T cells by surface-bound IL-7 on pretreated NALM-6 cells did not contribute to the enhanced costimulatory function observed. Rather, these studies implicate IL-7-induced alterations of the NALM-6 cells themselves as the basis for the enhanced costimulatory activity observed. The most remarkable phenotypic differences between efficient costimulatory B lineage cells and deficient pre-B cells are a lower expression of ICAM-1 (CD54) and a lack of expression of the costimulatory B cell activation antigen B7/BB1. Upon activation with IL-7, the expression of ICAM-1 was increased, and the expression of B7/BB1 antigens was induced. The increased T cell responses in the presence of IL-7-treated NALM-6 cells could be inhibited by addition of anti-BB1 mAb. These results suggest that B7/BB1-negative pre-B cells may acquire the surface expression of B7/BB1 upon stimulation with IL-7, a process which is associated with increased costimulatory function for T cell proliferation.

Dyshematopoiesis in combined immune deficiency with congenital neutropenia.

This report describes a patient with combined immune deficiency associated with congenital neutropenia (CID/CN) and reports a partial characterization of his hematopoietic abnormalities. The CID/CN syndrome described is characterized by neutropenia and by deficiencies in B-lymphoid and T-lymphoid cell number and function. Red cell and platelet counts were normal. In vitro assays indicate that the myeloid lineage was developmentally arrested at the level of the committed monocyte/granulocyte progenitor (CFU-GM), while precursors to the CFU-GM progenitor were normal. In vitro studies showed that the defect in myeloid development was not corrected with G-CSF or GM-CSF. However, combinations of cytokines present in conditioned media from the T-cell lines MO or C5MJ, or defined multiple cytokine combinations containing IL-1, IL-3, GM-CSF, kit ligand, IL-6, and IL-9, restored myelopoiesis in-vitro. In contrast, C5MJ-conditioned media did not correct deficiencies in immune function in the patient's lymphocytes and accessory cells. No abnormalities in the production of G-CSF, GM-CSF, M-CSF, or IL-1 from the patient could be identified to account for the defects in myelopoiesis orimmune function.

Dissection of the function of HLA class II and costimulation in B cell-mediated and toxic shock syndrome toxin-1-induced T cell proliferation.

In this study we have demonstrated that costimulation for TSST-1-induced T cell proliferation is provided by HLA class II negative accessory B cells. In addition, TSST-1 binding to these HLA class II negative accessory B cells could not be detected by immunocytofluorescence, or in a system in which T cell proliferation depended on TSST-1 pretreated HLA class II negative B cells. These findings suggest that accessory signals can be supplied by B cells that are not involved in the activation of T cells via Ag-HLA/TCR-CD3 complex interaction: TCR ligation by a TSST-1/HLA complex and costimulation need not be delivered by the same cell. In the presence of HLA class II negative B cells, T cell proliferation in response to TSST-1 could be achieved despite addition of a mAb that can block HLA class II binding sites for TSST-1. These findings suggest the possibility that TSST-1 may directly ligate TCR. In the presence of anti-BB1-treated HLA class II negative accessory cells, TSST-1-induced T cell proliferation was inhibited by over 50% indicating the involvement of the BB1/CD28 pathway. The leukemic pre-B cell lines NALM-6, and BLIN-1, both of which express surface HLA class II Ag, but not the costimulatory B cell activation Ag BB1, were deficient in their accessory function for T cell proliferation in response to the superantigen TSST-1 and the mitogen Con A.

Anti-histone autoantibodies react specifically with the B cell surface.

In an attempt to induce an immune response against Mls-1a antigens by immunizing C57B1/6 mouse (Mls-1b) with purified B cells from DBA/2 mouse (Mls-1a), we generated a panel of monoclonal antibodies from which the 5B9.6 mAb, taken as a representative antibody, was thoroughly investigated. This antibody specifically reacts with B cells from all mouse strains studied including C57Bl/6 mice as shown by FACS analysis of double-antibody labelled spleen cells. Using enzyme immunoassays and immunoprecipitation techniques, 5B9.6 mAb was found to be specific for histones. Amino acid sequence analysis of a peptide derived from a 5B9.6-immunoprecipitated polypeptide from DBA/2 B cells showed a 100% homology with a sequence within H2B histones. Furthermore, 5B9.6 mAb was able to interact with the cell surface of 7OZ/3 cell line, known as a typical pre-B cell line. The presence of histones can be modulated on the surface of 7OZ/3 cells since this antigen was upregulated after exposure of these cells to a cocktail of IL-1 and cAMP. Finally, 5B9.6 mAb was shown to interact with freshly isolated B cells from human peripheral blood.

