Presence Of Adherent Cells Research Articles

More of the same? Voxelotor spawns a successor, but on what success does it build?

More of the same? Voxelotor spawns a successor, but on what success does it build?

Real time remodeling of cellular morphology using optical imprinting of cell-culture substrates

Development of materials that can dynamically alter the topographic experience of cultured cells is key to accurately modeling in vivo processes such as tissue development and repair. Current technologies have largely focused on smart substrate materials that can be programmed to undergo topographical transformations in the presence of adhered cells, but such materials provide limited spatiotemporal resolution and generally are triggered by undesirable changes to environmental conditions. Here, we present an approach for investigating cellular responses to dynamic topographical cues in which multiphoton photochemistry is used to remotely imprint a substrate with arbitrary topographical patterns in real time (i.e., in the presence of adherent cells). In these studies, fibroblastic NIH3T3 cells were plated on a planar protein hydrogel substrate, where they generally established stereotypical polygonal morphology before the underlying substrate was dynamically transformed to a grooved topography. Elongation and alignment of cells exposed to low-micrometer-pitch grooves imprinted in this manner occurred less rapidly than for cells directly deposited on substrates having pre-formed grooves. Further, cell alignment on dynamically imprinted grooves was notably delayed relative to elongation, suggesting that the structural attributes of a cell at the time it first experiences a grooved topography may differentially influence these two processes. This minimally invasive approach for subjecting cells to dynamic topographical experiences represents a versatile means to model evolving conditions within in vivo systems and to systematically explore mechanisms of cellular morphology and behavior.

A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles.

Cellular labeling with inorganic nanoparticles such as magnetic iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles is an important method for the noninvasive visualization of cells using various imaging modalities. Currently, this is mainly achieved through the incubation of cultured cells with the nanoparticles that eventually reach the intracellular compartment through specific or nonspecific internalization. This classic method is advantageous in terms of simplicity and convenience, but it suffers from issues such as difficulties in fully removing free nanoparticles (suspended in solution) and the lack of selectivity on cell types. This article reports an innovative strategy for the specific labeling of adherent cells without the concern of freely suspended nanoparticles. This method relies on a nanocomposite film that is prepared by homogeneously dispersing nanoparticles within a biodegradable polymeric film. When adherent cells are seeded on the film, they adhere, spread, and filtrate into the film through the micropores formed during the film fabrication. The pre-embedded nanoparticles are thus internalized by the cells during this infiltration process. As an example, fluorescent silica nanoparticles were homogeneously distributed within a polycaprolactone film by utilizing cryomilling and heat pressing. Upon incubation within physiological buffer, no silica nanoparticles were released from the nanocomposite film even after 20 d of incubation. However, when adherent cells (e.g., human mesenchymal stem cells) were grown on the film, they became fluorescent after 3 d, which suggests internalization of silica nanoparticles by cells. In comparison, the suspension cells (e.g., monocytes) in the medium remained nonfluorescent no matter whether there was the presence of adherent cells or not. This strategy eventually allowed the selective and concomitant labeling of mesenchymal stem cells during their harvest from bone marrow aspiration.

Effects of sarcolectin (SCL) on human peripheral blood mononuclear cells

Sarcolectin (SCL) is a tissue growth factor found in various human or animal tissues, functioning in balance with interferons (IFNs) that can inhibit growth and affect cell differentiation. Like somatotropin, SCL is found in the pituitary gland. In humans, the SCL gene is located on chromosome 12 (q12-q13) and expressed as a 55 kDa protein consisting of 469 amino-acids. After a single activation of peripheral blood mononuclear cells (PBMC) obtained from more than 30 individuals, highly significant cell proliferation was found to peak after 7 days in culture. The presence of adherent cells was necessary for cell proliferation. SCL induced over-expression of α-IL-2 receptor (CD25) leading to proliferation of CD3+/CD4+/CD45RO+ T cells. Thus in PBMC, SCL induced CD4+ T cell growth and expression of inflammatory cytokine genes, including TNF-α, IL-1β, IL-6 and IL-8. IFNs are also produced following activation as a feedback response which is maintained for about 20 days.

