Non-sensitized Cells Research Articles

Lectin-dependent cell-mediated cytotoxicity in hamsters bearing syngeneic tumors

Spleen cells from LSH hamsters inoculated with xenogeneic, allogeneic, or syngeneic (PARA-7) tumor cells were assayed for their ability to mediate direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) in a 4-hr chromium release assay. Spleen cells from animals immune to xenogeneic or allogeneic cells demonstrated specific DCMC against homologous target cells in the absence of Con A and nonspecific LDCC against both homologous and heterologous target cells in the presence of Con A. Spleen cells from animals bearing syngeneic PARA-7 tumors (TBA) failed to express DCMC against homologous or heterologous target cells; however, significant lysis of all target cells occurred in the presence of Con A. LDCC was not detectable when nonsensitized spleen cells from normal animals were employed. The LDCC reaction was dependent on the concentration of Con A and the number of effector cells present in the reaction. The development of LDCC effector cells in the TBA appeared to parallel the development of both DCMC and LDCC effector cells in immune animals.

RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486)

RNA prepared from the lymphoid tissues of guinea pigs specifically sensitized to mono( p-azobenzene-arsonate)- N-chloroacetyl- l-tyrosine (ARSNAT) (MW = 486) was fractionated by oligo(dT)-cellulose affinity chromatography. Two fractions designated as I and II were eluted from the column. Fraction II, binding to the column and containing polyadenylic acid sequences, transferred ARSNAT or keyhole-limpet-hemocyanin (KLH) sensitivity to nonsensitized guinea pig peritoneal exudate cells (GP-PEC) in 14 experiments. Fraction I was unable to transfer KLH or ARSNAT sensitivity to GP-PEC. The amount of Fraction II needed to transfer ARSNAT sensitivity was 10 times less than previously reported. Synthetic nonlymphoid cell poly(A) tested in this system failed to transfer ARSNAT or KLH sensitivity to GP-PEC. Both Fractions I and II were inactivated by ribonuclease. The results suggest a possible messenger function for the poly (A)-containing RNA fractions.

Macrophage-Agglutinating Factor Produced In Vitro by BCG-Sensitized Lymphocytes

Supernatant fluids from cultures of BCG-sensitized rabbit lymph node and spleen cells contained a factor that strongly agglutinated normal rabbit alveolar macrophages within 3 min at room temperature. In contrast, fluids from nonsensitized cell cultures did not agglutinate normal rabbit alveolar macrophages. This factor was designated macrophage-agglutinating factor (MAgF) because it is similar to the previously described factor found in lung lavages of rabbits exhibiting a BCG-induced pulmonary delayed hypersensitivity reaction. The kinetics of MAgF production in vitro by sensitized lymph node cells and its inhibition by puromycin and actinomycin D suggest active synthesis; sensitized spleen cells exhibited kinetics resembling release rather than synthesis. Studies on purified lymphocyte and macrophage populations from sensitized spleen and lymph nodes indicated that lymphocytes are responsible for MAgF production. However, MAgF production was not induced in normal cells incubated in vitro with concanavalin A or phytohemagglutinin. Fractionation of cell culture supernatant fluids in Sephadex G-100 or Ultrogel AcA-34 clearly separated MAgF from migration inhibition factor; MAgF was present in the void volume of the eluates, suggesting a molecular weight of over 400,000, whereas migration inhibition factor was recovered in the same peak as albumin. The role of MAgF in vivo is unknown, but it is postulated that it may cause the adherence of macrophages during granuloma formation.

The Fc receptor on thymus-derived lymphocytes. III. Mixed lymphocyte reactivity and cell-mediated lympholytic activity of Fc- and Fc+ T lymphocytes.

