Addition Of PHA Research Articles

Evolutionary conservation in various mammalian species of the human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody.

The human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody (MAb) was detected in proliferating normal and neoplastic cells of many mammalian species (lamb, calf, dog, rabbit, rat) besides human. In contrast, Ki-67 stained proliferating cells from other species weakly (mouse) or not at all (swine, cat, chicken, pigeon). The immunostaining pattern of Ki-67 in animal tissues was identical to that previously described in human: Ki-67 reacted only with cells known to proliferate (e.g., germinal center cells, cortical thymocytes) but not with resting cells (e.g., hepatocytes, brain cells, renal cells); this MAb produced a characteristic nuclear staining pattern (e.g., stronger labeling of nucleoli than of the rest of the nuclei and staining of chromosomes in mitotic figures); and Ki-67 crossreacted with the squamous epithelium in both animal and human tissues. In vitro studies showed that when quiescent (Ki-67-negative) NIH 3T3 fibroblasts or bovine peripheral blood lymphocytes were induced to proliferate, the appearance of Ki-67-positive cells paralleled the induction of cell proliferation caused by addition of fetal calf serum or PHA, respectively, to the cultures, and in both human and rat proliferating cells the Ki-67 expression closely paralleled the incorporation of [3H]-thymidine. These findings indicate that the epitope recognized by the Ki-67 MAb in human and animal species is the same. The widespread evolutionary conservation of the human proliferation-associated epitope recognized by the Ki-67 MAb suggests that it and/or its carrier molecule may play an important role in regulation of cell proliferation.

Histamine regulates the generation of human cytolytic T lymphocytes

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10 −8–10 −4 M) suppressed the generation of class I specific CTL but, at 10 −4 M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H 2 antagonist ranitidine but not with the H 1 antagonist pyrilamine. These data suggest that suppression was mediated by the H 2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T Cells.

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells.

Abstract The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.

HIV-induced immunodeficiency. Relatively preserved phytohemagglutinin as opposed to decreased pokeweed mitogen responses may be due to possibly preserved responses via CD2/phytohemagglutinin pathway.

We studied the proliferative response of PBL to the mitogens PHA and PWM and Candida albicans Ag in 301 HIV seropositive homosexual men, of whom 55 had AIDS. The responses to PHA were reduced only in the clinically ill HIV seropositive subjects. In contrast, the responses to PWM were profoundly reduced in most HIV seropositive subjects including the asymptomatic group. Further analysis of 16 HIV seropositive subjects showed that the proliferative responses were reduced in both CD4 and CD8 T cell subsets. A total of 15 HIV seropositive individuals with low responses to PWM, of whom seven had AIDS and eight controls were chosen for the following studies. Expression of T3, Ti, delta receptors, and CD2 was investigated and showed an increased percentage of CD2 receptors positive cells in HIV seropositive subjects without AIDS. The proliferative responses of PBL to stimulation with PHA, PWM, antibodies to CD3, or antibodies to CD2 were investigated and showed significant correlation in controls, whereas in contrast, only the responses to PHA and CD2ab correlated in patients with AIDS. The proliferative responses to CD2ab and CD3ab in controls were larger than the responses to both PHA and PWM. In patients, these responses were less suppressed than the responses to PWM indicating that stimulation with mitogens is more complex than a simple stimulation of Ti/T3 and CD2 receptors. Further investigations were done on resting T cells, i.e., lymphocytes depleted of macrophages and pre-activated cells. Addition of PHA to these cells resulted in preactivation with expression of IL-2R (CD25) but not in proliferation. In contrast, addition of PHA plus SRBC, which bind to the CD2 receptors caused IL-2R expression, IL-2 production, and proliferation. Addition of PWM + SRBC did not result in proliferation. A comparison of the responses to PHA + SRBC of resting T cells from 26 HIV seropositive individuals, of whom seven had AIDS and 12 seronegative controls, showed that these responses were normal or only slightly decreased in the 19 seropositive men without AIDS whereas it was decreased in AIDS patients. Nevertheless, all AIDS patients showed clear-cut responses in this assay. Thus, the discrepancy between responses to PHA and PWM may be explained by an at least partially preserved function of the PHA/CD2-dependent pathway. We suggest that the defect induced by the HIV infection primarily concerns T3/Ti-induced responses.

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): Studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production and proliferation

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbolester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca 2+] 1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/ PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.