Monocytes affect cell‐cycle progression but not baseline frequency of sister chromatid exchanges of pig lymphocytes

The effect of pig monocytes (MNs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression of pig lymphocytes was studied in plasma leukocyte (PLCs) and whole blood leukocyte cultures (WBCs). No variation in SCE frequency was observed between control WBC and PLC, nor did the addition of pig MNs to PLCs modify the baseline frequency of SCEs. Cell proliferation was slower in PLCs than in WBCs. Variations in cell-cycle progression of pig lymphocytes from PLC were observed both in the absence and presence of adherent cells in the culture. In MN-free cultures, lymphocytes proliferated faster than in parallel PLC cultures. However, when MNs were seeded into the cultures, cell-cycle progression gradually slowed as a function of the concentration of adherent cells present in the cultures. This finding shows that pig MNs modulate the in vitro cell-cycle progression of pig lymphocytes in a dose-dependent manner and that the low baseline SCE frequency of pig lymphocytes is independent of the presence or absence of MNs in the culture. Environ. Mol. Mutagen. 33:86–90, 1999 © 1999 Wiley-Liss, Inc.

Monocytes affect cell‐cycle progression but not baseline frequency of sister chromatid exchanges of pig lymphocytes

The effect of pig monocytes (MNs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression of pig lymphocytes was studied in plasma leukocyte (PLCs) and whole blood leukocyte cultures (WBCs). No variation in SCE frequency was observed between control WBC and PLC, nor did the addition of pig MNs to PLCs modify the baseline frequency of SCEs. Cell proliferation was slower in PLCs than in WBCs. Variations in cell-cycle progression of pig lymphocytes from PLC were observed both in the absence and presence of adherent cells in the culture. In MN-free cultures, lymphocytes proliferated foster than in parallel PLC cultures. However, when MNs were seeded into the cultures, cell-cycle progression gradually slowed as a function of the concentration of adherent cells present in the cultures. This finding shows that pig MNs modulate the in vitro cell-cycle progression of pig lymphocytes in a dose-dependent manner and that the low baseline SCE frequency of pig lymphocytes is independent of the presence or absence of MNs in the culture.

Erythroid accelerating factor detected in serum from rats with drug induced hemolysis.

We have previously observed that an erythroid enhancing activity presents in rat serum in the early stage of drug induced hemolytic anemia. The further studies on biological and physicochemical aspects of this erythroid accelerating factor (EAF) is described in this paper. Hemolytic anemia was induced in rats by single intraperitoneal injection of acetylphenylhydrazine (APH) and serum was obtained from the rats on day 1 after APH injection. It was first fractionated by ultrafiltration on Amicon Diaflo membranes to give a series of fractions lying in the following ranges of molecular weight: 10-30 kDa, 30-50 kDa, 50-100 kDa, and >100 kDa. Among those fractions, largest increase in the number of colony forming unit erythroid CFU-E) colonies was shown in the fraction of >100 kDa that was subsequently fractionated by fast protein liquid chromatography (FPLC) system. EAF activity for CFU-E proliferation was detected in a FPLC fraction corresponding to a molecular weight of about 160 kDa. An addition of EAF significantly increased with dose dependent manner in the number of CFU-E colonies from rat bone marrow mononuclear cells. EAF alone had no burst promoting activity and exhibited no distinct activity to proliferate burst forming unit-erythroid even when interleukin-3 (IL-3) and high concentration (2 U/ml) of erythropoietin (Epo) were added together to the culture. The stimulating effect of EAF on CFU-E was markedly dependent on the presence of adherent cells in the culture. Partially purified protein was relatively heat-unstable (60% at 75 degrees C, 30 minutes) and sensitive to treatment with trypsin and alpha-galactosidase. These results suggest that EAF is a novel factor, possible glycoprotein to reinforce Epo function and is different from various cytokines previously documented because of differences of approximate molecular weight.

Trypanosoma cruzi cytosolic alkaline antigens (FI) induce polyclonal activation in murine normal B cells.