The involvement of Fc- and Fc+ T cells, separated on the fluorescence-activated cell sorter, in proliferative and cytotoxic responses to alloantigens was examined. The cytotoxic lymphocytes generated by in vivo exposure to allogeneic tumor cells were shown to express the Fc receptor. The proliferative responses to alloantigen exposure in mixed lymphocyte cultures was equivalent in intensity for unseparated T cells, the Fc+ T-cell fraction, and the Fc- T-cell fraction isolated from nonsensitized spleen cells. In contrast, the cytotoxic responses generated by the Fc- T-cell fraction (less than 1% Fc+) were much weaker than the cytotoxic responses generated by the Fc+ T-cell fraction (80-90% Fc+), and the responses of the Fc+ T-cell fraction were generally weaker than, or equal to the responses of unseparated T cells (Fc- T less than Fc+ T less than or equal to unseparated T). Mixtures of the Fc- and Fc+ T-cell fractions mounted stronger cytotoxic responses than the sum of the responses of either fraction alone. Examination of the Ly phenotypes of the synergizing populations revealed that the CL precursor activity (Ly-2+ T cells) resided in the Fc- T-cell population, and that the amplifier T-cell activity (Ly-1+ T cells) resided in the Fc+ T-cell population. The data are discussed in terms of T-cell heterogeneity, differentiation, and intercellular interaction.

Graft-versus-host response, measured by splenomegaly assay, by populations of allosensitized mouse lymphocytes

The graft-vs-host (G-v-H) reactivity of sensitized or nonsensitized mouse lymphoid cell populations was measured using a splenomegaly assay. Sensitized populations were obtained either from the local lymph nodes of alloimmunized animals or from the spleens of heavily irradiated mice previously infused iv with allogeneic lymphocytes (educated cells). Immunization of animals resulted in increased G-v-H responses of the cells in their local lymph nodes. This effect was more pronounced when the immunizing cells differed only at non-H-2 transplantation antigens than when H-2-disparate strain combinations were tested. There was no evidence of a changed doseresponse profile of lymphocytes obtained from immunized mice. The G-v-H reactivity of educated cell populations was complex. The slopes of the dose-response lines obtained for lymph node cells or thymic cells educated in an H-2-disparate strain were generally lower than those obtained for nonsensitized cells. This difference was particularly evident when testing educated thymocytes. By studying the G-v-H indices obtained in A/Sn × C57B1 hybrids after inoculation of nonsensitized C57B1 lymph node cells or specifically educated C57BL lymph node cells, it was observed that the latter cells were approximately 30 times more reactive when small cell inocula were compared. On the contrary, education of lymphocytes in H-2-compatible allogeneic hosts did not result in any increment of their G-v-H reactivity. The results indicate that different methods of sensitizing lymphocyte populations against alloantigens may lead to activation of different subclasses of T-cells which differ in their mode of antigen reactivity.

In vitro adherence of nonsensitized cells to antibody-coated thyroid tissue in autoimmune thyroiditis

In vitro studies on patients' sera with comparably high thyroglobulin antibody titers indicated the existence of two groups of sera, one of which binds normal lymphocytes to thyroid-tissue sections whereas the other does not. This so-called coopting activity of the sera appeared to be complement independent and related to the IgG fraction of the sera and could also be absorbed with pure human thyroglobulin, thus suggesting the coopting factor to be thyroglobulin antibody. A mononuclear cell population, different from monocytes and B lymphocytes, is involved in the coopting phenomenon. The participation of B cells could not, however, be excluded. On the basis of the diagnoses in the present series, no clinical significance of the presence or absence of coopting antibodies in the patients could be established.

Studies of lymphocyte stimulation by intact tumor-cell and solubilized tumor antigen.

BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.

