Lymphoid Cell Membranes Research Articles

Daratumumab Decreases Treg-Mediated Immunosuppression and Potentiates CD8+ T-Cell-Induced Killing of Chronic Lymphocytic Leukemia (CLL) Cells Ex Vivo

Introduction: CD38 is expressed on the plasma membrane of lymphoid cells and in chronic lymphocytic leukemia (CLL) pts. increased CD38 expression is associated with a poor clinical outcome. While its role as a prognostic marker has been well established, the approval of daratumumab (Dara, first-in-class anti-CD38 mAb) offers a new role for CD38 as a potential therapeutic target in CLL. Matas-Cespedes et al have reported that Dara induces CLL cell death through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). While CD38 is expressed on CLL cells, it is also expressed on T-cell subsets, such as Tregs. We hypothesized that in addition to its direct immune-mediated activity, Dara modulates the immune cellular environment; skewing T-cell populations from an immunosuppressive towards immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.Methods: CD19+ cells were isolated from PBMCs of CLL pts. (n=30). Pts. with ≥30% CD38+ CLL cells were classified as CD38+ (n=22) and pts. with <30% CD38+ cells as CD38- (n=8). Dara-induced ADCC was assessed in Calcein AM labelled CLL cells cocultured with healthy donor PBMCs (E:T ratio, 50:1); CDC using 10% serum from a healthy donor and ADCP using human macrophages (E:T ratio, 2:1). Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Regulatory T-cells (Treg; CD3+CD4+CD25+CD127loFoxP3+) and cytotoxic effector T-cells (cTL, CD3+CD8+) were identified and isolated by FACS. Data are represented as mean±SEM.Results: Dara (0.1ug/mL) induced significantly greater ADCC in CD38+ (24.63±3.46%) vs. CD38- cells (14.11±1.11%) (p<0.001), whereas CDC (18.22±2.41 vs. 13.58±0.95) and ADCP (9.37±0.77% vs. 8.79±0.92%) were marginally, but insignificantly, increased in CD38+ vs. CD38- CLL cases. We also observed significantly greater apoptosis in CD38+ (36.41±3.34%) vs. CD38- CLL cells (23.39±1.70%) (p<0.005) (Fig 1A). Immune repertoire analysis revealed lower CD3+ T-cell numbers in CLL pts. (11.45±3.25%) compared to those in healthy donor PBMCs (23.64±7.36%); but with a higher percentage of Tregs (10.71±2.47 in CLL pts. vs. 1.96±0.60 in healthy donors). To determine whether Dara could decrease CLL-Tregs, we treated CLL-PBMCs withDara (10µg/ml; 5 days) ex vivo, and noted significant decrease in Treg cells from 12.75±1.33 (Isotype control) to 7.17±0.91, (n=5, p<0.001). As downregulation in Tregs can consequentially lead to a rise in other T-cell subtypes, we observed increased effector cTLs: from 50.08±5.41 to 64.53±3.19 (p<0.02) (Fig 1B). Further analysis on CFSE-labeled CLL-cTLs (primed or unprimed with Dara), showed cTLs proliferated significantly more so when co-cultured with Dara-treated apoptotic CLL (51.75±1.16) cells vs. ibrutinib (31.44±0.36) or rituximab-treated apoptotic CLL cells (36.25±3.77) or live CLL cells (7.04±3.34) (Fig 1C, representative CLL pt.). To determine if Dara-primed cTLs had enhanced cytolytic aptitude towards autologous CLL cells, we cocultured primed cTLs with CLL cells. Compared to unprimed cTLs, specific lysis of target CLL cells was greater in the primed cTL-CLL coculture (12.61±1.75% vs. 28.27±0.82%, respectively). And this effect was noted to be patient-specific as cTLs cultured with JVM13 (CD38+) cells showed lower specific lysis (Fig 1D, E).Conclusion: Here we demonstrate for the first time that Dara modulates CLL-Treg levels and increases effector cTL proliferation. Moreover, Dara-primed cTLs display heightened CLL-specific cytolytic activity than compared to Dara-naïve cTLs. Dara also induces direct apoptosis of CLL cells and exposure of Dara-killed CLL cells to cTLs increases cTL proliferation significantly more so than cTL exposure to CLL cells apoptosed using ibrutinib or rituximab. Altogether, our findings highlight the multiple mechanisms by which Dara (and potentially other anti-CD38 agents, currently being investigated by us) can induce CLL cell death and potentially revert dysfunctional cTL activity from that of a pro-CLL to an anti-CLL composure. [Display omitted] DisclosuresParikh:AstraZeneca: Honoraria; Pharmacyclics: Research Funding; Pharmacyclics: Honoraria. Ding:Merck: Research Funding. Sher:LAM Therapeutics, Inc: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Sex‐specific influence of the vacuolar adenosine triphosphatase a2 isoform on outcome in twin pregnancies

