Murine Spleen Cell Cultures Research Articles

Enhanced adoptive transfer of immunity to Listeria monocytogenes after in vitro culture of murine spleen cells with concanavalin A.

In vitro incubation of spleen cells with T-cell mitogens has been shown to augment cytotoxic and cytolytic T-cell function. The plant lectins Concanavalin A and phytohemagglutinin were employed in a similar fashion to investigate their abilities to enhance cell-mediated immunity to the microbial pathogen Listeria monocytogenes. Spleen cells from immune mice were incubated in vitro with Concanavalin A or phytohemagglutinin before passive transfer into normal recipients. Results indicated a 10- to 100-fold enhancement in the ability of these cells stimulated in vitro to transfer antilisterial resistance, as assayed by changes in 50% lethal dose values and enumeration of splenic bacterial proliferation.

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS): I. Analysis of the PITSβ factor

Previously it has been shown that T cells cultured with prostaglandin E 2 are induced to release at least two peptide-containing lymphokines. These factors, the prostaglandin-induced T-cell-derived suppressors (PITS), were shown to be potent inhibitors of phytohemagglutinin (PHA)- and lipopolysaccharide (LPS)-induced blastogenesis of murine spleen cell cultures. It is now reported that the 2000-dalton factor (PITSβ) also suppresses the T-cell dependent mixed lymphocyte reaction (MLR). Moreover, the antibody responses to both T-dependent (SRBC) and T-independent (Dnp-polyvinylpyrrolidone and Dnp-Ficoll) antigens are also suppressed by PITSβ. Further experiments indicate that culture with PITSβ for 2–4 hr is sufficient for suppresion of PHA-induced blastogenesis, mixed-lymphocyte reactions, and antibody responses to both T-dependent and T-independent antigens. These results, along with previous reports, suggest that PITSβ, a T-cell product, exerts broad-range suppressive effects within 6–8 hr of stimulation by prostaglandin E 2.

Immunomodulating properties of 5,20 alpha (R)-dihydroxy-6 alpha,7 alpha-epoxy-1-oxo-(5 alpha)-whitha-2,24-dienolide and solasodine.

5,20α (R)-Dihydroxy-6α,7α-epoxy-1-oxo-(5a)-witha-2,24-dienolide and solasodine inhibit proliferation of murine spleen cell cultures. The steroidal lactone occurs in the medicinal plants WITHANIA SOMNIFERA and LYCIUM CHINENSE. The steroidal alkaloid is contained inter alia in SOLANUM NIGRUM.

Taenia solium: mitogenic effect of larval extracts on murine B lymphocytes.

The effect of an extract (CE) obtained from Cysticercus cellulosae on the proliferation of lymphocytes was studied in cultures of murine spleen cells. The addition of CE to the cultures resulted in the highly significant uptake and incorporation of tritiated thymidine (3H-TdR) into DNA. This phenomena was dose-dependent, with doses lower and higher than the optimal concentration causing less marked effects. The kinetic peak of this response was found to occur on day 2 of culture. CE evoked a proliferative response in cultures of spleen cells from congenitally athymic (nu/nu) BALB/c mice. Cultures of bone marrow-derived (B) lymphocytes, generated by treatment of spleen cells with rabbit antithymocyte serum and complement, incorporated 3H-TdR to a degree similar to that of normal spleen cell cultures. CE did not induce the proliferation of thymocytes. To eliminate the possibility that the mitogenic effect of CE was due to LPS, we carried out experiments using Polymyxin B (PB). CE was mitogenic after treatment with PB which inactivated the LPD effect. In addition, CE elicited 3H-TdR uptake in spleen cells from the LPS nonresponsive C3H/HeJ mouse strain.

Studies of the Producer Cell of Herpes Simplex Virus-Induced Interferon in Mouse Spleen Cell Cultures

The producer cell of type I interferon was studied in spleen cell cultures of C57BL/6 mice stimulated by inactivated Herpes Simplex Virus (HSV). Interferon production was not abolished by pretreatment of the spleen cells by anti-theta serum plus complement. The producer cell of interfere on was not removed by plastic adherence and was not destroyed by the addition of silica. It was present in spleens of 3 day old C57BL/6 mice and in spleens of nu/nu mice. It was not inactivated by treatment of nu/nu spleen cells by anti-theta serum plus complement. HSV-induced interferon production was abolished by passage of the spleen cells through nylon wool columns and by irradiation (1000 R) of the spleen cells. Collectively these data suggest that in murine spleen cell cultures type I interferon is produced by B cells. However, our data do not allow to rule out that the interferon producing cell may be an immature macrophage or an immature T cell. References

