Blast Cell Formation Research Articles

611 Introduction of specificity into cytotoxic drugs and improvement of therapeutic index by kinase-mediated trapping

The polyamines putrescine, spermidine, and spermine are oxidized by the enzyme diamine oxidase to form the corresponding aldehyde derivatives. These aldehydes have been shown to undergo a variety of spontaneous reactions, some of which result in polycationic addition compounds. We have chemically synthesized some spermine-derived polycations by reaction with the dialdehyde glutaraldehyde followed by reduction of the resulting Schiff base with sodium borohydride. Their migration on ion exchange and gel filtration columns was consistent with the formation of polycations with properties similar to those reported for the spontaneous reaction products. When added to cultures of alloantigen or mitogen stimulated lymphocytes, these polycations were potent inhibitors of the incorporation of tritiated thymidine and blast cell formation. This inhibition was reversible, non-cytotoxic, and only apparent if the polycation was added early in the culture period. The concentration of polycation necessary to achieve 50% inhibition of the lymphocyte response decreased as the cationic nature relative to spermine increased.

Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection.

Normal levels of CD4(+) regulatory T cells are critical for the maintenance of immunological homeostasis and the prevention of autoimmune diseases. However, we now show that the expansion of CD4(+) regulatory T cells in response to a chronic viral infection can lead to immunosuppression. Mice persistently infected with Friend retrovirus develop approximately twice the normal percentage of splenic CD4(+) regulatory T cells and lose their ability to reject certain tumor transplants. The role of CD4(+) regulatory T cells was demonstrated by the transmission of immunosuppression to uninfected mice by adoptive transfers of CD4(+) T cells. CD4(+) T cells from chronically infected mice were also immunosuppressive in vitro, inhibiting the generation of cytolytic T lymphocytes in mixed lymphocyte cultures. Inhibition occurred at the level of blast-cell formation through a mechanism or mechanisms involving transforming growth factor-beta and the cell surface molecule CTLA-4 (CD152). These results suggest a possible explanation for HIV- and human T cell leukemia virus-I-induced immunosuppression in the absence of T cell depletion.

Secretion of Neutral and Acid DNases in Cultivated Human Lymphocytes after Incubation with DNA; Possible Consequences for Inhalation Anesthesia

After incubation with DNA human lymphocytes release neutral and acid DNase activities into the culture medium; the release depends on DNA concentration and time of cultivation. The electrophoretic mobility of the released neutral DNase activity is in accordance with DNase I and the electrophoretic mobility of the released acid DNase activity with DNase II. The released DNase activities do not originate from dead cells and are not influenced by blast cell formation. The anesthetic halothane can inhibit the released neutral and acid DNase activities. Inhalation anesthesia can possibly disturb the correlation between DNA and DNases in human blood.

Corneal endothelial cells block T cell proliferation, but not T cell activation or responsiveness to exogenous IL-2.

We previously reported that Ia+ monolayers of LEW rat corneal endothelial (CE) cells were unable to stimulate proliferation of MHC compatible T cell lines or IL-2 release from hybridomas, and inhibited [3H]-thymidine incorporation when added to conventional lymphocyte proliferation assays. Our purpose was to further analyze the mechanism of the inhibitory activity of CE cells on T lymphocyte activation. Mitogen-induced proliferative responses of splenocytes were found to be as susceptible to inhibition by CE cells as previously reported for antigen-specific activation of T cell lines. Antigen presenting cell (APC) antigen pulsing experiments showed that CE cells did not inhibit antigen processing. Flow cytometry and microscopic observation of the co-cultures revealed that T cells became activated in the presence of antigen, APC and CE cells, exhibiting morphologic changes of blast cell formation, although they did not divide unless given exogenous IL-2. However, if T cells were preactivated in the absence of CE cells, they were no longer susceptible to inhibition if subsequently transferred into CE cell-conditioned medium or onto CE cells. Evidence for an inhibitory factor in CE cell culture supernatant was revealed by two approaches: 1) addition of conditioned medium from CE cell cultures led to inhibition of lymphocyte proliferation assays, and 2) split-well assays also demonstrated the existence of a cell-free immunosuppressive factor produced by the CE cells. However, the inhibition mediated by supernatant alone was less potent than that by direct T cell contact with CE cells, implying that cell-cell interaction contributed to the inhibition. Indomethacin, a prostaglandin synthetase inhibitor, did not reverse CE cell-mediated inhibition. Neutralizing antibodies to TGF-beta 1 and 2 did not reverse the inhibition by CE cells. In summary, T cells received activation signals from APC in the presence of CE cells, but proliferation was inhibited unless exogenous lymphokine was added.