Analysis of Sera from SV40-Immunized and Tumor-Bearing Hosts for Blocking Activity and Antibody-Dependent Cellular Cytotoxicity

The role of serum factors in tumor immunity to cells transformed by PARA-(defective SV40)-adenovirus 7 was investigated. It was found that sera from SV40-sensitized hosts did not block the specific cytotoxicity of SV40-sensitized spleen cells for PARA-7 cells. However, such sera could collaborate with nonsensitized spleen cells to produce specific killing. This activity could be absorbed out by PARA-7 cells but not by cells transformed by cytomegalovirus. The activity of sera from hamsters bearing tumor isografts depended upon when, after transplantation, the specimens were obtained. Sera collected greater than or equal to 10 days after grafting completely blocked immune spleen cell cytotoxicity and did not mediate target cell killing in the presence of normal spleen cells. Sera obtained at an earlier time, i.e., 3 to 6 days after transplantation, consistently were active in the antibody-dependent cellular cytotoxicity test and exhibited reduced or no blocking of antibody-independent cellular cytotoxicity. Thus, there appears to be an inverse correlation in the capacity of serum from tumor bearing hosts to block effector cell cytotoxicity and mediate antibody-dependent cellular cytotoxicity.

Tumor regression at an untreated site during immunotherapy of an identical distant tumor.

The effects of two immunotherapy regimens on the development of an untreated, uniformly lethal transplantable line-10 hepatoma in strain 2 guinea pigs were monitored during treatment of an identical tumor 10 cm away. Line-10 cells were injected intradermally simultaneously at each of two sites. When one site was treated 6 and 16 days later with rabbit antibody against guinea pig fibrin fragment E, the complete regression of the treated tumor, a 25--30% depression in the development of the untreated tumor, and an increased survival time were observed. In another group of animals, when one site was treated 5 days after tumor challenge with syngeneic or xenogeneic "tumor-immune" RNA in a regimen including syngeneic nonsensitive lymphoid cells and tumor-specific antigen, all animals survived after complete and apparently specific regression of the tumors at both the treated and untreated sites. For the RNA regimen, we have shown that immunotherapy of an intradermally established line-10 tumor results in complete abrogation of both the treated and a distant untreated tumor; i.e., demonstrating a systemic effect.

Ultrastructural characteristics of the non-immune rosette-forming cell.

Non-immune rosette formation, where a non-sensitized lymphocyte is surrounded by three or more sheep red blood cells, is an in vitro technique which is thought to be specific in the identification of thymus-dependent (T) lymphocytes. Using the electron microscope and tissue culture techniques, we have studied the ultrastructure of the rosette-forming lymphocyte which has been stimulated by various cellular mitogens. Non-sensitized but stimulated lymphocytes, which form rosettes, have a morphology similar to that of previously identified T cells, adding further credence to the concept that non-sensitized rosette-forming cells represent a population of T cells. Changes in the binding pattern of the sheep red cell membrane to the lymphocyte have been identified which may represent an early phase of cell 'activation'. This marker of activation offers a potential method of studying membrane responses in attempts to localize cellular defects involving the T lymphocyte. In addition, preliminary studies using fluorescein-labelled sheep red blood cell fragments suggest specific attachment of labelled membrane to the T lymphocyte, allowing rapid identification of T cells in smears or tissue preparations.

Fractionation of Functional Lymphocytes Sensitized to Basic Encephalitogen on Derivatized Collagen and Gelatin Gels

The lymph node cells of basic encephalitogen (BE)-sensitized guinea pigs were fractionated on derivatized collagen and gelatin gels. The population of cells specifically reactive to this antigen can be isolated from derivatized gelatin gels and retain their viability and functionality as assayed in vitro. The specific binding of BE-sensitized cells to BE-derivatized gels comprised between 1 and 2% of the cells applied per plate. The ratio of sensitized cells bound to non-sensitized cells bound ranged between 4 and 6. The viability and functionality of adherent cells detached from collagen gels after enzymatic degradation were impaired. In contrast, the responses obtained with the adherent cell population released from the gelatin gels, by melting at 37 degrees C, were equal or greater than those of the original unfractionated lymph node cell cultures. Furthermore, it was possible to obtain a nonadherent cell population which was virtually completely depleted of the capacity to respond to the sensitizing antigen.