Sister-chromatid exchanges in human lymphocytes exposed to 1- p-(3-methyltriazeno)benzoic acid potassium salt

The 1- p-(3-methyltriazeno) benzoic acid potassium salt (MTBA) is a triazeno analogue of dacarbazine, an antineoplastic agent capable of mediating the appearance of new antigenic specificities on cancer cells in mice, a phenomenon described as ‘chemical xenogenization’ (CX). Recently we reported the clastogenic potential of MTBA on human lymphocytes. Since sister-chromatid exchange (SCE) assay is more sensitive than clastogenic tests, at least at low drug concentrations, we assessed SCE frequencies induced by MTBA on human lymphocytes stimulated by PHA. Drug treatment at 2–500 μg/ml was performed in vitro prior to or after PHA addition. SCE values increased significantly in a dose-dependent manner up to 200 μg/ml. However, SCE frequencies, as well as chromosome breaks, did not increase dramatically. These data indicate that MTBA concentrations used for CX do not cause severe cytogenetic damage to immune cells at least in vitro.

Relative cloning efficiencies and long-term propagation capacity for T cell clones of highly purified natural interleukin 2 compared to recombinant interleukin 2 in man

Highly purified (HP) natural (n) and recombinant (r) interleukin 2 (IL 2) have beencompared for their ability to support clonal outgrowth and long-term clonal propagation of alloactivated human T lymphocytes. The frequency of outgrowth of T cell colonies was as high as 1:2 to 1:4 when HPnIL 2 was employed for limiting dilution cloning. Separated CD4 + and CD8 + activated populations produced similar cloning efficiencies. Recloning of established CD4 + lines in HPnIL 2 suggested that a clonable cell frequency of 1:2–1:4 was the maximum possible that could be achieved in this system. In contrast, purified rIL 2 allowed outgrowth of only 1:10–1:20 cells from alloactivated populations. Again, CD4 + and CD8 + fractions generated similar cloning efficiencies. In terms of the fraction of derived clones, which could be propagated to > 1 × 10 6 and > 1 × 10 8 cells, nIL 2 again proved superior to rIL 2. In either source of IL 2, the proportion of CD4 + clones which could be extensively propagated was greater than in the CD8 + population. Surprisingly, although the addition of PHA to these lectin-free IL 2 preparations reduced the frequency of clonable cells, the proportion of clones which could be extensively propagated was increased. These results suggest that rIL 2, consisting of three differently glycosylated molecular species, may be preferable to rIL 2, which consists only of a single non-glycosylated species, for the cloning and long-term propagation of human T cells. These results may have some bearing on the choice of rIL 2 versus nIL 2 for therapeutic applications.

Cell growth associated regulation of c-myc and c-fos in normal human T cells.

The mechanism whereby a quiescent lymphocyte is activated to proliferate and express differentiated functions is a fundamental question for understanding immune regulation and growth control. The binding of ligands such as antigen, mitogenic lectins, and some cell surface-specific antibodies to their membrane receptors on lymphocytes generates a transmembrane signal resulting in a variety of metabolic changes culminating after 24 hours in DNA synthesis (1,2). One initial consequence of transmembrane signalling is the expression of mRNA species novel to activated cells. For example, the cellular proto-oncogene c-myc (3), the T cell growth factor, IL-2 (4), and the IL-2 receptor (5,6) increase expression within approximately 1, 12, and 6 hours, respectively, following mitogenic stimulation of T cells. In additional mRNA species, the proto-oncogene c-fos, between 30 and 90 minutes after PHA addition to human T cells. Such increases occur in the presence of protein syrithesis inhibitors (3,6), indicating that mRNA induction is a direct result of the signals that follow receptor binding and as such probably represent primary transcriptional events.

Differentiation of myeloid cells in liquid culture. 1. Progenitor cells found in normal peripheral blood.

In order to establish a method for studying myeloid differentiation, light density, non-adherent, T-cell depleted mononuclear cells prepared from 26 normal peripheral blood buffy-coats were cultured in McCoy'5A medium supplemented with 15 per cent fetal calf serum (FCS) for three weeks at 37 degrees C in a humidified 5 per cent CO2 atmosphere. The total number of viable cells in the cultures on weeks 1 and 2 represented 73 +/- 10 per cent and 98 +/- 41 per cent of the initial number of viable cells seeded. After one week, blasts represented 26 +/- 10 per cent of the initial number of viable cells while all the initially contaminating mature granulocytes had disappeared. After two weeks, granulocytic differentiation was noted in most cultures and viable myelocytes and more mature cells represented 45 +/- 26 per cent of the initial number of viable cells. The differentiation was independent on the lot of FCS used. The addition of PHA stimulated leukocyte conditioned medium to the cultures did not enhance granulocytic differentiation. The granulocytic differentiation observed in the absence of exogenous CSF persisted after removing the cells adhering to the bottom of the flasks on day 2 of the culture. An endogenous colony stimulating activity was detected in the cultures on week 3 but its intensity did not clearly correlate with the degree of granulocytic differentiation.

