Immunosuppressive factors from human breast carcinoma cell lines that affect initiation of lymphocyte proliferation

Summary Supernatants from two human breast carcinoma cell lines, 734B and 231, have been shown to inhibit lymphocyte activation by mitogens and antigens. This inhibition appears to be specific for lymphocytes or recently stimulated cells, while having no effect on the growth of established cell lines. Studies of the mechanism of inhibition revealed that the factors inhibit lymphocyte activation and that the factors must be present at the initiation of lymphocyte stimulation for inhibition to occur. The supernatants do not inhibit lymphocyte activation by blocking binding of PHA to lymphocytes. Preliminary purification steps have shown that the inhibitory factors present in the tissue culture supernatants are precipitated at 50% ammonium sulfate saturation and their molecular weights are greater than 100 000. The inhibitory capacity of the 734B supernatants was destroyed by heating at 70° C, while the factors present in the 231 supernatants were only partially destroyed by heating to 90° C. The possible mechanism of action of the inhibitory substances released by tumors and their relevance to tumor growth are important to understanding of immune responses to neoplasia.     

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.     

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

HTLV-III large envelope protein (gp120) suppresses PHA-induced lymphocyte blastogenesis

The addition of inactivated preparations of purified human T cell lymphotropic virus (HTLV-III) was found to inhibit normal human lymphocyte phytohemagglutinin (PHA)-induced blastogenesis but had no effect on concanavalin A (Con A), pokeweek mitogen, or allogeneic stimulation. The inhibition was concentration-dependent and also dependent on adding the virus preparation before or at the same time as PHA. The CD4 molecule is the receptor for HTLV-III binding. Immunopurified large envelope protein (gp120) from HTLV-III was found to bind to the CD4 molecule and also inhibited PHA-induced lymphocyte blastogenesis. These results suggest that the gp120 viral protein may alter immune function by binding to the CD4 molecule, which in turn serves as an "off" signal to lymphocyte response to PHA stimulation. Alternatively, by binding to the CD4 molecule, gp120 may interfere with the interaction of this molecule with class II histocompatibility antigens on accessory cells, thus selectively suppressing PHA response.     

Functional analysis of macrophage hybridomas. III. Inhibition of lymphocyte blastogenesis and tumor proliferation

Four cloned macrophage hybridoma cells prepared by fusion of splenic adherent cells with a P388D1 macrophage tumor markedly inhibited the growth of lymphocytes and tumor cells. Macrophage clones 5, 8, 63, and 64 are strong inhibitors of B-cell blastogenesis, T-cell blastogenesis, and tumor proliferation, while the P388D1, tumor line and clones 13, 59, and 67 demonstrated little inhibitory activity in all three systems. The inhibitory effect of macrophage hybridomas can be detected within 8 hr, although greater inhibition was noted following longer incubation. The correlations among these three assay systems suggest that similar mechanisms may be involved. The data indicate that the inhibition of cell proliferation was not due to cell lysis. Furthermore, the inhibition of lymphocyte proliferation in Con A-stimulated cultures was not accompanied by inhibition of lymphokine production in the same cultures. Neither prostaglandins nor hydrogen peroxide appear to be primarily responsible for growth inhibition. The inhibitory properties of these macrophage hybridoma lines represent a stable phenotype and provide a homogeneous source of cells for further analysis of the phenomenon.     

Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro

Summary The co-culture of human peripheral blood mononuclear cells (PBMC) with high concentrations of interleukin 2 normally generates lymphokine-activated killer (LAK) cells capable of indiscriminate lysis of tumor targets. However, the addition of certain cell-line-derived tumor cells to the LAK generation cultures within the first 48 h of culture initiation resulted in the suppression of the LAK cytotoxicity measured after 3–4 days of culture. Suppression could be achieved with tumor cell:PBMC ratios as low as 1:50 when tumor cells were derived from melanoma and colorectal cancer (G361, COLO320, HT-29), but suppression was not observed with cells from the breast cancer cell line SKBr3. No suppression of LAK generation was observed with normal epithelial cells from colon or breast, with autologous or allogeneic lymphoblasts, or with allogeneic vascular endothelial cells. Suppression was independent of the removal of adherent cells from PBMC, could not be prevented by indomethacin and was not attributable to interleukin 2 absorption/adsorption by tumor cells. The suppressive activity of some tumor cells could be augmented by preculture in recombinant gamma interferon. Serum-free supernatants from G361, COLO320 and HT-29 (but not SKBr3 or endothelial cells) were also highly suppressive towards the generation of LAK cells. The elaboration by tumor cells of fractors capable of inhibiting LAK generation may partially explain the failure of LAK/interleukin 2 therapy in some experimental and clinical protocols.     

Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of tumor cells and their supernatants on proliferative responses of lymphocytes

Tumor-infiltrating lymphocytes (TIL) were obtained from 22 humans with solid tumors. In three cases only, one colon and two lung carcinomas, TIL which contained from 3 to 10% of T cells expressing the interleukin 2 receptor (IL 2R) were obtained, and these proliferated in the presence of exogenous IL 2. In most TIL preparations, however, the T lymphocytes did not express the IL 2R and failed to proliferate in response to IL 2. In contrast, TIL were able to proliferate in response to irradiated allogeneic spleen cells in mixed lymphocyte culture. Proliferative responses of autologous PBL were not inhibited by the addition of TIL. In most tumors, the TIL showed no response or had significantly lower (p less than 0.01) responses to PHA, Con A, and the phorbol ester TPA than did autologous peripheral blood lymphocytes (PBL). A limiting-dilution microculture system which allows clonal growth of every T cell was used to demonstrate decreased responses of the TIL to PHA at a single-cell level. In contrast to normal PBL-T with proliferating frequencies from 0.46 to 1.0, those for T cells in three TIL preparations were zero, 0.005, and 0.01. Normal PBL exposed in vitro to tumor cells or their supernatants lost the ability to respond to mitogens and to clone normally (e.g., proliferating frequency of 0.147 vs 0.863 in control). The TIL isolated from solid tumors resemble normal PBL exposed in vitro to tumor cells or their supernatants in terms of decreased responses to mitogens and poor clonogenicity in the PHA-dependent microculture system. It is possible that tumor cells may inhibit certain functions of the TIL in human solid tumors.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Anticancer activities of novel chalcone and bis-chalcone derivatives

A series of novel chalcones and bis-chalcones containing boronic acid moieties has been synthesized and evaluated for antitumor activity against the human breast cancer MDA-MB-231 (estrogen receptor-negative) and MCF7 (estrogen receptor-positive) cell lines and against two normal breast epithelial cell lines, MCF-10A and MCF-12A. These molecules inhibited the growth of the human breast cancer cell lines at low micromolar to nanomolar concentrations, with five of them (1-4, 9) showing preferential inhibition of the human breast cancer cell lines. Furthermore, bis-chalcone 8 exhibited a more potent inhibition of colon cancer cells expressing wild-type p53 than of an isogenic cell line that was p53-null.     

The synthesis of a proteinase inhibitor, α-1-antichymotrypsin,by human breast epithelial cells

The synthesis and release of glycoproteins were studied in organ cultures of human breast surgical specimens and in established breast epithelial cell lines, MCF-7 and MDA-MB-231. Biosynthesis was monitored by the incorporation of ¹⁴C-glucosamine. Labeled macromolecules in the culture supernatants were analyzed by biochemical and immunological techniques. One to 8% of the labeled glycoproteins from benign breast and infiltrating ductal carcinoma specimens was precipitated by antibodies produced against human serum 7α-1-antichymotrypsin. Twelve percent of the total glycoproteins from the culture supernatants of the MCF-7 cell line was identified as α-1-antichymo-trypsin. Both the normal serum and the human breast epithelia-derived proteinase inhibitor can be resolved into similar subclasses by two-dimensional gel electrophoresis. MDA-MB-231 and MCF-7 cells which were extensively washed with EDTA, serum-free medium, and phosphate-buffered saline retain this proteinase inhibitor on their cell surfaces. Three to 4% of the total cell-surface iodinated components was immunoprecipitated by these specific antibodies. Since α-1-antichymotrypsin is a potent inhibitor of neutral proteinases such as cathepsin G, the demonstration of its synthesis by benign and malignant breast epithelial cells is of considerable interest. This glycoprotein may represent the epithelia's own protective shield of cell surface components and the cell's attempt to moderate the effects of invading leukocytes. In addition, it may play a regulatory role in the maintenance of three-dimensional glandular structures.     