Interferon Production by Corynebacterium Parvum and BCG-Activated Murine Spleen Macrophages

Macrophages were identified to be a major source of interferon produced in murine spleen cell cultures after intravenous injection of Corynebacterium parvum (C. parvum), strain CN 6134 or Bacille Calmette Guérin (BCG). Another strain of C. parvum, CN 5888, which lacks RES stimulating activity and adjuvant activity in vivo, was not effective when injected intravenously. Protein synthesis was required for interferon activity to be produced and protein synthesis was also required for the antiviral state to be expressed. The antiviral activity was relatively stable to pH 2 and neutralized by an antiserum against virus-induced fibroblast interferon, thus exhibiting some properties of type I interferon. In vitro only CN 6134, the biologically active strain, could induce small amounts of interferon in spleen macrophage cultures. Macrophages from CN 6134 or BCG-infected athymic nu/nu mice produced similar interferon titers as their controls. It is concluded that infection with certain immunomodulators can activate splenic macrophages via a predominantly T-cell independent mechanism. Interferon in turn may operate locally as a mediator of immunoregulation.

Inhibition of mitogen-induced polyclonal activation by by a synthetic adjuvant, muramyl dipeptide (MDP).

A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), was previously shown to enhance polyclonal antibody response in murine spleen cell cultures. When MDP was added to the culture together with a potent murine B cell mitogen (such as bacterial lipopolysaccharide (LPS)), it inhibited completely the LPS-induced polyclonal activation without affecting either the 3H-thymidine incorporation or the number of blast cells in cultures. Strong suppression of mitogen-induced polyclonal activation by MDP was obtained by using a large range of cell concentrations in cultures and over various dosage levels of the stimulating mitogens (LPS and NWSM). An inhibition could be obtained even when MDP was added 24 hr after the addition of the mitogen, and highly significant suppression was observed in the absence of cell division in the cultures.

Comparison of the Binding of Radiolabelled Human IgG and Fc Fragments to Murine Spleen Cells

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.

Lipopolysaccharide‐Induced Mediators Assisting the Proliferative Response of C3H/HeJ Thymocytes to Concanavalin A

3H-Thymidine uptake of thymocytes from LPS-responder Balb/c mice in the presence of a submitogenic dose (0.5 microgram/ml) of con A in vitro was significantly enhanced by adding LPS (0.1 to 2.5 microgram/ml), while the uptake of thymocytes from LPS-nonresponder C3H/HeJ was not enhanced by LPS. However, "endotoxin soups," which were prepared from the supernatants of LPS-responder murine spleen cell cultures in the presence of LPS, clearly increased the incorporation of 3H-thymidine into C3H/HeJ thymocytes in the presence of this small amount of con A. The soup prepared from C3H/HeJ spleen cell cultures did not show any synergistic effect with con A. Even if the major histocompatibility between soup-producer cells and responder cells to con A was different, the soups were still effective. The active substance in the "endotoxin soups" was eluted through a Sepharose CL-4B column, and its molecular size was estimated to be about 20,000 daltons. The activity of the soups was destroyed by heating at 70 C for 30 min or at 80 C for 10 min. Digestion with trypsin destroyed the activity of the soups, but digestion with DNase or RNase did not. The role of the active substance in the soups in synergy with con A and its relation to the synergistic effect of con A and LPS are discussed.

Generation of H-2 Restricted Cytotoxic T Cells by Trinitrophenylated Proteins in Vitro: Specificity and Requirements