The influence of fetal sex on immune responses in multifetal pregnancies remains incompletely elucidated. The a2 isoform of vacuolar adenosine triphosphatase (a2V) is expressed on the cell membrane of maternal lymphoid cells and contributes to down-regulation of pro-inflammatory immune responses during gestation. The association between fetal sex and a2V expression on peripheral blood mononuclear cells (PBMCs) from mothers with twin gestations was assessed. Patients in this prospective study were 93 women with twin pregnancies in their mid-second or early third trimester-27 with two male, 30 with two female and 36 with one male and one female fetus. PBMCs were isolated and a2V was measured by ELISA in cell lysates. Demographic and clinical data were subsequently obtained and correlations between a2V and fetal sex, birthweight and pregnancy outcome were assessed by the Mann-Whitney and Spearman rank correlation tests. The mean a2V level was highest when both fetuses were male (2.0ng/mL) and lowest when both were female (1.5ng/mL; P=0.0184). Only when both fetuses were female did the a2V concentration negatively correlate with birthweight of the 1st (P=0.0011) and 2nd (P=0.0044) born fetus and with gestational age at delivery (P=0.0018). There were no associations between a2V and these outcomes in male only or mixed twin pregnancies. We conclude that the a2V-mediated regulation of maternal immunity during twin pregnancies is influenced by fetal sex.

Cellular and Molecular Bases of Changes to Neuroimmune Interactions in Stress

This article presents current views on the cellular and molecular bases of the central mechanisms of neuroimmune interactions, along with data on the effects of destabilizing factors (painful electrical stimulation, cold, psychoemotional stress) on the functions of neurons and immunocompetent cells. It is important to emphasize that stress is associated with changes in ligand-receptor interactions on the membranes of lymphoid cells which respond to the interleukin-1 regulatory signal; these changes consist either of enhanced responses to this regulatory signal (in mild stress situations) or sharp decreases in responses (on exposure to strongly stressful factors). Special attention is paid to the role of the orexinergic system in the mechanisms mediating the brain’s responses to administration of antigens and exposure to stressors. We consider possible means of correcting the imbalance in the functional interaction between the nervous and immune systems occurring as a result of various destabilizing factors.

199 - Oxygen Effect on Protection of Human Lymphoid Cells Against Free Radicals by the Carotenoid Lycopene

Carotenoids are known to act as dietary antioxidants and so are of wide interest for their health benefits. Dietary lycopene, the carotenoid pigment in tomatoes, has been shown to protect against human lymphoid cell membrane damage from free radicals produced by gamma radiation and also by the nitrogen dioxide radical, generated photolytically. This protective effect is dramatically reduced as the oxygen concentration increases, particularly for damage due to gamma radiolysis – becoming near zero at 100% oxygen from 5-fold protection at 20% oxygen and 50-fold protection at zero per-cent oxygen. The effect is less pronounced for nitrogen dioxide-induced cell killing falling from 17-fold protection in the absence of oxygen to 9-fold at 100% oxygen. Non-cellular gamma radiation and laser studies were also carried out to support the molecular mechanisms suggested for the effect of oxygen. The remarkable reduction in protection by lycopene against gamma radiation at high oxygen concentrations could be exploited to enhance radiation procedures for therapy and preliminary studies have also been undertaken for irradiations with high energy protons.

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis, we have developed previously, is applied to the analysis of Na+/Na+ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na+, K+ and Cl-, membrane potential and cell volume. It is shown that the Na+ input (I(Na/Na)) flux and output flux through the Na+/Na+ tract increased 4 times in 8 hours after disconnecting Na+/K+ -ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I(Na/Na) after 8 hours off pump by ouabain is 97% of the total Na+ input for both cell types. It is concluded that ouabain not only inhibits the Na+/K+ -ATPase, but also increases Na+ exchange fluxes through the Na+/Na+ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na+/Na+ equivalent exchange.