Guinea pigs immunized with xenogeneic cells: Skin test, in vitro stimulation; cytotoxicity of lymph node cells

Guinea pigs were immunized with cells of a human lymphoblastoid line. While nonsensitized lymph node cells were not influenced, those derived from immunized animals were markedly stimulated in vitro by the human cells. Maximal activity was found with lymphocytes harvested on day 6. The response was paralleled with the strength of skin induration elicited by the human cells and with the efficiency of the cytotoxic effect of lymphocytes on human target cells. The stimulation and cytotoxicity was specific while the skin test demonstrated cross reactivity with mouse cells.

Mitogenic properties of the thymus-independent antigen pneumococcal polysaccharide S3.

AbstractPneumococcal polysaccharide type SIII was found to stimulate DNA synthesis in nonsensitized spleen cells cultivated in vitro, peak activation occurring 2 days after the addition of 150 μg/ml of SIII. Cortisone‐resistant thymocytes did not respond, whereas spleen cells from congenitally athymic (nude) mice reacted as normal spleen cells.SIII induced polyclonal antibody synthesis in normal spleen cells, which was detected against a variety of red cell antigens and haptens. It was able to substitute for T cells, as demonstrated by the ability of sheep red cells to induce a primary immune response in cultures of nude spleen cells in the presence, but not in the absence, of SIII.It is concluded that SIII is a B cell mitogen capable of directly activating DNA and antibody synthesis in these cells. This property is presumably responsible for the thymus‐independent nature of the immune response to SIII and lipopolysac‐charide. It is suggested that other thymus‐independent antigens also are B cell mitogens, although the degree of mitogenicity may vary.

Effects of Carrageenan and High Serum Dilution on Synergistic Cytotoxicity to Tumor Cells

Summary Neither the C1 inhibitor carrageenan nor extensive dilution of the test sera (to 1:2560) abolished the specific killing of tumor cells in the presence of nonsensitized lymphoid cells and sera from tumor-bearing mice (i.e., synergistic cytotoxicity). The two lines of evidence indicate that conventional complement-dependent cytotoxicity does not play a role in synergistic cytotoxicity to tumor cells.

The Effect of Antibody on Antigen-Induced Lymphocyte Transformation

Abstract The influence of antibody on the antigen-induced in vitro uptake of tritiated thymidine by sensitive lymphocytes was determined. The cellular response of ovalbumin (OA)-sensitive lymphocytes was consistently depressed when OA was mixed with anti-OA antisera at concentrations equal to equivalence and 2 times antigen excess with one antiserum and 50 times antibody excess with another antiserum. At five other concentrations of these antisera, involving either antigen excess or antibody excess, the reaction of animals varied between depression and augmentation. Similar increases and decreases in stimulation occurred with cells sensitive to sheep RBC (SRBC) when the antigen was in the presence of anti-SRBC antiserum. Non-sensitive cells were not stimulated by immune complexes formed with OA or SRBC. Depressive complexes did not affect cell viability or response to phytohemagglutinin. Complexes composed of non-sensitizing antigen did not influence stimulation by another antigen to which the cells were sensitive. Depression also occurred when antibody was added to cultures composed of at least 97% lymphocytes. The experiments show that the in vitro response of sensitive lymphocytes may be specifically depressed or augmented by antibody.

Detection of Migration Inhibitory Activity in Human Mixed Lymphocyte Cultures

Abstract The macrophage migration test represents an in vitro correlate of delayed type hypersensitivity (1). The technique has subsequently been adapted to human peripheral blood leukocyte migration with regard to particulate (2) and water soluble (3, 4) antigens. Inhibition of migration of human peripheral leukocytes has been used as an indication of immunologic reactivity after liver transplantation (5) and after skin grafting (6, 7). Migration is inhibited in mixtures of two cell populations when one of these is sensitized to the transplantation antigens of the other, but direct migration does not demonstrate histoincompatibility since allogeneic mixtures of nonsensitized cells migrate normally (7, 8). It has been proposed, however, that degrees of histoincompatibility between nonsensitized cell types may be measurable by the production of migration inhibition factors (MIF)1 when such cells are cultured together (9).

Antigen-mediated fusion of specifically sensitized rabbit alveolar macrophages.