Abstract Addition of TNBS-conjugated soluble proteins to cultures of murine spleen cells, of the H-2k haplotype, led to the generation of MHC-restricted cytotoxic T cells. This phenomenon was effected by a wide range of proteins including ovalbumin, bovine γ-globulin, bovine serum albumin, human γ-globulin, human serum albumin (TNP-HSA), and keyhold limpet hemocyanin. Cytotoxic T lymphocytes (CTL) generated in such manner showed specificity for the hapten rather than for the protein carrier, in that they lysed cells bearing hapten coupled to either heterologous or homologous carriers. Spleen cell populations incubated with soluble HSA substituted to various degrees (8–40 moles TNP/mole HSA) displayed TNP on their surface as evidenced by a) C-mediated lysis, and b) spectrophotometric measurement of solubilized cell membranes. However, only those cells bearing HSA substituted at more than 26 moles TNP/mole HSA were susceptible to lysis by hapten-specific H-2 restricted CTL. Furthermore, TNP-HSA with lower substitution (<26 mole/mole) did not stimulate hapten-specific cytotoxic responses. These observations illustrate several features of target cell recognition by hapten-specific, H-2 restricted, cytotoxic T-cells: i) These CTL do not show specificity to the protein carrier. In this regard cytotoxic T cells differ from T cells responsible for delayed-type hypersensitivity (DTH). ii) Hapten and H-2 need not be covalently linked. iii) Hapten display on the cell surface, even in relatively large amounts, is not in itself sufficient. Rather, it seems that some stringent requirements of protein derivatization and/or distribution have to be met irrespective of whether the protein substituted is intrinsic to the cell membrane or exogenously added. These findings represent a reproducible system for further investigation of the exact molecular nature of the antigenic determinants recognized by CTL.

Tumor necrosis serum induces a serologically distinct population of NK cells.

Murine spleen cells generate nonspecific cytotoxic cells, referred to as natural killer (NK) cells, within 4 d of incubation in Mishell-Dutton cultures. This NK cell type does not arise in cultures of BALB/c.nu spleen cells or in cultures of T-cell depleted C57BL/6 spleen cells, indicating that its activation depends on T cells. Another type of NK cells is induced by tumor necrosis serum in murine spleen-cell cultures. It arises within 24 h and its activation does not depend on T cells. This cell type (and its precursor) expresses the recently discovered cell-surface marker Qa5 (controlled by the Q region of chromosome 17) that distinguishes this NK cell from the NK cell that depends for its activation on thymic function. Qa5+ NK cells are also induced by interferon.

A human monocyte function test: Release of B-cell differentiation factor (BDF)

The test described here determines a secretory monocyte function and differs in this respect from existing monocyte function tests; it measures the ability of monocytes to release a factor which induces differentiation of B cells (B-cell differentiation factor, BDF). BDF causes antigen-stimulated B cells to differentiate into antibody-producing cells. BDF released by human monocytes acts across species and can be assayed conveniently in murine spleen cell cultures. BDF is released by human monocytes in response to stimulation with bacterial lipopolysaccharide (LPS). Interference by residual LPS in the BDF preparation can be eliminated using for the assay of BDF-activity spleen cells from a mouse strain that is genetically unresponsive to LPS. An initial survey of some 150 individuals showed considerable variation in BDF production. Cancer patients as a group showed low activity. Low BDF production was also found in patients with aplastic anemia and common variable immune deficiency.

Growth of Trypanosoma musculi in cultures of murine spleen cells and analysis of the requirement for supportive spleen cells.

Growth of Trypanosoma musculi in vitro has been achieved. The number of parasites increased by more than 1,500-fold in less than 8 days under the most suitable conditions. The rate and magnitude of growth was comparable to that which occurs in inoculated murine hosts. Maximum growth was displayed in cultures composed of RPMI 1640 medium supplemented with fetal calf serum, murine spleen cells, and foreign erythrocytes (sheep). No growth occurred in the absence of spleen cells. The adherent, macrophage-rich population supported parasite growth much better than did the nonadherent population. Parasite growth was excellent in the presence of irradiated spleen cells or of cells from thymectomized, irradiated, bone marrow-reconstituted mice. The important cells appeared to be macrophages. The beneficial effect of sheep erythrocytes probably resulted from preoccupation or stimulation of phagocytes. Soluble substances released by spleen cell cultures promote parasite growth, as was shown by experiments with double-compartment culture vessels. The utility of this culture system for analysis of host immune responses against the trypanosome was demonstrated.

