Marie B. Snook

Redirecting Mouse CTL Against Colon Carcinoma: Superior Signaling Efficacy of Single-Chain Variable Domain Chimeras Containing TCR-ζ vs FcεRI-γ

The structurally related TCR-ζ and Fc receptor for IgE (FcεRI)-γ are critical signaling components of the TCR and FcεRI, respectively. Although chimeric Ab receptors containing ζ and γ signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the ζ and γ subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-ζ and scFv-γ receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-ζ chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-γ receptors. T cells expressing scFv-ζ chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the ζ signaling pathway.

Extracellular ATP causes loss of L-selectin from human lymphocytes via occupancy of P2Z purinoceptors

Lymphocytes from normal subjects or patients with chronic lymphocytic leukemia are known to possess receptors for extracellular ATP termed P2Z purinoceptors whose physiological role is undefined. Addition of extracellular ATP (50–500 μM) to both normal and leukemic lymphocytes caused loss of binding of monoclonal antibodies to L‐selectin (CD62L) on the cell surface. UTP, ADP, and adenosine (all at 500 μM) had no effect on L‐selectin expression. Several features of the ATP‐induced loss of L selectin indicate that this effect is mediated by lymphocyte P2Z purinoceptors. First the loss was attenuated in isotonic NaCl medium compared to 150 mM KCl medium. Second the loss of L‐selectin was immediately halted by addition of Mg2+ ions in molar excess of ATP. The most potent nucleotide causing L‐selectin loss was benzoylbenzoic ATP (>10 μM) which is also the most potent agonist for the P2Z purinoceptor. Finally preincubation of lymphocytes with oxidized ATP, an irreversible inhibitor of P2Z purinoceptors, also inhibited ATP induced loss of L‐selectin. Extracellular ATP is known to open an ion channel associated with the P2Z purinoceptor on B‐lymphocytes which allows influx of Ca2+. However, ATP‐induced loss of L‐selectin did not require extracellular Ca2+. Moreover addition of the calcium ionophore, ionomycin, had minimal effect on L‐selectin expression. Staurosporine (500 nM), an inhibitor of protein kinase C, inhibited only 10% of ATP induced loss of L‐selectin but completely inhibited the loss of L‐selectin caused by 50 nM PMA. Thus extracellular ATP interacts with lymphocyte P2Z purinoceptors which leads to shedding of L‐selectin via a pathway which requires neither Ca2+ influx nor activation of protein kinase C. ATP may have a physiological role in the loss of L‐selectin which occurs during the interactions of lymphocytes with other cells.

The P2Z‐purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP

1 Extracellular adenosine triphosphate (ATP) is known to open a receptor‐operated ion channel (P2Z class) in human lymphocytes which conducts a range of cationic permeants. The activity of a range of different agonists and inhibitors towards the P2Z‐purinoceptor was investigated by measuring the agonist‐induced influx of Ba2+ into fura‐2 loaded lymphocytes. 2 The most potent agonist was 2′ & 3′‐0‐(4‐benzoylbenzoyl)‐ATP (benzoylbenzoic ATP) which gave 2 fold greater maximum Ba2+ influx and had a 10 fold lower EC50 than for ATP. The rank order of agonist potency in K+‐media was benzoylbenzoic ATP>>ATP = 2‐methylthio ATP = 2‐chloro ATP>ATP‐γ‐S. ADP, UTP and α,β‐methylene ATP were unable to stimulate Ba2+ influx. 3 Extracellular Na+ inhibited the increment of Ba2+ influx induced by all concentrations of ATP, 2‐methylthio ATP, 2‐chloroATP and ATP‐γ‐S. This inhibitory effect of extracellular Na+ is also reflected in the different EC50s for benzoylbenzoic ATP (8 μm in K+‐media, 18 μm in Na+‐media) but the maximal response to this agonist was the same in the presence or absence of Na+. 4 Treatment of lymphocytes with 2,3 dialdehyde ATP (oxidized ATP) at 300 μm for 60 min gave total and irreversible inhibition of ATP‐induced Ba2+ influx. 5′‐p‐Fluorosulphonyl benzoyladenosine (FSBA) also was an irreversible inhibitor but the maximal inhibition achieved was 90%. 5 It is concluded that the P2Z‐purinoceptor of human lymphocytes has a rank order of agonist potency which clearly distinguishes it from other P2‐receptors and that oxidized ATP is a convenient irreversible inhibitor for the P2Z‐purinoceptor.

Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL.

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcε receptor I γ-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-γ following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-γ were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.

