Studies on the influence of liquid holding in Con-A stimulated human peripheral blood lymphocytes on mitosis and X-ray induced chromosome aberrations

Summary A method suitable for performance of liquid-holding (LH) experiments with human lymphocytes in vitro is described. The lymphocytes are stimulated with concanavalin A (ConA) and after washing off the ConA with a sugar-containing medium are liquid-held in ConA-free medium for different times, after which they are restimulated with ConA. The BUdR-labeling method was used to determine the proportion of first-, second-, and third-division cells. With this system we were able to show that the frequencies of X-ray-induced chromosomal aberrations are not influenced by liquid holding, supporting the idea that these aberrations are fixed shortly after irradiation by misrepair. This system may prove useful for investigation of specific problems in mutagenicity research, especially those involving mutagens, which induce delayed formation of chromosome aberrations.     

Mitogen response of peripheral blood and splenic lymphocytes and effect of 2-mercaptoethanol in tumor-bearing mice

Summary A murine osteosarcoma in which the number of tumor cells can be continually monitored by measuring the circulating plasma alkaline phosphatase levels was used to determine the effect of tumor burden on peripheral blood and splenic lymphocyte response to mitogens. In animals with tumors of different sizes, the pattern of response of the peripheral blood lymphocytes (PBL) to mitogens is different from that of splenic lymphocytes. PBL response to ConA, PHA, and LPS was initially depressed, but response to PHA and LPS recovered later, as the tumor burden exceeded 6%. However, the recovery of LPS response was not consistent, in that recovery was not seen when the tumor burden was 5%–6%. Response to ConA remained depressed. Splenic lymphocytes showed progressive decline of PHA response. Treatment of tumor-bearing mice with 2-mercaptoethanol (2-ME) restored the ConA response of PBL in 56% of mice. Treatment with 2-ME did not restore PBL response to PHA or LPS. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; peripheral blood lymphocytes; ConA, concanavalin A; PHA, phytohemagglutinin; LPS, lipopolysaccharide; 2-ME, 2-mercaptoethanol; FCS, fetal calf serum; AP, alkaline phosphatase; OS, osteosarcoma     

Cell surface changes in transformed human lymphocytes : I. ConA and E-PHA induced unique changes in surface topography

Binding studies with six purified plant lectins were used to investigate membrane alterations that occur in lymphocyte transformation. Normal human peripheral blood lymphocytes transformed with E-Phytohemagglutinin (E-PHA) or concanavalin-A (Con-A) generally possessed increased numbers of lectin receptors. When this increase was corrected for the expanded surface area of transformed lymphocytes, it appeared that E-PHA and ConA each produced a unique and complex reorganization of cell surface topography. Surface alterations occurred independently of DNA synthesis, cell proliferation, and microtubule or microfilament function. Puromycin inhibited emergence of new lectin receptors on cells transformed with E-PHA, but not with ConA. Lymphocytes incubated with either lectin showed increased incorporation of [¹⁴C]galactose into trypsin-sensitive cell surface glycoproteins. This incorporation was abolished by puromycin in cells stimulated by E-PHA but not by ConA. These studies demonstrate that although both lectins induce similar morphological alterations in human lymphocytes, at the molecular level the structural changes induced in the cell membrane by these two lectins differ considerably. Furthermore, these structural alterations are mediated via different mechanisms in the two groups of cells. De novo protein synthesis is required for cell surface reorganization in PHA-stimulated cells, but not in cells stimulated by ConA. The effect of ConA appears to be to enhance attachment of saccharide structures to pre-synthesized membrane proteins.     

