Gary P. Jamieson

Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein.

Mucin 1 (MUC1) is a large complex glycoprotein that is highly expressed in breast cancer, and as such could be a target for immunotherapy. In mice, human MUC1 is highly immunogenic, particularly when conjugated to mannan, where a high frequency of CD8(+) MHC-restricted cytotoxic T lymphocytes is induced, accompanied by tumor protection. On this basis, a clinical trial was performed in which 25 patients with advanced metastatic carcinoma of breast, colon, stomach, or rectum received mannan-MUC1 in increasing doses. After 4 to 8 injections, large amounts of IgG1 anti-MUC1 antibodies were produced in 13 out of 25 patients (with antibody titers by ELISA of 1/320-1/20,480). Most of the antibodies reacted to the epitopes STAPPAHG and PAPGSTAP. In addition, T cell proliferation was found in 4 out of 15 patients, and CTL responses were seen in 2 out of 10 patients. Mannan-MUC1 can immunize patients, particularly for antibody formation, and to a lesser extent, cellular responses. It remains to be seen whether such responses have antitumor activity.

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.

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.

Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.

Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis.

Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-x8)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds. with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-x8)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.

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.

Extracellular ATP stimulates an amiloride-sensitive sodium influx in human lymphocytes.

Extracellular ATP has been shown to increase the Na+ permeability of human lymphocytes by 3 to 12-fold. The kinetics of this ATP-induced response were studied by measuring 22Na+ influx into chronic lymphocytic leukemic lymphocytes incubated in low-sodium media without divalent cations. ATP-stimulated uptake of 22Na-ions was linear over 4 min incubation and this influx component showed a sigmoid dependence on ATP concentration. Hill analysis yielded a K1/2 of 160 microM and a n value of 2.5. The nucleotide ATP-gamma-S (1-2 mM) gave 30% of the permeability increase produced by ATP, but UTP (2 mM) and dTTP (2 mM) had no effect on 22Na influx. The amiloride analogs 5-(N-ethyl-N-isopropyl) amiloride and 5-(N,N-hexamethylene) amiloride, which are potent inhibitors of Na(+)-H+ countertransport, abolished 72-95% of the ATP-stimulated 22Na+ influx. However, the involvement of Na(+)-H+ countertransport in the ATP-stimulated Na+ influx was excluded by three lines of evidence. Sodium influx was stimulated 7-fold by extracellular ATP but only 2.4-fold by hypertonic conditions which are known to activate Na(+)-H+ countertransport. Addition of ATP to lymphocytes produced no change in intracellular pH when these cells were suspended in isotonic NaCl media. Finally ATP caused a membrane depolarization of lymphocytes which is inconsistent with stimulation of electroneutral Na(+)-H+ exchange. These data suggest that ATP acts cooperatively to induce the formation of membrane channels which allow increased Na+ influx by a pathway which is partially inhibited by amiloride and its analogs.

Characterization of activated lymphocyte-tumor cell adhesion.

This study demonstrates the variable expression of ICAM‐1 and leukocyte function antigen‐3 (LFA‐3) on four tumor cell lines (COLO526, K562, Daudi, and HT‐29). In addition, phorbol ester (PMA) activation of lymphocytes modulated LFA‐1 from a uniform to a clustered surface distribution; whereas after treatment with high levels of Mg2+ ions, the unique epitope for high‐affinity LFA‐1 was identified using clone Mab24. Using a flow cytometric adhesion assay it was demonstrated that PMA‐activated lymphocytes formed conjugates with COLO526 and Daudi, and that these conjugates were inhibited by anti‐CD2 with varying inhibition by LFA‐1 clones MHM24 and 25.3.1. When lymphocytes were induced to express the high‐affinity form of LFA‐1, conjugates were identified with COLO526, K562, and Daudi and these conjugates were sensitive to the presence of both CD2 and LFA‐1 antibodies. Further studies using confocal microscopy confirmed significant adhesion between peripheral blood lymphocytes pre‐treated with either PMA or high levels of Mg2+ and the adherent cell line COLO526. In conclusion, this unique study has demonstrated for the first time the important role of the active form of LFA‐1 on the lymphocyte cell surface for conjugate formation with an ICAM‐1‐expressing tumor cell; also, two pathways of cell signaling were identified for conjugate formation to occur. J. Leukoc. Biol. 67: 847–855; 2000.