Two-dimensional receptor patterns in the plasma membrane of cells. A critical evaluation of their identification, origin and information content

A concise review is presented on the nature, possible origin and functional significance of cell surface receptor patterns in the plasma membrane of lymphoid cells. A special emphasize has been laid on the available methodological approaches, their individual virtues and sources of errors. Fluorescence energy transfer is one of the oldest available means for studying non-randomized co-distribution patterns of cell surface receptors. A detailed and critical description is given on the generation of two-dimensional cell surface receptor patterns based on pair-wise energy transfer measurements. A second hierarchical-level of receptor clusters have been described by electron and scanning force microscopies after immuno-gold-labeling of distinct receptor kinds. The origin of these receptor islands at a nanometer scale and island groups at a higher hierarchical (μm) level, has been explained mostly by detergent insoluble glycolipid-enriched complexes known as rafts, or detergent insoluble glycolipids (DIGs). These rafts are the most-likely organizational forces behind at least some kind of receptor clustering [K. Simons et al., Nature 387 (1997) 569]. These models, which have great significance in trans-membrane signaling and intra-membrane and intracellular trafficking, are accentuating the necessity to revisit the Singer–Nicolson fluid mosaic membrane model and substitute the free protein diffusion with a restricted diffusion concept [S.J. Singer et al., Science 175 (1972) 720].

Correlation of Pasteurella haemolytica leukotoxin binding with susceptibility to intoxication of lymphoid cells from various species.

Pasteurella haemolytica, the causative agent of shipping fever pneumonia in cattle, produces a leukotoxin (LKT) which lyses ruminant leukocytes with high efficiency but is reputed to not affect leukocytes from nonruminant species. In this study, we tested the supposition that LKT binding correlates positively with susceptibility to intoxication of susceptible isolated bovine lymphocytes and lymphoma tissue culture cells (BL3 cells) and negatively with reputed nonsusceptible equine, porcine, and canine lymphocytes and human lymphoid tissue culture cells (Raji cells). Bovine lymphocytes and BL3 cells were highly susceptible to LKT intoxication, exhibiting both substantial increase in intracellular Ca(2+) concentration and marked leukolysis. Exposure of reputed LKT-nonsusceptible porcine lymphocytes and Raji cells to LKT caused a slightly increased intracellular Ca(2+) concentration but no leukolysis. No LKT effect was detected for equine and canine lymphocytes. LKT bound to lymphoid cells from all species tested. Intact 102-kDa LKT was recovered from exposed isolated lymphoid cell membranes. Pro-LKT acylation was not required for LKT binding to BL3 cells. LKT binding was rapid, with maximal binding occurring by 3 min, and was proportional to the LKT concentration in the range 0.04 to 4.0 microg/ml. For this LKT concentration range, BL3 cells bound more LKT than did porcine lymphocytes or Raji cells, suggesting that LKT binds to BL3 cells with higher affinity than to porcine lymphocytes or Raji cells. Above 4.0 microg/ml, LKT demonstrated saturable binding to BL3 cells. Neutralizing anti-LKT monoclonal antibody (MAb) MM601 diminished LKT binding to BL3 by 36% while decreasing leukolysis by 81%. In contrast, MM601 did not diminish LKT binding to Raji cells. Pretreatment of target cells with 120 microg of protease K per ml diminished LKT binding to BL3 cells by 75%, with only a 25% decrease in leukolysis. However, pretreatment with 150 microg of protease K per ml abolished the remaining 25% of LKT binding and 75% leukolysis. Therefore, P. haemolytica LKT binds rapidly to susceptible and to reputed nonsusceptible lymphoid cells. LKT binding resulting in species-specific leukolysis was characterized by high affinity, inhibition by MAb MM601, and relative resistance to protease K pretreatment of lymphoid cells. Two types of LKT binding to lymphoid cells are proposed. High-affinity binding leads to efficient leukolysis. In some lymphoid cells from reputed LKT-nonsusceptible species, low-affinity LKT binding may cause a low-efficiency increase in the intracellular Ca(2+) concentration without leading to leukolysis.

Morphological Analysis of the Interaction of Charged Surfactant Vesicles (SVs) with Human Cultured Cells

We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.