Functional properties in Sézary cells with an unusual phenotype

The immunological and functional characteristics of Sézary cells with an unusual phenotype are reported. The clinical, histologic, and hematologic picture was typical for Sézary syndrome. Studies with monoclonal antibodies showed that 80% Sézary cells had an CD3+, CD4+, CD5+, CD7−, CD8−, Leu-7+, Leu-8−, Leu-11−, OKM1− phenotype. By two-color immunofluorescence assay 80% FACS-sorted Leu-7+ cells coexpressed CD4 antigen and did not express the myeloid antigen OKM1, CD8, and antigens characteristic of immature T cells. The cells had no NK activity but did display a high helper activity. Unseparated and FACS-sorted Leu-7+ and Leu-7− Sézary cells did not respond to mitogens but were able to grow in the presence of exogenous IL-2. FACS sorted Leu-7− cells, cultured for 7 days in the presence of 20% IL-2, acquired the receptors for Leu-7. IL-2 and IFN-γ production was studied in unseparated Leu-7+ and Leu-7− FACS-sorted Sézary cells. IL-2 production was lower than in normal cells. The addition of PHA or PHA plus TPA led to an increase in IL-2 production. Also IFN-γ production was marked lower than in normal controls but increased after 7-day culture in exogenous IL-2. In conclusion in this case the Sézary cells may represent a neoplastic expansion of the CD3+, CD4+, CD5+, Leu-7+, Leu-11− subpopulation which is equivalent to the 2–4% of the Leu-7+ population in normal lymphocytes.

Cell-mediated lysis of murine target cells by nonimmune salmonid lymphoid preparations

Lymphoid preparations from nonimmune rainbow and brook trout were found to lyse murine tumor cells (EL-4 & P815Y) invitro in an 18 hr 51Cr-release assay conducted at 16–18°C. Lysis was proportional to the effector: target cell ratio, required direct cell to cell contact, and was not depleted by the removal of nylon wool adherent cells. Lymphoid populations from peripheral blood, the thymus, and the anterior kidney, but not the spleen, were active in the cytotoxicity assay. Individual fish varied considerably in their ability to lyse one or both target cells. These data and the results of unlabelled target cell inhibition studies suggest that the reaction is selective if not specific. The addition of PHA to the reaction mixture resulted in markedly enhanced cytotoxic reactivity. In the presence of PHA lysis was readily detectable at 4 hr. The data demonstrate that nonimmune Salmonids possess a cytolytic effector cell population which has considerable cytotoxic potential and may represent a heterogeneous “natural killer cell” population.

The control of transferrin receptor synthesis in mitogen-stimulated human lymphocytes

Human lymphocytes cultured in the presence of the plant mitogenic lectin phytohemagglutin in (PHA) become activated and leave the G0 phase of the cell cycle. In the presence of PHA and lymphokines produced in situ the cells will enter S phase and undergo cell division. We have determined the time course of appearance for the receptor for transferrin as an initial attempt to understand the molecular mechanisms regulating the onset of lymphocyte differentiation and proliferation in the 48 h following PHA addition. Using three different assay methods we have shown that the increase in the number of surface receptor molecules is due to the accumulation of newly synthesized receptor and not to the redistribution of a previously existing pool of receptor molecules. The total amount of transferrin receptor increased at least four-fold. In vitro translation of RNA from activated lymphocytes indicates that the new receptor synthesis is due, at least in part, to increased availability of mRNA encoding the transferrin receptor.

The effects of cyclosporin and hydrocortisone on human T lymphocyte colony formation

The immunosuppressive activities of two compounds, cyclosporin A and hydrocortisone, were evaluated using a human T lymphocyte colony assay. Both compounds produced a dose-related reduction in colony formation. With cyclosporin, concentrations of 1.0–100 μg/ml virtually abolished PHA-induced lymphocyte growth; as little as 0.01 μg/ml decreased clonal growth by 48%. Suppression was observed as late as 4 days after the addition of PHA. The addition of exogenous IL-2 did not completely restore clonal growth to normal. Similarly, hydrocortisone, in concentrations of 0.4 μg/ml, produced a 96% inhibition in clonal growth. Partial suppression could also be obtained if the drug was added as late as 4 days after PHA. Exogenous IL-2 completely reversed the inhibitory effects of hydrocortisone. By contrast, IL-1 was able to only partially restore growth potential. Parallel studies using TPA indicated that both immunosup-pressants inhibited responses to this mitogen. However, in plates containing both TPA and PHA, hydrocortisone failed to impair clonal growth.

The effect of alpha 1 antitrypsin on the proliferative response of human peripheral blood lymphocytes.