Production of a suppressor of lymphocyte proliferation by two human oral carcinoma cell lines.

This study examined the production of an immunosuppressive factor by the KB and H191 human oral squamous carcinoma cell lines. Conditioned media (CM) from both cell lines markedly inhibited mitogen- and alloantigen-induced proliferation of normal human and rat peripheral blood lymphocytes. By contrast, the proliferation of an exponentially-growing fibroblast cell line remained unchanged by CM. The immunosuppressive factor appeared to act after lymphocyte commitment as indicated by continued blast cell formation, the failure of CM to suppress resting lymphocytes and the fact that CM caused maximum inhibition of lymphocyte proliferation 72 h after the addition of PHA. The addition of exogenous IL-2 did not counteract lymphocyte suppression. Inclusion of indomethacin and isoniazid during cell culture did not significantly alter the degree of suppressive activity. Mycoplasma contamination was absent and CM did not act directly with the thymidine or mitogen. The factor was heat stable at 50 degrees C, acid labile and had a molecular weight in excess of 300 kDa. The results demonstrate that human oral squamous carcinoma cell lines produce an immunosuppressive factor that may have a role in tumour evasion of the host immune response.

T‐Cell Phenotypes after Stimulation of Human Mononuclear Cells by Pokeweed Mitogen or Pokeweed Mitogen Bound to Erythrocytes

Stimulation of human peripheral blood mononuclear cells (PBMC) by pokeweed mitogen bound to erythrocytes (E-PWM) has been found to result in an increased blast cell formation, lymphocyte proliferation, and enhanced immunoglobulin production, compared to stimulation of PBMC by PWM. Using flow cytometric analysis we compared the T helper/inducer (CD4+) to T suppressor/cytotoxic (CD8+) cell ratios of PBMC after stimulation by PWM and by E-PWM. E-PWM was found to induce significantly lower CD4+:CD8+ ratios on days 6 and 9 of the culture than PWM did. This effect was due predominantly to a relative increase in CD8+ T cells after stimulation of PBMC by E-PWM, compared with stimulation by PWM. However, the increase in T suppressor/cytotoxic cells on days 6 and 9 after E-PWM stimulation was accompanied by a simultaneous relative decrease in CD4+/2H4+ T cells (T suppressor-inducer cells) and by a relative decrease in CD8+/Leu-15+ T cells (CD4+/2H4+ independent T suppressor cells) on day 9, compared with stimulation by PWM. These results suggest that the greater immunoglobulin production after stimulation of PBMC by E-PWM than by PWM may be the result of a relative lack of suppression on fully differentiated B cells.

Potentiation of the stimulatory capacity of pokeweed mitogen via its binding to autologous erythrocytes.

The effect of human erythrocytes (E) on blastogenesis and immunoglobulin (Ig) production induced by pokeweed mitogen (PWM) in cultures of human peripheral blood mononuclear cells (PBMC) was investigated. The stimulation of PBMC with PWM was markedly enhanced in the presence of E, and PWM bound to E, followed by thorough washing (E-PWM), was even more effective in inducing blast cell formation and Ig production. Blast cell responses after stimulation with E-PWM were on average two times higher than those seen after stimulation with comparable dilutions of fluid-phase PWM. The PWM that remained in solution after incubation with E (Sup E-PWM) had little mitogenic capacity and inhibited the blast cell response induced by fluid-phase PWM. Transwell culture experiments demonstrated that the enhancement of the blast cell response of PBMC by E-PWM could be induced by PWM that was released from E-PWM, whereas the enhancement of Ig production was found to be dependent on the presence of PWM on E. Both for blast cell formation and Ig production, it was found that the enhanced stimulation with E-PWM depended on the presence of monocytes.