Fluorescent lipid probes 12-AS and TMA-DPH report on selective, purinergically induced fluidity changes in plasma membranes of lymphoid cells

The effect of extracellular ATP (ATP ex) on the anisotropy of 1-[4-trimethylamino) phenyl]-6-phenyl-hexa-3,5 triene (TMA-DPH) and 12-anthroyloxi-stearic acid (12-AS) fluorescence was investigated in Balb/C mouse thymocytes and in JY human lymphoblasts. These cells have been shown recently to be sensitive and resistant to ATP ex, respectively, in terms of cellular responses Extracellular ATP (1 mM) induced a time-dependent elevation in the emission anisotropy of both probes (indicating an increased lipid packing density) in the plasma membrane of thymocytes. The maximal effect, at 37°C, was observed between 20 and 60 min after ATP ex administration, and followed by a gradual decrease of fluorescence anisotropy at longer times (60–180 min). ATP ex did not change membrane fluidity of thymocytes below the phase transition temperature (at 18 °C). Oxidized ATP (oATP), a selective antagonist of P 2, purinoreceptors, blocked the ATP ex-induced decrease in membrane fluidity. Low ATP ex concentrations (100–300 μM)—which are known to induce distinct signals (changes in membrane potential and intracellular Ph)—slightly fluidized the plasma membrane of thymocytes. This effect was partially blocked by quinine, a blocker of Ca 2+-activated K + channels. Neither 12-AS nor TMA-DPH showed any change in their emission anisotropy upon ATP ex-treatment in the plasma membrane of the resistant human JY lymphoblast cells. No other signalling event (membrane potential change, Ca 2+ response) is elicited by ATP ex in this cell line. These data suggest that the changes in the membrane fluidity are likely consequences of specific, purinoreceptor-mediated signalling events, such as hyper- or depolarization of the plasma membrane or Ca 2+ influx. These signals may induce changes in the conformation or lateral organization of membrane proteins, perturbing protein-lipid interactions, as well.

Analysis of recombinant and native CD4 by one- and two-dimensional gel electrophoresis.

Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one- and two-dimensional (2-DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of approximately 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degrees C) of cell-(CEM-T4, THP-1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell extracts, however, revealed both monomeric and higher-order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1-DE and 2-DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.

Small virus-like particles bud from the cell membranes of normal as well as HIV-infected human lymphoid cells.

Electron microscopy (EM) of cell sections showed cell associated virus-like particles (VLP), 50-60 nm in diameter, budding from the membrane of human lymphoid cells in culture. The particles had an envelope continuous with the cell membrane and a dense core that almost filled the particle. Particles 70-80 nm in diameter with prominent external spikes were found in the culture medium by negative staining (medium-associated VLP). Cell-associated VLP were also present in cord lymphocytes, both on initial separation and after culture with or without foetal calf serum, and therefore were considered to be endogenous to the cells and were not bovine diarrhoea virus. VLP were observed in most of the lymphoid cell lines examined. VLP were also found less frequently in established human tumour and nontumour cell lines. Both cell-associated and medium-associated VLP were also present in HIV infected cell cultures, and they could be distinguished from HIV by their characteristic morphology and smaller size. It was not determined whether the 2 types of particle represented the same entity, or whether they were defective virus particles or a cellular secretory product.

Potential of Diet Therapy on Murine AIDS ,

Recently, a murine retrovirus (LpBM5 MuLV), which induces immunodeficiency syndrome in mice, termed MAIDS, has been found to have several features similar to those seen in human acquired immunodeficiency syndrome (AIDS). The present study was undertaken to compare the effects of 40% energy restriction (R) and/or ad libitum (AL) diets with vegetable [corn oil, (CO) (n-6)] or marine oil [menhaden fish oil (FO), (n-3)] as a source of dietary fats on the immune function and survival rate of C57BL/6 mice injected with the LpBM5 MuLV virus. Weanling mice were fed, throughout the study, either a 5% CO-, 5% CO(R)-, 20% CO- or 20% FO-based diet and 8 wk later the mice were injected with the LpBM5 MuLV (5 × 105 plaque-forming units). The results revealed a significantly prolonged postinjected survival rate in the mice fed 20% FO and 5% CO(R) diets [5% CO = 131 ± 7 d; 5% CO(R) = 161 ± 13 d; 20% CO = 125 ± 6 d; 20% FO = 164 ± 14 d]. Immunological studies conducted 4 wk after injection revealed decline in both interleukin-2 production and proliferative response to mitogens in spleen cells of mice in all four dietary groups. However, this decline was less apparent in mice fed 5% CO(R) and 20% FO diets. The levels of prostaglandin E2 (PGE2) were found to be significantly lower only in animals fed 20% FO diets, whereas the levels of arachidonic acid (20:4) were found to be significantly lower in the spleen cells of groups fed 5% CO(R) and 20% FO diets and were higher in mice fed 5% CO and 20% CO diets. In conclusion, these findings indicate that changes induced by dietary (n-3) lipids and/or moderate energy restriction on the composition of lymphoid cell membranes can modulate the production or response of lymphokines, including immunosuppressive PGE2, which appears to delay progression of the retroviral infection.