Several reports have described polyclonal activation in mice acutely infected with Trypanosoma cruzi. The aim of this work was to analyse the participation of one T. cruzi antigenic fraction in this immunological event. The antigen selected was FI, an antigenic fraction of pI 7-9 obtained from T. cruzi cytosol separated by isoelectricfocusing. FI is constituted by molecules with molecular weights of around 60 and 20 KDa. The authors assayed the ability of this antigenic fraction to induce polyclonal activation of spleen mononuclear cells from normal (NSMC) BALB/c mice. NSMC showed a marked lymphoproliferative response measured by 3H-thymidine incorporation after 3 days of culture in presence of FI. The values reached by FI-stimulated cells were 10 times higher than the controls (non-stimulated cells). This effect was dose-dependent. Furthermore, the authors observed that a purified T-cell population in the presence of adherent cells was unaffected by FI. Additionally, in a culture of NSMC, FI stimulated the proliferation of B cells as observed by the increase of the percentage of B220+ cells determined by FACS using FITC-conjugated anti-mouse B220. The authors noticed that the percentage of B220+Ly1+(CD5) populations in the presence of FI did not change with respect to the control (non-stimulated cells), indicating that FI expanded both conventional and CD5+ B cells. The isotypic pattern of the antibodies produced after 6 days of culture of NSMC in the presence of FI was predominantly IgM, which reacted with highly conserved antigens such as actin, myosin, myoglobin, thyroglobulin and carbonic anhydrase, but did not react with FI. A slight increase of IgG1 and IgG3 with respect to the control was observed but no changes on the levels of IgG2 was noticed. These results indicate that FI promotes activation, proliferation and differentiation in antibody-secreting cells of normal murine B lymphocytes.

In VitroEnhancement of Antibody Responses by Tδ Cells Expressing IgD Receptors

Previousin vivostudies have shown that murine T cells induced to express receptors specific for IgD (IgD-R) have humoral immunoaugmenting properties with respect to both primary and secondary antibody responses to various antigens. Such murine IgD-R+T cells (Tδ cells) belong to the CD4+T cell population in contrast with human Tδ cells which also include CD8+cells. The purpose of these studies was to develop anin vitroassay system to examine the mechanism by which Tδ cells facilitate enhanced antibody responses. Our studies demonstrate that B cell responses to both soluble and particulate antigens can, indeed, be enhancedin vitroby coculture with T cells previously induced to express IgD-R. Thisin vitroeffect requires cognate interaction between T and B cells and is dependent on the presence of adherent cells or IL-1. T cell priming with antigen, while not a prerequisite, was found to result in more effective Tδ–B cell interactions compared with naive Tδ cells. Finally, evidence was obtained in support of an adhesion-mediated Tδ–B cell interaction since the immunoaugmenting properties of T cells expressing IgD-R are completely blocked by the addition of very low doses of monomeric IgD to the cultures.

Inhibition of Plasmodium falciparum growth in vitro by CD4+ and CD8+ T cells from non‐exposed donors

T cells from most adult non-exposed donors, which express a memory phenotype (CD45RO+), can respond by proliferation to P. falciparum asexual stages in vitro. Such cells may have arisen from exposure to environmental organisms. To address the efficacy of such cells in eliminating parasites and investigate the mechanisms involved, we have used an in vitro assay where parasite growth can be precisely monitored in the presence of different cell preparations. Unfractionated peripheral blood mononuclear cells (PBMC) from both malaria-exposed and non-exposed donors inhibited parasite growth by up to 62% in a two day assay. Purified T cells in the presence of adherent cells had a similar effect, but purified T cells alone or adherent cells alone had minimal effect. Antigens released at the time of schizont rupture were maximally effective in stimulating interferon-gamma (IFN gamma) production. Neutralizing antibodies to IFN gamma showed a partial reduction of growth inhibition in some individuals tested suggesting that different mechanisms may be operative. Neutralizing antibody to TNF alpha had a partial effect in combination with anti-IFN gamma. Antibodies to IL-1 and IL-4 had no effect. T cell fractionation experiments showed that while purified CD4+ T cells from some donors produced IFN gamma and inhibited parasite growth, purified CD8+ T cells could inhibit parasite growth to a greater extent without production of detectable IFN gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

The polybacterial lysate Olimunostim modulates lymphocyte function in vitro and restores depressed cellular immunity in vivo.