Saturation of intracellular cytosine arabinoside triphosphate accumulation in human leukemic blast cells

Accumulation of cytosine arabinoside triphosphate (araCTP) from a range of cytosine arabinoside (araC) concentrations (1-50 microM) was measured during incubations of leukemic cells freshly isolated from patients with acute leukemia. In all but one patient, increments in extracellular araC above 10 microM did not increase intracellular araCTP levels. This maximal level of araCTP accumulation ranged from 254 to 1607 pmol/10(7) cells attained after 1 h incubation and did not correlate with either the number of nucleoside transporters on the cell membrane or the Vmax of araC phosphorylation in cell free extracts. Extremely low araCTP accumulation (103 pmol/10(7) cells/h at 50 microM araC) was observed in an AML patient with the unusual finding of micromyeloblasts. These cells also had very low numbers of nucleoside transport sites (less than 500 sites/cell) and were mitotically inactive. The unique feature of the myeloblasts from this patient was that intracellular araCTP accumulation showed a linear dependence on extracellular araC up to 50 microM with no evidence of saturation.

Nucleoside transport in acute leukaemia and lymphoma: close relation to proliferative rate

Summary The proliferation of mammalian cells requires nucleosides which are provided either by de novo synthesis or by influx of nucleosides via membrane transporters with subsequent metabolic trapping. In this study the density of nucleoside transporters in freshly‐isolated blast cells from patients with leukaemias and lymphomas was quantitated by equilibrium binding of 3H‐nitrobenzylmercaptopurine riboside (NBMPR). In acute myeloid leukaemia (AML) the density of NBMPR binding sites on blast cells ranged from 3800 to 24 200 sites/cell and this value correlated with the 3H‐thymidine labelling index (1–20%) which was used to measure proliferative rate (r=0.80, P < 0.001). Cells from patients with Burkitts lymphoma, other B‐cell lymphomas, T‐lymphoblastic lymphoma and large cell lymphoma gave a 20‐fold range of NBMPR site densities (from 3700 to 75 300 sites/cell) and site numbers correlated closely with the labelling index (r=0.87, P < 0.001). Non‐proliferating cells from patients with chronic lymphocytic leukaemia expressed the lowest density of NBMPR binding sites (850–2900 sites/cell). Comparison of bone marrow and peripheral blood blasts confirmed the positive correlation between NBMPR binding sites and labelling index for four individual patients. In contrast, the density of NBMPR binding sites on lymphoblasts from non‐T acute lymphoblastic leukaemia (ALL) was low (2300–7400 sites/cell) and showed little dependence on proliferation over a wide range of labelling indices (1–20%). No correlation was observed between NBMPR site density and cell size measured by the intracellular water space. Thus an increased proliferative rate of AML or lymphoma is associated with higher numbers of nucleoside transporters in the cell membrane.

cDNA cloning of granzyme J.

Granule-mediated killing by cytotoxic lymphocytes requires the exocytosis of dense cytoplasmic granules (Tschopp and Jongeneel 1988). These granules contain the pore-forming protein perforin (Lowin et al. 1995), and a number of serine proteases termed granzymes (Hudig et al. 1993). The cDNAs for over ten different granzymes have been isolated and sequenced by several laboratories. Cytolytic granules contain at least five serine protease activities, but it has not been determined how many enzymes exist. Recently, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) technique to obtain cDNA clones for additional granzymes. We cloned and sequenced two new rat granzymes, RNKP-4 and RNKP-7(Ewoldt et al. 1997). Here we report the isolation of the cDNA of an additional new granzyme and term it granzymeJ (Gr J). The Gr J cDNA gene was obtained in three steps: 1) degenerate RT-PCR to obtain several gene-specific cDNA fragments, 2) 3 9 RACE with a gene-specific primer to obtain most of the cDNA clone, and 3) isolation of a fulllength ratGr J clone from a cDNA library to obtain the 5 9

SAENTA-fluoresceins: New Flow Cytometry Reagents for Assessing Transport of Nucleoside Drugs in Acute Leukemia

New strategies are needed to improve the outlook for patients with acute myeloid leukemia (AML). The use of granulocyte-macrophage colony stimulating factor (GM-CSF) given prior to and in combination with chemotherapy represents such a new approach and its safety and efficacy have been established [1, 2]. GM-CSF is a growth promoter of leukemic colony-forming cells in vitro [3, 4] and can render such cells more susceptible to the cytotoxic action of cytosine arabinoside (araC). When given in vivo to patients with AML, GMCSF usually increases the proliferative activity of myeloblasts and in a small study is associated with a high remission rate to subsequent chemotherapy incorporating araC.