Combined immunocytochemical staining and image analysis for the study of lymphocyte specificity and function in situ

Immunocytochemical staining methods were combined with computer-aided image analysis to quantitate relative intracytoplasmic and cell membrane products within single lymphoid cells. Lymphoid cells fixed to microscope slides were immunocytochemically stained and the densities of immunocytochemical stains for individual lymphoid cells were determined on video-camera-captured microscopic images to quantitate synthesized and fully assembled antibody molecules detected by an anti-idiotypic antibody. Similarly, expression of a lymphoid cell membrane molecule, Ly-1, was comparatively quantitated in individual lymphoid cells. Densitometric analysis of tissue sections was utilized to measure tissue areas and quantitate cell nuclei in tissues. Two-color immunocytochemical staining and sequential image analyzer-determined locations of individual cells in tissues allowed unambiguous identification of doubly stained cells in their native tissue environment. This analysis was applied to the in situ identification of antibody forming cells producing the CRIc idiotype of the BALB/c anti-arsonate response and a determination of the mu chain usage by those individual cells. Applications of combined immunocytochemistry and computerized image analysis to studies of in vivo immunologic functions are discussed.

Detergent extraction of the human alpha-beta interferon receptor: a soluble from capable of binding interferon

After examining a variety of detergents we find that receptors for human alpha interferon can be solubilized, in active form, from plasma membranes of lymphoid cells using the detergent CHAPS. The complexes formed, in solution, with interferon are stable enough to be separated chromatographically.

B cell-stimulating activity of lymphoid cell membrane fractions.

We had previously found that a mutagenized subline of the mouse thymoma EL4 very efficiently stimulates B cells via direct cell-cell contact, thereby inducing the responsiveness of B cells to cytokines. In the present study, we investigated whether this effect could also be mediated by plasma membranes of EL4 (and other) cells. By equilibrium centrifugation of cell homogenates, four cell membrane fractions of different densities were obtained. These were tested for (a) stimulation of B cell proliferation in conjunction with EL4 supernatant as source of cytokines, and (b) enhancement of B cell proliferation at suboptimal concentration of lipopolysaccharide. It turned out that all membrane fractions from a variety of T lineage cells (mutant EL4, parent EL4, BW5147, P198 thymomas, normal T cells) and B lineage cells (BCL1 lymphoma, X63Ag8 cytoplasma, normal B cells) exhibited similar B cell stimulating activity in both assays. Interleukin 1 activity was not detected in the membrane fractions. Heat treatment abolished all activity showing that protein at least was involved. Either protease treatment or extraction with detergent abolished the activity of subcellular fractions rich in intracellular membranes but not that of fractions most enriched in surface membranes. Finally, erythrocyte membranes also displayed B cell-stimulating activity sensitive to protease and detergent extraction. In contrast, and in confirmation of a previous study, liver cell membrane was inhibitory in the B cell proliferation assay with lipopolysaccharide. In conclusion, the effects of cell membranes did not reflect the unique activity of intact mutant EL4 cells. However, with respect to our data it is conceivable that membrane proteins with relatively nonspecific activity and wide distribution among lymphoid cells could play a role in T cell help together with molecules specialized in cell adhesion and cell triggering.

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols > HDL-phospholipids > plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatrine. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.

Vesicle-to-cell protein transfer: insertion of band 3, the erythrocyte anion transporter, into lymphoid cells.