Immunobiological activity of the polybacterial lysate Olimunostim (P. acnes, K. pneumoniae, S. aureus) was examined by investigating its effects on murine lymphocytes. When added to in vitro lymphocyte cultures, Olimunostim induced interleukin-2 (IL-2) biological activity (in a 2-d culture) and subsequently potentiated lymphocyte proliferation (on day 3); the latter effect was dependent on the presence of adherent cells. In vivo, significant enhancement of lymphocyte reactivity to T-mitogens and increase of CD4+ helper-inducer T lymphocytes were observed 3 d after a subcutaneous application of Olimunostim to mice with cellular immune deficiency. These results confirm the modulatory properties of Olimunostim towards lymphocytes both in vitro and in vivo, which may form a basis for its clinical application.

Human tonsil, blood and bone marrow in vivo‐induced B cells capable of spontaneous and high‐rate immunoglobulin secretion in vitro: Differences in the requirements for factors and for adherent and bone marrow stromal cells, as well as distinctive adhesion molecule expression

Human B cells capable of spontaneous IgG secretion are commonly found in circulation and in lymphoid tissues such as tonsil and bone marrow (BM). The present study compares the mechanisms that regulate tonsil, blood and BM B cells capable of spontaneous IgG secretion. The BM cell subset produced IgG during a markedly longer period of time (14 days) than did tonsil and blood cell subsets (2-3 days). Blood and BM, but not tonsil, B cell IgG secretion depended on the presence of adherent cells, as demonstrated by adherent cell depletion and re-addition experiments. Stromal BM cells supported linear IgG secretion by non-adherent BM cells for 2 weeks, but were unable to prolong the short-term IgG secretion by tonsil and blood cells. Different factors induced IgG secretion in each of the three B cell populations as optimal IgG secretion by tonsil, blood or BM cell subsets required either tumor necrosis factor-alpha, interleukin-6 or fibronectin + interleukin-6, respectively. Finally, these populations also showed differences in the expression of adhesion molecules; the tonsilar cell subset was PNA+/- CD44+ CD49d+ CD49e- Leu-8+/-, the blood cell subset was PNA- CD44+/- CD49d+ CD49e- Leu-8+ and the BM cell subset was PNA- CD44+/- CD49d+ CD49e- Leu-8-. These results suggest that the mechanisms controlling the final differentiation and the expression of adhesion molecules in these B lymphocytes exhibit territorial specificity.

Prolactin-Induced Mitogenesis of Lymphocytes from Ovariectomized Rats*

PRL can induce interleukin-2 (IL-2) receptor expression in splenocytes from ovariectomized (OVX) female rats. In this further study of the effects of PRL on lymphocytes in vitro we found that PRL induced IL-2 receptor expression, IL-2 production, and proliferation of splenocytes and thymocytes from OVX rats. Cells from male rats were not affected. The proliferative response, as measured by [3H]thymidine incorporation, depended on the concentration of PRL and the presence of adherent cells in the culture. After a 48-h incubation with PRL (1 microgram/ml), splenocytes from OVX rats incorporated essentially the same amount of [3H]thymidine as cells incubated with the polyclonal T-cell mitogen Concanavalin-A (ConA). As determined by autoradiography, approximately 40% of the splenocytes responded to PRL or to ConA. After incubation of splenocytes and thymocytes with PRL, bioactive IL-2 was detected in culture medium only from cells of OVX female rats, while incubation with ConA caused IL-2 production by lymphocytes from both male and OVX rats. However, ConA induced IL-2 activity sooner than PRL. Immunofluorescent-flow cytometric analysis revealed time-dependent increases in percentages of IL-2 receptor-positive splenocytes as well as increases in percentages of total T-cells and cells of the CD8 and, to a lesser extent, the CD4 subclass after PRL stimulation.