Modulation of the Antibody Response to Sheep Erythrocytes in Murine Spleen Cell Cultures by a T Cell Mitogen Extracted from Bordetella pertussis

Mailard and Bloom have reported that the adjuvant effect of Bordetella pertussis vaccine (BPV) on antibody response in vitro to sheep erythrocytes (SRBC) was observed when BPV (5.1 •~ 105 to 6.4 •~ 106 organisms) and SRBC were added to cell cultures prepared from the spleen of BPV-primed mice, whereas this effect of BPV was not observed in cultures prepared from the spleen of normal mice (8). Recently, Murgo and Athanassiades showed that the adjuvant effect of BPV was observed on the antibody response in vitro to SRBC using spleen cells obtained from normal mice when large doses (1.0-1.5 •~ 108 organisms) of BPV had been added to the culture

Mechanism of adjuvant activity of dental plaque: in vitro activation of residual helper T-cell precursors in T-cell-deficient murine spleen cell cultures

The immunoenhancing activity of a water-soluble extract of dental plaque (DP), which contains a mixture of microbial antigens, has been investigated. DP was tested for its capacity to augment the in vitro antibody-forming cell (AFC) response to sheep erythrocyte by adherent spleen cells from thymectomized, lethally irradiated, and bone marrow-transplanted (TxB) mice. Although DP was found to induce a small polyclonal AFC response, most of the increase in AFC induced by DP was antigen dependent. The latter enhancing effect is an indicator of the adjuvanticity of DP. This adjuvant activity of DP was T-cell-dependent, since removal of the residual prethymic and/or thymic-derived lymphocytes (T-cells) by anti-T-cell serum (anti-theta) and guinea pig complement abrogated the capacity of DP to augment the in vitro AFC response. This view was further supported by the synergistic restorative effect obtained by culturing anti-theta-treated adherent spleen cells with both DP and a population of unactivated T-cells that by themselves were unable to significantly enhance AFC responsiveness. Moreover, DP was found to be mitogenic for thymocytes. The cumulative results suggest that the adjuvant activity of DP is dependent on both the T- and B-cell-activating components present in DP.

In vitro generation of tumor-specific cytotoxic lymphocytes. Secondary allogeneic mixed tumor lymphocyte culture of normal murine spleen cells.

In vivo or in vitro immunity to murine leukemia virus (MuLV)-induced leukemia cells which do not effectively produce virus, has been difficult to demonstrate. Because immunizations with allogeneic murine leukemia cells have been used to confer syngeneic tumor immunity to virus- producing cells, we attempted to generate lymphocytes, cytotoxic to syngeneic nonproducer leukemia cells, by stimulating normal murine spleen cells with allogeneic nonproducer leukemia cells in mixed tumor lymphocyte culture (MTLC) reactions in vitro. Secondary allogeneic MTLC of normal C57BL/6 or DBA/2 spleen cells effectively produced syngeneic tumor-specific cytotoxic lymphocytes. Target cells lysed in lymphocyte- mediated cytolysis (LMC) assays, included both Friend and Rauscher virus- induced syngeneic murine leukemia cells and chemically-induced hematopoietic tumor cells. Syngeneic tumor cells were lysed regardless of whether they produced infectious MuLV or expressed viral antigens gp-71, p-30, or p-12 at the cell surface. Syngeneic normal cells (thymus, lymph node, or Concanavalin A-stimulated spleen cells) used as targets in LMC assays were uneffected by lymphocytes harvested from secondary allogeneic MTLC. Several other in vitro culture treatments including secondary syngeneic MTLC and repetitive mixed lymphocyte culture stimulations were incapable of generating tumor-specific cytotoxic lymphocytes. Based upon these results, we propose that secondary MTLC stimulation of normal spleen cells with allogeneic nonproducer leukemia cells selects for the proliferation of two subpopulations of antigen-specific cytotoxic lymphocytes. The population capable of effecting syngeneic tumor cell lysis is directed against tumor-associated cell surface antigens which may be distinct from viral structural proteins or glycoproteins. The growth of these tumor-specific cytotoxic lymphocytes may be enhanced by a soluble allogeneic effect factor produced by the proliferation of the second subpopulation of lymphocytes generated in repetitive allogeneic MTLC, namely those lymphocytes with specificities directed against differing histocompatibility antigens.

Synthetic polyamines added to cultures containing bovine sera reversibly inhibit in vitro parameters of immunity

POLYAMINES are present in high concentration in foetal1 and neoplastic tissue2 and seminal fluid3—all representing antigenic challenges which usually fail to elicit the appropriate immune response. This has prompted us to investigate the influence of polyamines on in vitro parameters of immunity. We report here that micromolar quantities of spermine and spermidine reversibly inhibit three responses of primary cultures of murine spleen cells. These are (1) DNA synthetic response stimulated by phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (con A) or bacterial lipopolysaccharide (LPS); (2) mixed lymphocyte response (MLC) stimulated by irradiated allogeneic cells; and (3) the induction but not the expression of the cytolytic lymphocyte response. The inhibition does not seem to be due to toxicity but occurs only in the presence of calf or foetal calf sera.

The Xenogeneic Effect

Abstract Studies were conducted on the mechanism of action of a soluble mediator which was generated in human mixed lymphocyte cultures and assayed for helper activity in T cell-deficient murine spleen cell cultures. The mediator, termed xenogeneic reconstitution factor or XRF, restored the anti-sheep erythrocyte plaque-forming cell response of spleen cells from thymectomized, lethally irradiated, and syngeneic bone marrow transplanted (TxB) mice. It was found that in order to obtain a maximal antibody response, the XRF had to be present sometime during the first 24 to 40 hr of the induction process. XRF was also required during the last 24 hr of the incubation period. A striking synergistic effect was obtained by combined exposure of the T cell-deficient spleen cell cultures to XRF during the first and last days of culture. These data suggested a bi-modal mechanism of action of XRF, and raised the possibility that in addition to providing a signal to the B cells, XRF-mediated activation of the residual TxB splenic T cells was crucial to the successful restoration of the antibody response. Treatment of the nonadherent splenocytes from the TxB mice with anti-T cell serum and guinea pig complement completely abrogated the antibody response of these cells in the presence of adherent spleen cells, sheep erythrocytes, and the helper factor XRF. The antibody response of this combination of cells, antigen, and XRF was reconstituted by a population of unimmunized murine T cells which, in the absence of XRF, were totally unable to restore responsiveness. These results suggest that B cell activation to the formation of antibody involves the mandatory co-participation of two functionally distinct helper activities.

Biological Expressions of Lymphocyte Activation

Abstract Supernatant fluids from murine spleen cell cultures incubated with concanavalin A for 48 hr contain a factor(s), soluble immune response suppressor (SIRS), which suppresses plaque-forming cell responses to sheep erythrocytes by murine spleen cells in vitro. In the present studies, some of the biochemical and biophysical properties of SIRS were investigated. SIRS was non-dialysable; the suppressive activity was stable at 56°C for 30 min, but was destroyed by treatment at 70°C for 30 min, 80°C for 10 min, or at pH 2. The suppressive activity was not absorbed by the stimulating antigen, SRBC, or antisera against murine IgG or µ-chain, suggesting that SIRS does not contain immunoglobulin determinants. Murine spleen and thymus, but not kidney cells, however, absorbed SIRS activity. Enzyme treatments revealed that SIRS was resistant to DNase and RNase, but was destroyed by trypsin and chymotrypsin. In gel filtration with Sephadex G-100, SIRS activity eluted in the fraction corresponding to m.w. in the range between 48,000 and 67,000. With polyacrylamide gel electrophoresis, SIRS activity migrated in the region cathodal to albumin. Isopycnic centrifugation in a cesium chloride gradient suggested that SIRS is a glycoprotein. These supernatant fluids with SIRS activity were also found to contain macrophage migration inhibitory factor (MIF). In the experiments using gel filtration, polyacrylamide gel electrophoresis, and isopycnic centrifugation to fractionate supernatant fluids, SIRS and MIF activity were found in the same fractions, and to date we have been unable to dissociate definitively SIRS activity from MIF activity.

The Effect of Serum Inhibitor on the Antigenic and Mitogenic Responses to E. Coli Bacteria

The effect of serum inhibitory factors on the induction of the in vitro immune response and mitogenic response to Escherichia coli antigens in murine spleen cell cultures was investigated. Both normal and congenitally athymic mice served as spleen cell and serum donors. Cultured CBA/J spleen cells produced anti-bacterial antibody in the presence of mouse serum, but the mitogenic response to the bacterial antigen was suppressed by the addition of mouse serum. The addition of mouse serum from either CBA/J or nude mice to nude spleen cell cultures led to suppression of both the antigenic and mitogenic responses to bacterial antigen. The polyclonal antibody response by CBA/J spleen cell cultures to SRBC induced by bacteria was significantly enhanced by the addition of either CBA/J or nude mouse serum. Conversely, the nude spleen cell polyclonal response to SRBC was significantly inhibited by high concentrations of nude or CBA/J mouse serum. These data indicate that inhibition of the specific E. coli and nonspecific polyclonal antibody responses by mouse serum occurs only in cultures of spleen cells taken from mice lacking a population of T lymphocytes.