The effects of plasma from guinea pigs with tumor on PHA stimulated lymphocyte cultures.

Strain 13 guinea pigs with a transplanted, 3-methylcholanthrene induced fibrosarcoma produced a plasma factor that inhibited PHA induced blastogenesis of lymphocyte cultures from normal and tumor animals. The suppressive activity was not cytotoxic, was not adsorbed from plasma by lymph node cells, and was a noncompetitive inhibitor of PHA activation. The tumor plasma could not inhibit the uptake of [³H]thymidine if added during the period of maximal DNA synthesis, which suggested that the suppressive factor acts most effectively on metabolically resting cells. This nonspecific suppressive factor may represent an important mechanism for allowing rapidly progressive tumors to escape immunological destruction.     

The effects of corticosteroid concentrations on lymphocyte blastogenesis

High concentrations of methylprednisolone added for prolonged periods in vitro to lymphocyte cell cultures inhibited allogeneic-cell-induced or phytohemagglutinin (PHA)-induced blastogenesis in contrast to lower concentrations which were inhibitory only if added before or within several hours of blastogenic induction.     

Immunomodulation of human leukocytes by staphylococcal enterotoxin A: augmentation of natural killer cells and induction of suppressor cells

Staphylococcal enterotoxin A (SEA), a protein isolated from culture supernatants of Staphylococcus aureus, is a potent T-cell mitogen and an inducer of interferon-gamma (IFN-gamma). We report here that SEA exhibits a number of significant in vitro immunomodulatory functions. In vitro treatment of human peripheral blood monocyte-depleted lymphocytes with SEA resulted in significant augmentation of their natural killer cytotoxicity against target cells from hemopoietic (K562, Daudi) or solid (melanoma, lung, colon) human tumor cell lines. SEA was found to be more effective than interferons-alpha (natural or Escherichia coli-derived) in augmenting natural killer (NK) cytotoxicity of peripheral blood lymphocytes. Studies on the kinetics of the augmentation revealed a significant increase of NK within 3 hr of in vitro treatment with SEA at 37 degrees C. A neutralizing monoclonal antibody specific for human IFN-gamma did not affect the augmentation of natural killer cytotoxicity by SEA, suggesting that SEA augmented natural killer cytotoxicity primarily by a mechanism not involving induction of interferon-gamma. Furthermore, in vitro treatment with SEA resulted in significant augmentation of antibody-dependent cell-mediated cytotoxicity and of natural killer-like cytotoxicity, generated in mixed lymphocyte culture, against the K562 targets. Induction of suppressor cells to proliferative responses of autologous or allogeneic mononuclear cells to phytohemagglutinin (PHA) or to allogeneic cells in mixed lymphocyte culture was observed after in vitro treatment of peripheral blood mononuclear leukocytes with SEA for 24 or 48 hr at 37 degrees C. In addition, the presence of SEA in mixed lymphocyte cultures (MLC) resulted in significant inhibition of the generation of specific T-cell-mediated cytotoxicity in MLC. These results suggest that SEA, which may be involved in S. aureus infections and in treatment with extracorporeal perfusion systems over S. aureus columns, can regulate a number of significant lymphoid functions.     