A signal transduction through a unique rat T cell specific antigen, 8H3

A signal transduction was detected in various rat T cells by cross-linking of 8H3 antigen by 8H3 antibody. A rat T cell proliferative response induced by 8H3 antibody is dependent on the presence of adherent cells. When spleen cells were cultured in the presence of 8H3 antibody, only CD4-positive T cell proliferation was induced. These proliferative CD4-positive T cells express rat interleukin 2 receptor, alpha chain. Cross-linking of 8H3 antigen resulted in an increase in cytoplasmic free Ca2+ in T cells and phosphorylation of a 120 kDa component of the 8H3 antigen. It should be noted that cross-linking of 8H3 antigen together with CD4 antigen but not with CD8 antigen by suboptimal doses of 8H3 and corresponding antibodies initiated the mobilization of [Ca2+]i. Furthermore, physical association of 8H3 antigen with TCR was demonstrated by comodulation of these two molecular complexes in some T cells. Thus, 8H3 antigen is involved in rat T cell transmembrane signal transduction.

Thrombocytopenia after bone marrow transplantation for leukaemia: changes in megakaryocyte growth and growth‐promoting activity

Megakaryocyte growth-promoting activity (MK-GPA) was scored on a scale of 0-3 in the serum of 23 patients up to 120 d following bone marrow transplantation (BMT) for leukaemia. Nine of 19 allografts and two of four autografts had thrombocytopenia requiring platelet transfusion more than 30 d after BMT. There was a close correlation between MK-GPA and platelet count. MK-GPA reached a maximum before day 30 after BMT but remained elevated in patients with persisting thrombocytopenia secondary to poor engraftment, graft-versus-host disease (GVHD) or relapse. Recent platelet transfusion did not suppress serum MK-GPA. Two of four patients undergoing autologous BMT for acute myeloid leukaemia (AML) showed delayed platelet recovery and persistence of MK-GPA in the serum. Seven further AML remission marrows were tested for megakaryocyte production before or after autologous BMT, using pooled sera with known MK-GPA activity. Megakaryocyte generation was reduced before BMT and absent in post transplant samples. This failure of MK production was not corrected by T-cell depletion or by the presence of adherent cells from normal marrow. We conclude that thrombocytopenia after BMT is associated with an appropriate increase in MK-GPA levels in response to a reduction in the megakaryocyte pool rather than the platelet pool, and that persisting thrombocytopenia after autologous BMT is due to decreased numbers of available megakaryocyte precursors.

Polyclonal in vitro proliferative responses from nonimmune donors to Plasmodium falciparum malaria antigens require UCHL1+ (memory) T cells*

The in vitro polyclonal proliferative responses of peripheral blood mononuclear cells to whole blood stage parasites or fractionated antigens from the human malaria parasite Plasmodium falciparum were studied. Cells from healthy laboratory donors who had never been exposed to malaria antigens in vivo consistently proliferated to P. falciparum antigens, as did cord blood mononuclear cells. This response was only observed in sheep rosette-positive cells in the presence of adherent cells and was inhibited by NH4Cl, indicating a requirement for antigen processing. The proliferative response was strongest at day 6 and was dependent on the presence of cells expressing high levels of CD45 180-kD isomer (UCHL1 monoclonal antibody), a marker for activated or memory cells, but not for CD45R (SN130 monoclonal antibody) a marker for naive or unprimed T cells. This suggests a similarity to the recall response to tuberculin antigen. These results suggest that the proliferative response to malaria antigens observed previously and described as a nonspecific mitogenic response may be a cross-reactive response to epitopes shared between P. falciparum and other common immunogens. This would explain the establishment of T cell clones to malaria antigens from such donors, but might suggest that the epitopes to which such clones are specific may be of questionable protective or diagnostic use.

Enhancement of Colony-Forming Activity of Granulocyte-Macrophage Colony-Stimulating Factor by Monocytes In Vitro

Human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) stimulated granulocyte-macrophage (GM) colony formation from human marrow mononuclear cells (MMCs) in a dose-dependent manner in methylcellulose culture. When phagocytes were depleted from MMCs, GM colony formation from the phagocyte-depleted (PD) MMCs by hrGM-CSF markedly decreased. Experiments in which PD-MMCs were cultured with hrGM-CSF and adherent cells showed that 94% (on day 7 and day 14) of the colonies from PD-MMCs were dependent on the presence of adherent cells. In contrast, the ability of granulocyte colony-stimulating factor (G-CSF) to form colonies was not affected by phagocyte depletion. To check for the presence or absence of progenitors that could form GM colonies in direct response to hrGM-CSF, single-cell culture of hematopoietic progenitor cell surface antigen (My-10)-positive PD-MMCs was carried out using a flow cytometer and an Autoclone System. In duplicate experiments, 0.7% and 3.5% (day 7) or 3.6% and 3.9% (day 14) of My-10-positive PD-MMCs formed GM colonies in response to hrGM-CSF and 5.1% and 6.0% (day 7) of My-10-positive PD-MMCs formed GM colonies in response to G-CSF. This was clear evidence for the presence of progenitors directly responding to hrGM-CSF. Also observed was a synergistic effect on GM colony formation in which more My-10-positive PD-MMCs stimulated by hrGM-CSF and G-CSF could form GM colonies than the sum of those stimulated by each separately. This enhancing effect of colony-forming activity of hrGM-CSF by adherent cells and the single cell culture experiment were reproduced in serum-free culture system.

Enhancement of colony-forming activity of granulocyte-macrophage colony- stimulating factor by monocytes in vitro

Human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) stimulated granulocyte-macrophage (GM) colony formation from human marrow mononuclear cells (MMCs) in a dose-dependent manner in methylcellulose culture. When phagocytes were depleted from MMCs, GM colony formation from the phagocyte-depleted (PD) MMCs by hrGM-CSF markedly decreased. Experiments in which PD-MMCs were cultured with hrGM-CSF and adherent cells showed that 94% (on day 7 and day 14) of the colonies from PD-MMCs were dependent on the presence of adherent cells. In contrast, the ability of granulocyte colony-stimulating factor (G-CSF) to form colonies was not affected by phagocyte depletion. To check for the presence or absence of progenitors that could form GM colonies in direct response to hrGM-CSF, single-cell culture of hematopoietic progenitor cell surface antigen (My-10)- positive PD-MMCs was carried out using a flow cytometer and an Autoclone System. In duplicate experiments, 0.7% and 3.5% (day 7) or 3.6% and 3.9% (day 14) of My-10-positive PD-MMCs formed GM colonies in response to hrGM-CSF and 5.1% and 6.0% (day 7) of My-10-positive PD- MMCs formed GM colonies in response to G-CSF. This was clear evidence for the presence of progenitors directly responding to hrGM-CSF. Also observed was a synergistic effect on GM colony formation in which more My-10-positive PD-MMCs stimulated by hrGM-CSF and G-CSF could form GM colonies than the sum of those stimulated by each separately. This enhancing effect of colony-forming activity of hrGM-CSF by adherent cells and the single cell culture experiment were reproduced in serum- free culture system.

Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery.

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.

Glycoproteic nature of surface molecules of effector cells with lymphokine-activated killer (LAK) activity. Evidence that T11, T8 or T3 molecules are not involved in tumor-cell lysis by LAK effector T cells.

Human peripheral blood mononuclear cells cultured in the presence of interleukin-2 (IL-2) acquire the capability of lysing NK-resistant fresh tumor target cells. In an attempt to delineate the surface structure(s) present on the effector cells, the latter were first treated with different amounts of pronase and neuraminidase. The effect of the enzymes on cytolytic activity against fresh melanoma cells was evaluated and compared with the NK-like activity against K562 target cells of the same effector population. At a pronase concentration of 0.01 mg/ml, no inhibition of NK-like activity was detected, whereas LAK activity was inhibited by more than 75%. In addition, neuraminidase had no effect on NK-like activity, even at 1 U/ml, whereas as little as 0.03 U/ml inhibited LAK activity by more than 75%. Metabolic inhibition of N-linked glycosylation with Tunicamycin prevented the generation of LAK activity, even when added late (18 hr before termination of the culture). Tunicamycin, on the other hand, had no effect on the boost of NK activity induced by IL-2. Provided that LAK activity can also be generated in T-cell (E-rosetting) populations, in the presence of adherent cells, we analyzed the inhibitory activity of monoclonal antibodies (MAbs) to T11, T3 and T8 molecules. While all these MAbs strongly inhibited the specific target cell lysis by alloreactive CTLs, they had no effect on the LAK activity.