Anti-pokeweed mitogen antiserum inhibits and enhances blastogenesis of mononuclear cells induced by pokeweed mitogen.

The interaction between pokeweed mitogen (PWM) and peripheral blood mononuclear cells (PBMC) was investigated using rabbit anti-PWM antiserum (anti-PWM) and 125I-PWM. Incubation of PBMC with PWM in the presence of anti-PWM resulted in an inhibition of the mitogenic effect of PWM. Anti-PWM predominantly blocked the interaction of PWM with monocytes, which is essential for optimal stimulation of lymphoid cells with PWM. Addition of anti-PWM to PBMC at several time-points after incubation with PWM showed inhibition of mitogenic activity when anti-PWM was added within 8 hours. However, enhancement of PWM-induced blast cell formation was found when anti-PWM was added after 48 hours. Further analysis revealed that the inhibition of PWM stimulation was mediated by the F(ab')2 part of anti-PWM IgG. On the other hand F(ab')2-anti-PWM was not able to enhance the effect of PWM. Incubation of PBMC with 125I-PWM and anti-PWM simultaneously, decreased the binding of PWM to both lymphocytes and monocytes. In contrast, addition of anti-PWM 48 hours after the incubation of PBMC with PWM resulted in an increased binding of PWM to monocytes. These results show that anti-PWM can modulate the lymphocyte reaction to PWM and suggest two possible mechanisms by which PWM can stimulate PBMC, both of which are dependent on the interaction of PWM with monocytes.

Replication of infectious bursal disease virus in lymphoid cells.

Replication of infectious bursal disease virus in lymphoid cells from the bursa of Fabricius occurs preferentially in a population of proliferating cells characterized by increased thymidine incorporation and by blast cell formation. This was demonstrated by enrichment of virus producing cells in gradients of bovine serum albumin and by mitogenic stimulation of cells with fetal calf serum or an extract prepared from bursae of susceptible chickens. Susceptibility of lymphoid bursa cells is not correlated with the expression of immunoglobulins on their surface. This conclusion was reached after the enrichment of Ig-bearing cells by rosetting techniques and by separation through a cell sorter.

Inhibition of lymphocyte DNA-synthetic responses by spermine-derived polycations

The polyamines putrescine, spermidine, and spermine are oxidized by the enzyme diamine oxidase to form the corresponding aldehyde derivatives. These aldehydes have been shown to undergo a variety of spontaneous reactions, some of which result in polycationic addition compounds. We have chemically synthesized some spermine-derived polycations by reaction with the dialdehyde glutaraldehyde followed by reduction of the resulting Schiff base with sodium borohydride. Their migration on ion exchange and gel filtration columns was consistent with the formation of polycations with properties similar to those reported for the spontaneous reaction products. When added to cultures of alloantigen or mitogen stimulated lymphocytes, these polycations were potent inhibitors of the incorporation of tritiated thymidine and blast cell formation. This inhibition was reversible, non-cytotoxic, and only apparent if the polycation was added early in the culture period. The concentration of polycation necessary to achieve 50% inhibition of the lymphocyte response decreased as the cationic nature relative to spermine increased.

Inhibitory effect of leukemic plasma on periodate-induced lymphocyte transformation

Plasma from nine out of 18 patients with untreated acute lymphoblastic leukemia (ALL) depressed the transformation of normal blood lymphocytes induced by sodium periodate (Na10 4) as judged by reduction of blast cell formation and [ 3H]thymidine and [ 3H]uridine incorporation into DNA and RNA respectively. The depressed mitogen responsiveness of lymphocytes cultured in the presence of leukemic plasma was due to the presence of inhibitory factor(s) present in the plasma rather than the absence of components present in normal plasma. The inhibitory effect of leukemic plasma on periodate-induced cell stimulation indicated that the leukemic plasma inhibitory factor(s) exert their action very rapidly and directly on the cell. Transfer of the inhibitory factor(s) from the leukemic plasma to the cultured cells was supported by the finding that the depressive action of leukemic plasma on lectin and non-lectin mitogen-induced transformation of lymphocytes was reduced if the leukemic plasma was preincubated with either resting or mitogen-treated lymphocytes prior to testing with lymphocytes not previously exposed to leukemic plasma or mitogen.