Band 3, the erythrocyte anion transporter, transfers spontaneously between human red cells and model membranes. During incubation of intact erythrocytes with sonicated dimyristoylphosphatidylcholine vesicles, the transporter inserts in functional form and native orientation into the liposome bilayer, with the cytoplasmic segment of the protein contacting the lumen of the vesicle [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117; Huestis, W. H., & Newton, A. C. (1986) J. Biol. Chem. 261, 16274-16278]. When band 3-vesicle complexes are incubated with erythrocytes whose native band 3 has been inhibited irreversibly, reverse transfer of the protein restores anion transport capacity to the cells [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117]. Here we report the vesicle-mediated transfer of band 3 to human peripheral blood lymphocytes and to cultured murine lymphoma cells (BL/VL3). Subsequent to incubation with protein-vesicle complexes, both lymphoid cell types exhibit a 2-4-fold increase in the rate of chloride uptake. This enhanced permeability is inhibited greater than or equal to 98% by the exofacial band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, consistent with right-side-out insertion of functional band 3 into the lymphoid cell membrane.

The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts.

Thy-1 is a plasma membrane protein, but its primary structure lacks the typical membrane-spanning sequence. Recent studies revealed that a glycophospholipid is covalently bound to the carboxyl terminus, suggesting that the protein is integrated into the plasma membrane by this lipid moiety. Lateral diffusion of Thy-1 was measured in mouse thymocytes, lymphoma cells, and fibroblasts by the fluorescence recovery after photobleaching technique. Thy-1 was labeled with rhodamine-conjugated anti-Thy-1 monoclonal antibodies. Diffusion coefficients of 2-4 X 10(-9) cm2/sec were obtained for the antigen-antibody complex in all the cell types. About 50% of the Thy-1 was mobile. The diffusion coefficient for the mobile fraction of Thy-1 is considerably larger than the diffusion coefficients of many other plasma membrane proteins. Rather, the diffusion coefficient of Thy-1 is similar to those of lipid analogs embedded in the same membrane, providing strong support for the suggested lipid anchoring of this antigen.

Unique Proliferation-Associated Marker Expressed on Activated and Transformed Human Cells Defined by Monoclonal Antibody&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

The expression, tissue distribution, and preliminary characterization of a cell surface molecule, apparently a glycolipid, recognized by a monoclonal antibody, anti-PAA, were described. This antibody (anti-PAA) was produced by the fusion of myeloma cells NS-1 with spleen cells from a BALB/c mouse, which were sensitized with activated human T-cells generated by allogeneic stimulation in mixed-lymphocyte culture (MLC). Resting human peripheral blood T-cells, B-cells, and monocytes demonstrated weak anti-PAA binding. Binding of proliferating T-cells (phytohemagglutinin- and MLC-activated T-cells) and thymocytes to anti-PAA was two to six times greater than that of resting T-cells. A fifteenfold-increased binding was observed with acute lymphocytic leukemia T-cell lines. Epstein-Barr virus-transformed B-cell lines bound anti-PAA up to sixteenfold greater than resting B-cells. Tumor cell lines of various nonlymphoid origins demonstrated marked reactivity with this antibody. Both benign and malignant cells in hyperplastic tissues, of various origins, bound anti-PAA, whereas their normal, nonproliferating counterparts did not. Normal proliferating cells in these tissues, including cells of the placental chorionic villi and trophoblasts, also bound anti-PAA. Of all lymphoid and nonlymphoid cell lines examined, only chronic lymphocytic leukemia (CLL) cells and some cell lines derived from Burkitt's lymphoma showed weak or no binding. This antibody also failed to react with a variety of nonprimate cell lines. Anti-PAA antibody did not immunoprecipitate any protein from lymphoid tumor cell lines to which it demonstrated a quantitatively high degree of binding, nor did protease treatment of these lines decrease antibody binding. Anti-PAA did, however, bind to glycolipids extracted from these cell lines. Binding of this monoclonal antibody to a minor neutral glycolipid, isolated from the erythroleukemia cell line K562, was about sixfold greater than that of any other K562 neutral glycolipid or ganglioside. Anti-PAA demonstrated weak or undetectable binding to purified, predominant, lymphoid cell membrane's neutral glycolipids and gangliosides. The monoclonal antibody anti-PAA appeared, therefore, to recognize a unique, proliferation-associated, neutral glycolipid present on normal as well as on benign and malignant proliferating cells. The antigen appeared to be universally expressed on proliferating cells from all human tissues with the exception of some Burkitt's cell lines and CLL cells. Nonhuman cell lines, except those for closely related primates, did not express PAA.

The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts.

The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts.