Nude Mouse Spleen Cells Research Articles

Lymphoproliferation induced in mouse spleen cells by Mycoplasma arthritidis mitogen. Reversal of the defect of nonresponder mice.

Spleen cells of various mouse strains (e.g. BALB/c, C311, and CBA) reacted towards MAS (a mitogen derived from supernatants of cultured Mycoplasma arthritidis) with a marked lymphoproliferative response. This reactivity was T-cell-dependent. It was reduced by 90% after removal of macrophages by passage of the spleen cells through Sephadex G-10 columns. Addition of 2-mercaptoethanol (2-ME) to macrophage-depleted CBA spleen cells completely restored the response to MAS. Spleen cells of C57BL/6 and C57BL/10 mice were unreactive to MAS, even in the presence of macrophages, and this non-reactivity was controlled by the I-region of H-2. Other mouse strains that, similarly to C57BL/6, lack the expression of I-E on the cell surface (that is, mice of the haplotype H-2f, H-2q, and H-2s) were also non-responsive to MAS. However, the addition of 2-ME to spleen cells of non-responder mice resulted in high lymphoproliferative responses to MAS, which were as high as those of CBA spleen cells. The reaction of C57BL/6 spleen cells to MAS in the presence of 2-ME again was T-cell-dependent, as shown by data with spleen cells of homozygous nude mice and spleen cells treated by anti-thy-1 and C. A macrophage dependency of this response was also evident. When C57BL/6 spleen cells were vigorously freed of accessory cells by the use of nylon wool columns, the MAS response could no longer be restored by 2-ME.

Increase of natural killer (NK) activity of mouse lymphocytes following in vitro treatment with cytosine-arabinoside

The in vitro influence of cytosine-arabinoside (Ara-C) on mouse NK activity was studied treating effector cells, target cells or effector and target mixture with graded concentrations of the drug. Ara-C increased the NK efficiency of mouse splenocytes without enhancing the susceptibility of target cells or the cytolytic events when added to effector - target mixture. This phenomenon was confirmed with splenocytes collected from congenitally athymic (nude) or conventional donors of different ages, untreated or depressed or boosted for NK activity by various agents. In addition Ara-C increased the NK activity of spleen cells of nude mice deprived of nylon-adherent cells, and did not affect suppressor cells capable of inhibiting the lytic phase of NK process. The drug was able to significantly augment the binding ability of spleen cells to the NK-sensitive YAC-1 target. It was concluded that Ara-C would increase the efficiency of natural cytotoxicity presumably through a direct influence on effector lymphocytes.

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol.

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.

Role in nude mice of interferon and natural killer cells in inhibiting the tumorigenicity of human hepatocellular carcinoma cells infected with hepatitis B virus

The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 10(6) cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 10(6) cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 10(6) PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response. The nature of the response was delineated by treating nude mice challenged with tumor cells with sheep anti-mouse interferon globulin (anti-IFN). When 2 X 10(6) cells were injected, tumor growth occurred in 75% of anti-IFN-treated mice, whereas controls injected with the same number of cells, but not receiving anti-IFN, failed to develop tumors. The tumors in the anti-IFN-treated mice were highly invasive and the latency period until tumor appearance was reduced to 3-5 d. An inverse correlation was found between susceptibility of the hepatoma cells to natural killer (NK) activity in vitro and resistance to tumor growth in vivo. In vitro cytotoxicity for PLC/PRF/5 cells was eliminated by anti-NK 1.1 and complement, establishing the effector cell as an NK cell. NK cell activity 14 d after inoculation of mice with PLC/PRF/5 cells was augmented against PLC/PRF/5 target cells but not against SK-HEP-1 cells. Treatment of mice with ALS, irradiation, or anti-IFN abolished NK activity against PLC/PRF/5 cells. Co-cultivation of nude mouse spleen cells with PLC/PRF/5 but not with HBsAg or SK-HEP-1 cells induced secretion of murine IFNalpha. These results suggest that the IFN/NK cell system may play a role in limiting tumorigenicity and invasiveness of HBV-infected human hepatocellular carcinoma cells by a mechanism similar to that found for other cells persistently infected with viruses.

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF).

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.

Suppression of T cell cytotoxicity by nude mouse spleen cells: reversal by monosaccharides and interleukin 2.

The effects of monosaccharides on the suppression of cytotoxic T cell generation by spleen cells from nu/nu mice were examined. Suppression of the B6 anti-BALB/c response and the B6 anti-C3H response was reversed by alpha-methyl-D-galactoside (alpha MG) but not other sugars, including beta MG. Suppression was associated with a decrease in the level of IL 2, which suggests competition; this decrease was also reversed by alpha MG.

Stimulation of chemiluminescence by synthetic muramyl dipeptide and analogs.