3H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12- O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12- O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [ 3H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [ 3H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [ 3H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.

In vitro effect of DDS on phytohaemagglutinin (PHA) induced lymphocyte transformation

A study to find out the in vitro effect of diamino diphenyl sulphone (DDS) on phytohaemagglutinin (PHA) induced lymphocyte transformation was carried out in three phases using a wide range of DDS concentrations. Lymphocytes from healthy volunteers were investigated. Volunteers were divided into three groups to conduct the study in three phases. In each phase in addition to 0.02 ml of PHA, 4 different concentrations of DDS were added per 106 lymphocytes in tissue culture system. A statistically significant depression (P<0.05) in the per cent of blast cell formation induced by PHA was observed in the cultures with all the concentrations of DDS except with the lowest concentration (0.01 μg) of DDS. While the depression observed in the first and the third phase was found to be dose dependent, no significant correlation was noted between the DDS concentration and depression in PHA induced blastogenesis in the second phase. The significance of these observations are discussed.

Investigations into the in vitro and in vivo immunosuppressant activity of [formula omitted] methyl ester, a potential l-asparagine antimetabolite

N- Acetyl-3- sulphonamoyl- l-alanine methyl ester (Asame) has been previously reported to inhibit mitogen-induced DNA synthesis by human lymphocytes in vitro and to exhibit immunosuppressant activity in monkeys in vivo without overt signs of toxicity. We have confirmed and extended the in vitro studies by demonstrating that Asame inhibits concanavalin A-induced DNA synthesis and blast cell formation by mouse spleen lymphocytes. However, a requirement for l-asparagine by cultured lymphocytes could not be demonstrated and the inhibitory effects of Asame were not reversed by l-asparagine. Thus, the in vitro immunosuppressant activity of Asame could not be attributed to interference with l-asparagine metabolism. Furthermore, no in vitro immunosuppressant activity could be demonstrated for Asame in mice. These results cast doubt on the concept that l-asparagine anti-metabolites have potential as immunosuppressants.

Human plasma-derived immunosuppressive factors having anti-lymphoma activity

Extraction of Cohn IV-1, an α-globulin enriched fraction of human plasma, with a high-salt, low-pH solution, followed by sequential ultrafiltration steps yielded an immunosuppressive preparation (UM05R) of mol.wt. 500–10,000. UM05R inhibited antibody formation in the mouse in vivo and transformation in vitro of lymphocytes treated with either T-or B-cell stimulants. Suppression of lymphocyte transformation, indicated by inhibition of 3H-thymidine incorporation into DNA, was confirmed by inhibition of blast cell formation. From dose-response curves the UM05R concentration to produce 50% suppression of lymphocyte blast transformation was 15–50 μg protein/ml. Selectivity for lymphoid cells was suggested by growth inhibition in vitro of L1210 and P1798 leukemias but not murine neuroblastoma or human fetal fibroblasts. This observation also rules out the presence of an agent which is broadly cytotoxic. Fractionation of UM05R on Sephadex G-25 in 10% acetic acid yielded an early-emerging fraction, mol. wt. 5,000–10,000, containing B-cell inhibitor, and a late fraction, mol. wt. 1,400, inhibitory for both T- and B-cell transformation and growth of L1210. The inhibitory activity for B cells was removed from the other two activities by 5% trichloroacetic acid (TCA). The possibility is raised that the inhibitory activity for T cells and L1210 may reside in the same molecule. Sensitivity of the early-emerging B-cell inhibitor to carboxypeptidase B suggests that it is a polypeptide, but resistance of the T-cell inhibitor to various treatments leaves its nature uncertain. The properties of these factors suggest consideration of them as lymphocyte chalones occurring in plasma complexed to high-molecular-weight components.

Lectin-driven maturation of cytotoxic effector cells: the nature of effector memory.