Rabbits sensitized intravenously with heat-killed Mycobacterium tuberculosis (strain H37Ra) suspended in mineral oil developed a strong pulmonary granulomatous response which reached its peak about 3 to 4 weeks after injection. Alveolar cells (4 x 10(6) cells/ml of tissue culture medium 199) procured 6 weeks after sensitization showed extensive development of multinucleated giant cells after 12 hr of incubation in tissue culture flasks containing heat-killed H37Ra (5 mug/ml). Giant cells measured 80 mum to 2.5 mm in length and contained between 30 and 700 nuclei. In contrast, no giant cells were observed when similar samples of the same cell populations were incubated in flasks containing: (i) no mycobacteria; (ii) heat-killed Escherichia coli; (iii) heat-killed Bacillus subtilis; (iv) latex particles; (v) ovalbumin; or (vi) phytohemagglutinin. The addition of immune (anti-H37Ra) sera potentiated the phenomenon of giant cell formation. In addition, supernatant fluids obtained from sensitive alveolar cells incubated with H37Ra were capable of inducing giant cell formation when incubated with nonsensitized alveolar cells. The results suggest that fusion of alveolar macrophages is mediated by an immunological mechanism.

The Early Stimulation of Protein Synthesis in Sensitized Guinea Pig Lymph Node Cells by Antigen

Abstract Lymph node cells from immunized guinea pigs were studied in short-term cell culture to determine the effect of specific antigen on protein and RNA synthesis. Both RNA and protein synthesis were stimulated early after the addition of antigen to these cultures. Pulse-labeling techniques disclosed a stimulatory effect on protein synthesis by antigen as early as 4 hr after its addition. This stimulatory effect was associated with the specific release of a soluble non-dialyzable mediator which recruited nonsensitized cells to increase their rate of protein synthesis. The stimulatory effect of antigen on protein synthesis was abolished by the prior addition of actinomycin D.

Cell-mediated immunity in polyoma oncogenesis

Primary polyoma virus-induced tumors in rats were prevented by the transfer of sensitized lymphoid cells. Sensitized lymphoid cells from donors infected with the virus or immunized with allogeneic polyoma tumors were both effective. The efficacy of the transfer gradually decreased with the increase in the period of time between the virus inoculation and the injection of the immune cells. Non-sensitized lymphoid cells were only effective when given before the virus inoculation. By this in vivo transfer assay the gradual development of the cell-mediated immunity directed against polyoma TSTA could be demonstrated, indicating the absence of tolerance or unresponsiveness to the tumor antigens.

Degranulation and histamine release in focal antigen-antibody reaction by means of microelectrophoresis in a single rat mesentery mast cell.

Several observations (1-5) have suggested that an antigen-antibody combination takes place on the mast cell surface this triggering the cellular processes leading to histamine release and morphological changes in the mast cells. It is as yet unknown whether or not intracellular antigen-antibody-induced histamine release can be precluded, in any type of immunologic reaction in mast cells. Even though an antigen-antibody combination takes place on the mast cell surface, a question arises as to whether the reaction occurs over the whole cell membrane in all-or-none fashion or at a limited portion where antigen is applied locally. Recent findings (6-9) indicate that both degranulation and histamine release from a single rat mast cell can be provoked locally on the cell surface at the site of topical application of chemical histamine releasers by means of microelectrophoresis, while these reactions are not induced by their intracellular applications (6, 7, 9). The present experiments were undertaken to see whether antigen-antibody reaction in mast cell is a local reaction or a kind of integral reaction spreading over the whole cell, and also to determine whether the site of the reaction is inside or outside of the mast cell, using two types of antigen-antibody reaction: 1) applying antigen to the mast cell of an actively sensitized rat, or to the mast cell sensitized passively in vitro with homologous antibody, and 2) applying a rabbit anti-rat serum to the non-sensitized mast cell. Antigen and antiserum were applied by means of microelectrophoresis, topically or diffusely on the cell surface or intracellularly, to a single rat mesentery mast cell.