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

Allogeneic tumor-specific cytotoxic T lymphocytes

Summary We report the development of cytotoxic T lymphocytes specific for an allogeneic brain tumor in a rat model. DA strain cytotoxic T cell precursors stimulated by an allogeneic tumor (9L gliosarcoma) from the Fischer rat could generate a population of cytotoxic T lymphocytes that lysed the allogeneic 9L tumor but failed to lyse other targets, including Fischer concanavalin-A(ConA)-stimulated lymphoid blast targets. DA T cells depleted of reactivity to the Fischer haplotype (DA-f) retained reactivity to the 9L tumor, demonstrating that T cell precursors with specificity for normal Fischer alloantigens were not required for the generation of a response to the 9L Fischer tumor. The preferential lysis of the tumor target did not simply reflect a higher density of Fischer target antigens on the tumor than that found on normal Fischer ConA blast targets. First, the relative densities of class I antigen on the 9L tumor and normal Fischer ConA blasts were comparable. Second, cytotoxic T cells could not be generated from DA-f precursors when Fischer ConA blasts were used as stimulators. If DA-f T cells were simply responding to the higher density of Fischer antigen found on 9L tumor, it would have been expected that the ConA blasts expressing comparable levels of antigen to that found on the tumor would have generated cytotoxicity for both the 9L and ConA targets. We conclude that the cytotoxic T cells are specific for a determinant expressed only by the tumor. Such tumor-specific cytotoxic T cells could be useful in vivo for adoptive immunotherapy of brain tumors.     

Concanavalin A as an Inducer of Human Lymphocyte Mitogenic Factor

IT is likely that pharmacological products of antigen : lymphocyte interaction (“lymphokines”) act as mediators and regulators of a variety of cellular immune responses^1,2. This view is strengthened by demonstrations that phytomitogen lectins induce lymphocytes to generate products with similar biological activities^3,4 and physicochemical characteristics⁵, to the lymphokines. Increasing evidence suggests that mitogenic lymphokines may mediate lymphocyte transformation responses in vitro and facilitate lymphoid cell cooperation in vivo (refs. 1,2,6–9). The study of mitogenic factor production by phytomitogens which may predominantly activate thymus-dependent lymphocytes (Concanavalin A (ConA))^8,9 provides a model approach to the investigation of lymphokine function in man. Powles et al.⁴ have described a ConA-induced mitogenic factor which stimulated autologous human lymphocytes only, whereas antigen-induced lymphocyte factors generally stimulate both allogeneic and syngeneic lymphocytes^11–13. Interest in ConA as an inducer of human lymphocyte mitogenic factors would be widened if conditions were found in which ConA stimulated human lymphocytes to generate products which were mitogenic for both allogeneic and autologous lymphocytes. As a lymphokine stimulant, ConA has the advantage that it is largely removed from culture fluids by absorption to cross-linked dextrans (‘Sephadex G-50’) or serum glycoproteins¹⁴. Here we demonstrate that a ‘Sephadex’-binding fraction of ConA (ConA- V) induces human lymphocytes to generate a mitogenic factor which activates both allogeneic and autologous lymphocytes.     

Binding of concanavalin A to lymphocytes after storage at --196 degrees C.

A reported loss in the binding capacity to ConA of thawed human peripheral blood lymphocytes has been investigated using two methods. With acetyl-³H ConA there was an apparent loss in the total binding of ConA to 2 × 10⁵ dye-excluding cells thawed from liquid nitrogen, after cooling with a two-step procedure of 10 min at ?26 °C in 5% DMSO. Using the same cooling method, this apparent loss of binding capacity was not confirmed when a Fluorescence Activated Cell Sorter was used to measure the binding of fluorescent labelled ConA to thawed cells that are shown to be within the light scatter range of unfrozen lymphocytes. This second method, therefore, shows that a large population of lymphocytes can be recovered after thawing without any loss of receptors for ConA. The loss of binding measured by the radioactive method may be due to damaged lymphocytes and also to the loss of the small numbers of residual granulocytes.     

Progressive loss in vitro immune response with tumor growth.

Peripheral blood lymphocytes from rats carrying a transplantable hepatoma were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (ConA) or dextran sulfate (DS) at various times after tumor cell inoculation or after its surgical removal. Mitogen-induced lymphocyte transformation, measured by tritiated thymidine incorporation, declined as the tumor size increased, especially when cells were cultured in autologous serum. The response to PHA and ConA declined prior to the response to DS. This inhibition could not be removed by extensive washing of the cells, alteration of serum concentration, time of incubation or mitogen dose. Culture for 24 hr prior to the addition of high doses of mitogen resulted in partial restoration of the PHA and ConA, but not DS, responses. Previously inhibited responses also returned when the tumor was surgically removed. Spleen cells from animals with large tumors were also inhibited.     

Simulated microgravity conditions affect poly(ADP-ribose) polymerase activity in cultured human lymphocytes

Human peripheral blood lymphocytes (PBL), activated with concanavalin A (ConA), were used to determine the effects of simulated microgravity on poly(ADP-ribose) polymerase (PARP) activity. Results indicate that the ConA stimulation of human cultured PBL induces a partial but signitficant inhibition of PARP-1 acitvity (-30%). In control PBL, not exposed to ConA, after 24 hours, there was a clear decrease in PARP-1 acitivty (-40%). In PBL exposed to ConA and simulated weightlessness, activity decreased by -37%.     

On the mode of action of thymosin

Thymosin was administered to CBA mice which had been depleted of recirculating small lymphocytes by combining ALS and thymectomy or through lethal irradiation of thymectomised mice reconstituted with syngeneic bone marrow. The population of recirculating small lymphocytes was monitored by determining the numbers of “lymph node localising” cells in the lymphoid organs of treated animals. In no case was there any evidence that thymosin treatment accelerated the recovery of recirculating lymphocytes. Moreover, it was not possible to show that bone marrow cells incubated with thymosin acquired theta-positivity.We conclude that thymosin does not act by augmenting the production of mature recirculating small lymphocytes.     

Receptors for thymosin alpha 1 on mouse thymocytes

Thymosin alpha 1 is able to act in vitro to stimulate T-cell precursors and to induce surface markers. The initial mechanism by which alpha 1 activates T cells could be the binding of alpha 1 to cell membranes. Using a specific anti-alpha 1 antibody and an indirect immunofluorescence procedure it was found that thymosin alpha 1 binds to the surface of a large portion of murine lymphocytes. Furthermore, thymocytes have been fractionated into immature and mature subpopulations by using the peanut agglutinin (PNA) technique. It was found that PNA+, immature cells showed specific receptors for alpha 1 on the cell membrane.     

Lack of specificity in the mechanisms involved in the enhancement of the concanavalin A driven human T lymphocyte stimulation by beta-endorphin: studies on activation marker expression, cell cycle and interleukin release.

We report on the effects of a physiological concentration of Beta-Endorphin (BE) (10(-12)M) on Concanavalin A (ConA) stimulated human peripheral blood T-lymphocytes and monocytes. We evaluated the effect of timing of BE addition to the culture medium on thymidine uptake, the kinetics of expression of activation markers (CD69, CD25 and CD71) on CD4+ and CD8+ lymphocytes, and of class II MHC antigens on CD14+ cells (monocytes), the kinetics of interleukin-1 (IL-1), interleukin-2 (IL-2) and interferon gamma (IFN-gamma) release, and the cell cycle. Data show that BE is able to influence T lymphocyte only when added together with ConA at the beginning of culture, suggesting its major activity is on the early phases of the T cell response. BE did not increase the amount of class II MHC antigens on monocytes and did not preferentially stimulate CD69, CD25 and CD71 antigen expression on either CD4+ or CD8+ lymphocytes. After 24 hours, the relative proportions of CD4+ and CD8+ lymphocyte in S and G2-M phases were not affected by BE, although the opioid did augment the number of cells in the proliferative compartments of the cell cycle, S and G2-M, indicating an actual increase in the number of cells committed to proliferation. BE did not consistently influence the amount of IL-1, IL-2 and IFN-gamma found in the supernatant of ConA stimulated cultures. The mechanism of the enhancing effect on the proliferative response of normal human lymphocytes to ConA by BE, does not seem to be selective for or unique to specific lymphocyte subsets.     