The effect on respiratory burst of murine splenic cells after in vitro exposure to synthetic muramyl dipeptide (MDP) and 6-O-acyl and quinonyl derivatives was studied at an early phase of interaction by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. The MDP molecule enhanced CL, but the degree of CL response varied with the kinds of fatty acids introduced in the chemical structure of synthetic glycopeptide analogs. A 6-O-acyl derivative possessing an alpha-branched fatty acid chain, B30-MDP, stimulated maximum levels of CL activity. High CL responses were obtained with L8-MDP having a short chain of linear fatty acids and with QS-10-MDP-66 containing a ubiquinone compound. CL was also stimulated by MDP and its analogs in the spleen cells of nude mice lacking mature T lymphocytes, but the extent of stimulation was decreased compared with that of normal spleen cells.

Enhancement of growth of bovine leukemia cell line (FLK) by anti-asialo GM1 serum and the effect of the serum on NK and K cell activities in nude mice.

The enhancement of tumor growth in athymic nude mice transplanted with bovine leukemia cell line (FLK) was observed by inoculation of anti-asialo GM1 (gangliotetra-glycosylceramide) serum which has been shown to eliminate natural killer (NK) activity in vitro and in vivo. The biological mechanisms for the enhancement were investigated by examining NK activity and antibody-dependent cellular cytotoxicity (ADCC) of the spleen cells of the nude mice. FLK cells, that are weakly sensitive to NK cell-mediated immunolysis. were also killed by normal spleen cells of nude mice in the presence of specific antibodies to FLK cells. The ADCC was suppressed by inoculation of anti-asialo GM1 serum, which indicates that ADCC-functioning killer (K) cells too have asialo GM1 as NK cells. These results suggest that normal nude mice reject transplanted FLK cells by K cells as well as NK cells and inoculation of anti-asialo GM1 serum allows the tumor growth through elimination of both the NK and K cell activities.

The selection and maintenance of the V region determinant repertoire is germ-line encoded and T cell-independent.

The number of lipopolysaccharide-sensitive precursor cells synthesizing immunoglobulin (Ig) which reacts with the monoclonal anti-M460 antibody F6(51) has been determined in the spleen and in the bone marrow of different strains of mice. These precursor frequencies fall into two quantitatively different groups. The first group includes mice with the same Igh haplotype as BALB/c animals (Igha). In this group, spleen cells contained between 1:10(4) to 1:5 X 10(4) B cell precursors secreting Ig which bound F6(51). The second level of precursor was obtained with animals with allotypic haplotypes other than Igha. These values were too low to allow accurate frequency determinations. The frequency of these cells in mice of the latter group, however, increased dramatically when these animals were hyperimmunized with the monoclonal anti-M460 antibody. Similar results were obtained when the frequencies were determined using the Ig- fraction of bone marrow cells. Surprisingly, the numbers of lipopolysaccharide-sensitive B cell precursors secreting F6(51)-binding Ig in spleen cells of nude mice was found to be similar to the one in splenocytes of normal mice, and even in this case, the frequencies reflected the genetic background of the animals tested. Taken together these data support the notion that the establishment and the maintenance of the M460 idiotypic repertoire is germ-line encoded and independent of regulatory T cells.

Flow cytometric analysis of the effect of phenytoin and its major metabolite on mitogen stimulated mouse spleen cells

The in vitro immunopharmacological effects of phenytoin (PHT) and its major metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were investigated by flow cytometric analysis of the DNA content of mitogen stimulated mouse spleen cells. The qualitative effects of PHT and HPPH were similar in concanavalin A stimulated mouse spleen cells with both compounds causing an increase in the percentage of S phase cells. The data suggests that this effect is due to an augmentation of cell cycling as demonstrated by the significant increase in 4N cells in PHT treated cultures relative to control cultures following colcemid treatment. A PHT time course study revealed an increase in S phase cells and a subsequent increase in 4N cells. PHT had no significant effect on congenitally athymic nude mouse spleen cells stimulated with lipopolysaccharide (LPS) except at the highest concentration tested (80 μg/ml) where a depression of cell cycling was observed. HPPH caused a colcemid-like accumulation of 4N cells in the LPS stimulated nude mouse spleen cell cultures. PHT and HPPH were found to be effective in enhancing cell cycling in cultures containing a significant population of T-cells stimulated with a T-cell mitogen whereas an inhibitory effect was observed in cultures without T-cells stimulated with a B-cell mitogen. The capacity of PHT to enhance the mitogenic action of concanavalin A may relate to its capacity to induce immunologic abnormalities and lymphadenophathy in humans.

In vitro immunological activities of the polysaccharide fraction from Haemophilus influenzae type a endotoxin.

Mild hydrolysis of Haemophilus influenzae type a lipopolysaccharide by ion exchangers in the presence of chloroform, to remove the lipid moiety, yielded a nontoxic and immunogenic polysaccharide fraction. This polysaccharide selectively triggered murine B lymphocytes in vitro: (i) it induced enhancement of thymidine incorporation and stimulated antibody secretion in cultures of normal and nude mouse spleen cells; (ii) it did not stimulate splenic T lymphocytes; (iii) the activation of B lymphocytes was not absolutely dependent on the presence of macrophages. Sepharose 4B gel filtration showed that this polysaccharide consisted at least of two fractions: PS I (molecular weight [MW] 10(6)) and PS II (MW 10(4)). Only PS I was found to act as a polyclonal B cell activator. EDTA treatment dissociated the polysaccharide into PS III (MW 10(6)) and PS IV (MW 10(4)), which was not reassembled after the addition of 0.02 M CaCl2. Both fractions PS III and PS IV were unable to stimulate B lymphocytes. The immunological active fraction of H. influenzae polysaccharide is PS I. This fraction consists of a high-molecular-weight group (10(6)) and an association of 10(4)-MW aggregated units.