Interleukin-2 may enhance or inhibit antibody production by B cells depending on intracellular cAMP concentrations.

The primary IgM response of murine B lymphocytes against red blood cell-bound antigens can be induced by incubating antigen-reactive B cells either with the lymphokines interleukin-1 (IL-1) and IL-2 together with the nucleoside cAMP, or by the addition of antigen-specific helper T cells. The reactivity of B cells is strongly influenced by the T-cell lymphokine IL-2. IL-2 inhibits the cyclic adenosine 3',5'-phosphate (cAMP)-dependent B-cell response when it is allowed to act on the cells prior to cAMP. On the other hand, if IL-2 acts on B cells together with or after cAMP, it synergizes with the nucleoside and enhances the immune response. A similar effect of IL-2 is observed in the T-cell-mediated activation of B cells. If IL-2 is present before helper T cells interacted with B cells, it inhibits antibody production. The inhibitory IL-2 effect is reversed by the simultaneous addition of exogenous cAMP. The finding supports the hypothesis that Ia ligation by T cells results in B cells in the elevation of cAMP which acts as an important second messenger in B cells. The antagonism between cAMP and IL-2 was also examined in the pre-B-cell line 70Z/3. The nucleoside is highly toxic to 70Z/3 pre-B cells and a majority disintegrates within hours of exposure to the nucleoside. The surviving cells undergo phenotypic differentiation expressing surface Ig kappa chains and major histocompatibility complex (MHC) class II molecules, and increase the expression of IL-2 receptor (R). The phenotypic differentiation requires the presence of IL-1. IL-2 inhibits both of these B-cell responses to cAMP, the IL-1-independent cell death, and the IL-1-dependent phenotypic differentiation.

Recombinant human granulocyte-macrophage colony-stimulating factor stimulates superoxide anion and hydrogen peroxide production in human neutrophils.

The effect of purified recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the oxidative metabolism of human peripheral blood granulocytes was investigated. The respiratory burst of granulocytes was assessed in individual cells by flow cytometry utilizing the oxidation of the nonfluorescent 2',7'-dichlorofluorescein (DCFH) to the highly fluorescent DCF by hydrogen peroxide (H2O2). Treatment with GM-CSF caused granulocytes to produce H2O2 without addition of a second stimulus. The amount of H2O2 produced correlated with the concentration of GM-CSF administered. Also, GM-CSF did not prime the granulocytes for enhanced H2O2 production in response to N-formylmethionyl-leucyl-phenylalanine (f-MLP). Consecutive stimulation of granulocytes with GM-CSF and f-MLP resulted in additive production of H2O2. GM-CSF also induced granulocytes to release superoxide anion (O2-) in a dose-dependent manner, when the respiratory burst was assessed by a conventional cytochrome c reduction assay. In contrast to hydrogen superoxide production, GM-CSF significantly (p < 0.001) enhanced f-MLP-stimulated release of superoxide anion over that expected from the additive effects of the two agonists.

B cells control the aggregability of CD4 on T cells through continuous physical interactions.

It has previously been demonstrated that a gene on chromosome 1 in or near Mls-1 controls, on the surface of B cells, the mobility and aggregability of major histocompatibility complex (MHC) class II molecules but not the mobility or aggregability of other B-cell molecules, such as immunoglobulin (Ig) and class I antigens. We report here that this gene may also influence the aggregability of two class II antigen-reactive molecules on the surface of T cells, the T-cell receptor complex and CD4. The aggregability of the two membrane components is markedly higher on Mls-1+ T cells than on Mls-1- T cells. The properties of this phenomenon were examined in vitro as well as in vivo with particular emphasis on CD4 aggregability. It was found that, after removal of B cells, T cells lose the ability to aggregate CD4 in our standard CD4 aggregation assay. Similarly, T cells isolated from the B-cell-deficient environment of the thymus failed to aggregate CD4. Addition of B cells to either thymic T cells or B-cell-depleted peripheral T cells established CD4 aggregability within minutes. This process can be blocked with antibody against CD4 or antibody against Ia. The Mls-1 genotype predicts within the limited tests of this study the efficacy of the B-cell ability to impose a CD4 aggregation pattern on T cells: Mls-1+ B cells are markedly more effective in this respect than Mls-1- B cells. This can be demonstrated in tissue culture as well as in the animal. Similar to the Mls-1 response, this is a one-way process: Mls-1+ B cells confer to Mls-1- mice a CD4 aggregation pattern typical of the Mls-1+ mouse while Mls-1- B cells do not impose a Mls-1b-typical CD4 aggregation pattern in Mls-1a mice. Mls-1+ B cells also influence the composition of lymphocytes in the mouse. Mls-1+ mice or Mls-1- mice treated with Mls-1+ B cells have fewer T cells and more B cells in their spleen than Mls-1- animals. The gene that encodes stimulatory Mls-1 cell-surface structures has recently been identified as an endogenous mammary tumour virus (Mtv-7). We expect that the analysis of the virus genome will produce information whether the effects described here can be attributed to the virus or not.