Effects of zinc on cell-mediated immunity in chronic hemodialysis patients

Thirteen healthy subjects and 20 hemodialysis patients were studied to observe the delayed hypersensitivity skin tests (DHSTs) and phytohemagglutinin (PHA)-stimulating lymphocyte blastogenesis. Significant differences were observed between the groups. Controls had a higher proportion of positive skin reaction than hemodialysis patients in relation to Escherichia coli (p<0.01) and tuberculin (PPD) (p<0.05). Regarding lymphocyte blastogenesis stimulated by phytohemagglutinin (PHA), cell proliferation was more accentuated in controls than hemodialysis patients (p<0.05). On the other hand, serum zinc was elevated in controls (78 +/- 8 microg/dL) in comparison to hemodialysis patients (71 +/- 33 microg/dL) (p<0.05). Of the 20 hemodialysis patients, 8 patients were maintained on long-term hemodialysis before and after zinc therapy, with the aim of studying DHST and PHA-stimulating lymphocyte blastogenesis. There was a significant improvement of DHST response to E. coli antigen after 100 d of zinc treatment (p<0.01), and with the discontinuation of therapy, the DHST responses decreased back to the initial values (p<0.05). Zinc administration also increased the lymphocyte proliferation induced by PHA from 31386 +/- 3974 to 42480 +/- 5242 cpm (mean +/- SD) (p<0.05). These results indicated that zinc therapy improved in vivo and in vitro DHST and lymphocyte function of hemodialysis patients and that its discontinuation suppressed all of the benefits observed.     

INHIBITORY EFFECTS OF MITOGENS ON ADENOIDAL LYMPHOCYTES IN VITRO

Cultures of human adenoidal lymphocytes exposed briefly to either phytohemagglutinin (PHA), Staphylococcus filtrate (Staph-F), concanavalin-A(Con-A), or pokeweed mitogen (PWM) incorporate increased amounts of thymidine earlier than replicate cultures exposed continuously to the mitogens. These effects can begin in the first 24 hr of culture and are seen maximally between 36 and 72 hr. Once a blastogenic response is established, PHA or PWM can diminish that response. Inhibition with PWM requires that the initial stimulation was with this mitogen, while PHA can inhibit blastogenesis to both PHA and PWM-stimulated cells. Because these mitogens can have a paradoxical effect on adenoidal lymphocytes, being capable of both initiating and inhibiting DNA synthesis, this phenomena should be kept in mind when such systems are utilized for the evaluation of antigens and drug effects.     

Suppression of phytohemagglutinin-stimulated lymphocyte blastogenesis by ovine uterine milk protein

Two basic glycoproteins (UTM-P) with molecular weights of 57,000 and 59,000 were purified from ovine uterine milk collected on Days 125 and 130 of pregnancy. The UTM-P were evaluated for immunosuppressive activity in phytohemagglutinin (PHA)-treated, mixed lymphocyte (MLC) and resting lymphocyte (RLC) cultures. For PHA and RLC cultures, UTM-P (2.5 to 800 micrograms UTM-P/ml) were added to 1 X 10(6) lymphocytes and 0.8 micrograms of PHA (for PHA cultures only), while for the MLC, UTM-P (50 to 1600 micrograms UTM-P/ml) were added to 5 X 10(5) lymphocytes combined from each of two ewes. Following [3H] thymidine addition, cells were later harvested for determination of thymidine incorporation. Lymphocyte blastogenesis was suppressed by UTM-P in PHA (R2 = 0.32 to 0.92, P less than 0.01 to 0.001), MLC (R2 = 0.8, P less than 0.001) and RLC (R2 = 0.65, P les than 0.01) experiments. To determine reversibility, PHA-treated lymphocytes were incubated with UTM-P for 6, 12 or 24 h, then washed to remove surface UTM-P. Incubation was continued in the presence of PHA as with other experiments. Exposure of lymphocytes to UTM-P for 6 or 12 h did not result in suppression of blastogenesis, whereas exposure for 24 h was sufficient for suppression (P less than 0.01). In an additional experiment, UTM-P were added to PHA-treated cultures at 0, 6, 12 or 24 h. Suppression (P less than 0.01) of blastogenesis was observed for each time period. Immunosuppressive activity was not mediated by overall cytotoxicity and was not affected by routine handling and storage of UTM-P. Data from these experiments provide one explanation for tolerance of the conceptus allograft during defined stages of ovine pregnancy.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.