In evaluating immune aberrations in patients with alpha 1 antitrypsin (alpha 1 AT) deficiency, we have previously shown that they exhibit enhanced lymphocyte responsiveness to PHA that is serum mediated. In this study, we demonstrate suppression of the PHA response by using purified alpha 1 AT, and also similar but less marked suppression of the Con A response. alpha 1 AT, however, has no effect whatsoever on PWM-induced proliferation. This effect is demonstrable provided alpha 1 AT is added within 4 hr of mitogen activation and is mediated by its action on adherent cells rather than on proliferating lymphocytes. Adherent cells still exhibit this effect if pulsed with alpha 1 AT, then thoroughly washed before their activation. This suggests that it may be inhibiting a membrane serine esterase already activated before the addition of PHA. Thus alpha 1 AT may modulate the activation of T cells through its effect on monocytes, leading to abnormalities in immunoregulation, and hence a predisposition to the development of a variety of immunologic disorders in alpha 1 AT-deficient subjects.

Non-Immunological Recognition and Killing of Xenogeneic Cells by Macrophages: III Destruction of Fish Cells by Murine Macrophages

Non-immune non-activated murine peritoneal macrophages killed in vitro fish (Cyprinus carpio) PHA-induced lymphoblasts. Addition of PHA and WGA to effector-target cell cultures did not potentiate the killing. This killing (xenolysis) was expressed by non-elicited and thioglycollate-elicited macrophages as well as by macrophages depleted of lymphocytes. It is suggested that mammalian macrophages have a xenolytic potential towards phylogenetically distant species which is analogous to the capacity of invertebrate phagocytes to destroy xenografts.

Activation of human blood lymphocyte subsets for cytotoxic potential

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.

Radiosensitivity of lymphocytes separated from rat lymph nodes and human peripheral blood.

As a criterion for inactivation of lymphocytes after irradiation, loss of capacity for stimulation after addition of mitogens such as PHA is often used (Conrad, 1969; Carson et al., 1965). A simple measure of stimulating ability is the incorporation of radioactive thymidine into DNA, measured by liquid scintillation counting after addition of PHA. This method has been found useful in estimation of whole-body absorbed dosage in man. For human lymphocytes isolated from peripheral blood, the radiosensitivity has been well established (Ilberty et al., 1971 ; Baral and Blomgren, 1976; 1978).

908 SUPPRESSOR CELL ACTIVITY IN NEONATAL PERIPHERAL BLOOD

A technique for assessing suppressor cell activity in peripheral blood of healthy premature neonates was established using ConA induced suppression of the PHA response in vitro. Neonatal lymphocytes were separated in routine fashion and placed in preculture with ConA (1μg/ml). Cells were also precultured without ConA to provide information on spontaneous suppressor cell activity. After 48 hours incubation, cells were collected, washed and irradiated with 3000r. Effect of precultured cells on PHA stimulated blastogenesis was studied by microculture technique using 105 inactivated cells and an equal number of fresh, autologous lymphocytes (responder cells). Controls were provided by addition of PHA to responder cells alone. Normal adult cells were used for comparison cultures. Data were expressed as one minus the ratio (× 100) of the observed mean cpm of the precultured plus fresh cell blastogenic response to the observed mean cpm of the neonatal control response. Preculture of neonatal lymphocytes yielded suppression in the majority of samples, 73% (mean 33 ± 24) with and 55% (mean 23 ± 15) without ConA; conversely, stimulation was seen in 18% and 27% respectively. Adult studies showed suppression in 25%, unchanged by ConA, but showed stimulation in 50% (mean 45 ± 35) with and 58% (mean 35 ± 25) without ConA. We conclude that neonatal lymphocytes, as studied in this system, demonstrate a propensity to suppress PHA induced blastogenesis.

Inhibition of polypeptide synthesis by a factor isolated from ribosomes of resting human lymphocytes: Studies on the mechanism of action

Peripheral blood lymphocytes are non-dividing cells which synthesize small amounts of RNA and protein. These resting cells are stimulated when incubated in the presence of mitogens, and after a variety of metabolic changes which include a marked increase of RNA and protein synthesis, they start DNA replication and reach a state of active proliferation [ 1,2]. The enhancement of protein synthesis occurring during the first hours after the addition of PHA to a lymphocyte culture does not require the synthesis of RNA 13-51, indicating that during this period preexisting mRNA molecules and ribosomal particles are being utilized. Thus lymphocyte activation may involve regulation at the translational level besides other controls which might be occurring on transcription and DNA replication. Experiments with cell-free systems have indicated that the low rate of protein synthesis observed in unstimulated lymphocytes is probably due to the absence of initiation factors [6] and/or to the presence of cytoplasmic inhibitors which block the formation of initiation complexes [7,8]. The possibility of a more complex control of protein synthesis in lymphocytes has been reinforced by the isolation of a new ribosome-bound translational inhibitor (TI) obtained from resting human lymphocytes [9].

Cytidine 5′-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes

The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h.