In an attempt to define further the activation of effector T-cell function with concanavalin A (Con A), we examined the ability of Con A to generate effector memory in mouse lymph node cells in vitro. In the course of these studies, it became necessary to define parameters by which memory could be defined. These parameters include length of time of exposure to signal required to generate full cytotoxic function; time of onset and kinetics of blast cell formation; requirement for DNA synthesis; sensitivity to the drug 5'-bromodeoxyuridine; and kinetics of the cytotoxic response to alloantigen. By these criteria, mouse lymph node cells exposed 12 days previously to Con A behave qualitatively differently from unprimed mouse lymphocytes. We found that the time of onset and kinetics of DNA synthesis could not be used to distinguish primary and secondary cytotoxic responses. We propose that the parameters defined in these stidues can be applied generally in determining whether a given cytotoxic response involves primed (memory) or unprimed cells.

Effect of Phytohemagglutinin on Enzymes of Thymidine Salvage Pathway of Cultured Chronic Lymphatic Leukemic Lymphocytes

Abstract N-L and CLL-L prior to cell culture gave evidence of high activities of TdR phosphorylase, TMP and TDP kinases, while TdR kinase and phosphatase activities were relatively low. Cells cultured without PHA showed no appreciable alteration in enzyme activities during three to five days of culture. Response to PHA with blast-cell formation in cultures of N-L or CLL-L (low-count cases) was associated with increased TdR kinase activity which became prominent at the time of increased DNA polymerase activity and DNA synthesis during the second to third day of culture. Cells from cases of CLL-L with high WBC counts which failed to respond to PHA showed no increase in TdR kinase activity but did show increased phosphatase activity. Increased TdR activity and increased DNA polymerase activity may both be needed for increased synthesis of DNA to occur in lymphocytes responding to PHA.

DNA polymerase activity of cultured normal and leukemic lymphocytes: Response to phytohemagglutinin

Normal lymphocytes when cultured with PHA showed increased DNA polymerase activity in 24 h. This preceded the onset of DNA synthesis. Further large increases in enzyme activity occurred in the next day or two of culture accompanying blast-cell formation and the start of mitosis. Leukemic lymphocytes from cases with low peripheral WBC counts showed the greatest increases in DNA polymerase activity and produced the largest numbers of blast-cells, while cells from cases with high WBC counts produced few or no blast-cells and failed to increase their DNA polymerase content. A broad spectrum of intermediate results were obtained in a series of 21 cases ranging in cell count from 21,000 to 265,000 per mm 3. Initial studies indicate that a fluorimetric assay of cellular DNA may be a more satisfactory basis than initial cell count or protein determination for calculation of results of enzyme content of cells clumped by PHA. Increased DNA polymerase activity may be an essential preliminary to increased DNA synthesis in cells responding to PHA stimulation in cultures.

Effect of Phytohemagglutinin in Cultures on the Lactate Dehydrogenases of Lymphocytes from Chronic Lymphatic Leukemia

Abstract 1. Normal lymphocytes show significant increases in their total LDH content and proportion of M-LDH during the first 24 hours of culture with PHA, while most of them undergo blast-cell formation in three days. The changes in LDH occur before mitosis is a factor in altering the cell population and apparently is the result of PHA action at the genetic loci controlling the synthesis of M- and H-LDH polypeptide subunits. 2. The capacity of lymphocytes from patients with CLL to produce blast-cells and to increase their total LDH activity and M-LDH content varied inversely with the height of the peripheral WBC counts. Sometimes prolongation of incubation to five days permitted LDH and blast-cell changes to become discernible. This perhaps resulted from increase by mitosis in the numbers of cells capable of reacting to PHA. 3. Increase in total LDH and proportion of M-LDH promotes glycolysis which may be needed to supply increased energy requirements for both blast-cell formation and mitosis.

Vesicular stomatitis virus replication in human leukocyte cultures: enhancement by phytohemagglutinin.

The replication of vesicular stomatitis virus in human leukocyte cultures shows that virus yields can be enhanced 6-to 180-fold by treating the leukocyte cultures with phytohemagglutinin prior to virus inoculation. Data suggest that a substance is produced in phytohemagglutinin-treated leukocyte cultures which is capable, on transfer to fresh leukocytes, of inducing blast cell formation and enhancing virus replication.