Age and the response of peripheral blood lymphocytes to mitogens]

Reactions of the peripheral blood lymphocytes of normal persons from 19 to 49 years of age to the following mitogens was studied; phytohemagglutinin (PHA), concanavalin A (ConA), rabbit serum against human thymocytes (ATS). A significant reduction of the lymphocytes proliferative response to ConA was reported in persons above 30 years old. There was also demonstrated a significant negative correlation between the proliferative response indices and the donor's age (in ConA-stimulation-for the whole group examined, and in ATS-stimulation-for persons aged from 30 to 49 years only. Analysis of the intensity of thymidine-(3)H incorporation showed that with the advance of age there was a fall of the percentage of cells with an intensively labelled nuclei and an accumulation of cells with weakly labeled nuclei, this phenomenon was observed both when the proliferative response was decreased and when no significant differences were reported in these indices.     

Thymosin beta 4 induced phenotypic changes in Molt-4 leukemic cell line

Thymosin beta 4 was tested for its ability to induce phenotypic changes in the human T-cell line Molt-4. Cells were cultured with nanogram concentrations of thymosin beta 4 for up to 16 days and were analyzed with a panel of monoclonal antibodies, sheep erythrocyte rosetting, peanut agglutinin binding (PNA) and an antibody to the enzyme, terminal deoxynucleotidyl transferase (TdT). Thymosin beta 4 induced Molt-4 cells to reduce the expression of a T-cell lineage specific antigen, with preferential expression on T blast-cells, detected by WT 1 monoclonal antibody. Thymosin beta 4 also induced an increase in sheep erythrocyte rosettes and PNA binding as well as an increased expression of OKT 11 A and OKT 8 in Molt-4 cells. TdT was found to be unchanged, however. Analysis of thymosin beta 4-treated cells with other monoclonal antibodies (OKT 3, OKT 6, OKT 9) showed no change when compared to controls. These results showed that thymosin beta 4 is capable of inducing phenotypic changes in Molt-4 cells. Such changes may represent a differentiation process of these cells through the early stages of the maturation process of thymus-dependent lymphocytes, albeit not to the stage of mature T cells.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Characterization of a lympho-inhibitory peptide produced by Mycoplasma bovis

Mycoplasma bovis is able to inhibit the mitogen-induced proliferation of bovine lymphocytes. Herein is described the isolation of an immuno-inhibitory peptide from M. bovis. Using size exclusion chromatography, three lympho-suppressive fractions were isolated from M. bovis free supernatant. MALDI-TOF analysis revealed a common peak throughout the suppressive fractions. The purest of these fractions was subjected to N-terminal sequencing, revealing an 84% homologous match with the C-terminus of the M. bovis surface protein VspL (variable surface protein-L). A recombinant of the 26 amino acid peptide was also able to suppress Concanavalin A (ConA)-induced proliferation of bovine lymphocytes. This describes a unique immunosuppressive peptide produced by the bovine respiratory pathogen, M. bovis.     

Structure and immunological properties of thymosin beta 9 Met, a new analog of thymosin beta 4 isolated from porcine thymus

During the course for the studies of thymosin beta 4 and prothymosin alpha from porcine thymus, a new analog of thymosin beta 4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin beta 9 except that leucine at position 6 in beta 9 is substituted by methionine. Thus, this new peptide has been termed thymosin beta 9 Met. The recoveries of beta 9 Met, beta 4, and prothymosin alpha in porcine tissues have been determined (in micrograms/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin beta 9 Met was found to be more active than beta 4 in enhancing gamma-interferon production in cord blood lymphocytes. However, beta 4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.