Does interleukin-1 save B cells from paralysis? Studies with pre-B cells and antigen-reactive B cells.

The role of the second messenger cAMP in the differentiation of the pre-B cell line 70Z/3 and in the initial phase of the immune response of B cells, IgM antibody production is examined. Our data indicate that in these B cells, the elevation of intracellular cAMP levels can have two fundamentally different effects: it may dedifferentiate B cells and cause their disintegration (catabolic pathway) or it may induce their differentiation and maturation (anabolic pathway). The switch that determines which pathway B cells enter is set by interleukin-1. Our experiments indicate that intracellular cAMP levels rise in B cells when they interact with helper T cells in a cognate interaction. Given the dual effect that cAMP may have on the fate of B cells, activation or destruction, our data may be taken to suggest that helper T cells may direct B cells into catabolic or anabolic responses, depending on the availability of a costimulatory signal mediated by interleukin-1.

Immunogenic peptides require an undisturbed phospholipid cell membrane environment and must be amphipathic to immobilize Ia on B cells.

Ia-reactive immunogenic peptides have been shown to immobilize Ia molecules on the B cell surface and to facilitate their aggregation with specific alloantibody. We show that to immobilize Ia the peptide must be amphipathic. Polar peptides appear to bind to Ia molecules as judged by competitive inhibition, but do not immobilize the MHC molecule. This suggests the possibility that peptides establish the immobilizing membrane contact via a lipophilic group. Examining the B cell membrane lipid environment, we found that treatment of B cells with phospholipase C prevents peptide-mediated immobilization of Ia. The requirement of a lipophilic peptide portion as well as of phospholipase-sensitive membrane components for effective peptide-mediated Ia aggregation on B cell membranes suggests a role for membrane phospholipids in this process. We advance the speculation that immunodominant amphipathic peptides immobilize Ia molecules by attaching them to cell surface phospholipids which we tentatively refer to as immobilizing phospholipids.

Mls and the helper T-cell repertoire: II. Does the Mls gene product influence the formation of the T-cell repertoire? Studies with Mls tolerance.

In the formation of a repertoire, T cells are selected through a window of autoreactivity which is defined by a low boundary representing the minimal autoreactivity required to enter the T-cell compartment and a high boundary which determines the highest admissible autoreactivity. We propose that the Mls gene product down regulates autoreactivity and thus modifies the formation of the T-cell repertoire. Given the polymorphism of Mls and the assumption that Mlsb is more inhibitory than Mlsd, it is conceivable that Mlsb (responder) mice admit into their T-cell compartment T cells with higher autoreactivity than Mlsd mice. We suggest that it is this highly autoreactive fraction of Mlsb T cells which responds in the overt Mls response. We show that Mls tolerance eliminates T cells responding in the overt Mls response but not T cells responding in the latent Mls response. This is consistent with the finding that T cells responding in the latent Mls response occur in Mlsb as well as in Mlsd mice.

Chemoprophylaxis of bacterial infections in granulocytopenic patients with ciprofloxacin.

The trial was conducted to evaluate the antimicrobial prophylactic efficacy of ciprofloxacin in reducing the frequency of infections in granulocytopenic patients. The frequency of infections was evaluated in 34 patients with acute non-lymphoblastic leukemia, acute lymphoblastic leukemia, blast crisis of chronic myelogenous leukemia and other malignancies. 46 courses of oral prophylactic treatment with 500 mg ciprofloxacin twice daily were administered. While there was no infection in 61% of treatment courses, fever over 38 degrees C (axillary) occurred in 39%. 6 patients had a fungal pulmonary infection, one patient a supposed viral pneumonia, and only two patients had a documented bacterial infection. There were no severe side effects. We conclude that ciprofloxacin is a potent drug in prophylaxis of bacterial infections in cancer patients with therapy-induced granulocytopenia.