A B lymphoma cell line produces growth factors for hemopoietic, lymphoid, and mast cells

A cloned B lymphoma cell line (WEHI-231.1) released P-cell stimulating factor, a factor allowing the in vitro growth of presumptive mast cell precursors together with small amounts of T-cell growth factor and granulocyte-macrophage colony-stimulating factor. These factors were produced without deliberate stimulation of the B lymphoma cells. Lipopolysaccharide-stimulated nude mouse spleen cells also released low levels of P-cell stimulating factor and granulocyte-macrophage colony-stimulating factor. These results raise the possibility that B cells may produce regulatory factors.

Synthesis of lambda 1, lambda 2, and lambda 3 light chains by mouse spleen B cells.

To determine whether the infrequency of immunoglobulins with lambda 3 light chains is due to a corresponding scarcity of lambda 3 B cells, the production of the various lambda chain subtypes (lambda 1, lambda 2, and lambda 3) by normal spleen cells was compared. The results showed that lambda 1, lambda 2, and lambda 3 chains are produced in a ratio of about 1.0: 0.7 : 0.3, respectively. The argument is made that lambda 1, lambda 2, and lambda 3 B cells exist in the same ratio. Results obtained with neonatal and nude mouse spleen cells suggest that these small differences are not due to stimulatory effects of environmental antigens or regulatory T cells. The much greater disparity in the abundance of lambda subtypes in various antibody responses and serum Ig suggests that lambda 1 B cells may be more likely than lambda 2 or lambda 3 B cells to differentiate into antibody-secreting plasma cells.

Fatal response suggestive of graft-versus-host reaction following transplantation of spleen cells from allogeneic athymic (nude) donors.

Normal female SJL/J mice were exposed to 950 R of total body irradiation (TBI) and transplanted with allogeneic spleen or marrow cells from normal or nude (athymic) C57BL/10 donors. With nude mouse donor marrow, no evidence of graft-versus-host (GVH) response was seen and all SJL/J recipients survived for more than 75 days. In contrast, when spleen cells taken from the same nude C57BL/10 donors were engrafted into SJL/J mice the incidence of fatalities among the recipients was 70% by 60 days. Furthermore, all of the recipients of nude mouse spleen cells showed signs strongly suggestive of GVH response. Comparative fatalities among the recipients of cells from normal donors were 27% for marrow at 60 days and 100% for spleen at 11 days, and these were accompanied by the characteristic signs of GVH response usually seen after transplantation of cells from normal allogeneic donors. Transplantation of normal C57BL/10 marrow mixed with small numbers at normal spleen cells resulted in an increase in the number of fatalities among the SJL/J recipients, and an increase in the severity of the signs of GVH response as compared to that seen following engraftment of normal C57BL/10 marrow alone. However, no such increases in fatalities or severity were observed when similar amounts of nude C57BL/10 spleen cells were engrafted along with normal marrow cells into SJL/J recipients. The results suggest that a factor may exist in nude mouse spleen which in allogeneic transplantation can lead to a fatal response suggestive of GVH reaction, but that nude mouse spleen lacks the T cell-related ability to enhance GVH response that has been previously demonstrated following allogeneic transplantation using normal spleen and marrow donors.

Phorbol myristic acetate enhances the production of Interleukin 2

Interleukin 2 is an antigen-nonspecific factor produced by Concanavalin A (Con A)-activated mouse spleen cells that has a number of biologic activities including the capacity to stimulate thymocyte proliferation, support the growth of continuous T cell lines, augment the antibody response of nude mouse spleen cells, and support the generation of antigen-specific cytotoxic T cells. In order to obtain increased amounts of Interleukin 2 for further purification and biologic studies, we have examined the use of Phorbol Myristic Acetate (PMA) as a costimulant. In this report we demonstrate that the addition of PMA to Con A-induced mouse spleen cell cultures results in a 5- to 20-fold increase in the production of Interleukin 2 activity under serum-containing and serum-free culture conditions.

Immunoglobulin isotype expression. II. Frequency analysis in mitogen-reactive B cells.

The frequency of lipopolysaccharide (LPS)-reactive B cells developing into clones that secrete the various immunoglobulin (Ig) classes has been determined in vitro, in cells from BALB/c mice, under culture conditions which detect all growth-inducible cells. Secretion of the different Ig classes was assessed in the protein A plaque assay for Ig-secreting, plaque-forming cells by using developing antisera specific for either IgM, IgG1, IgG2a, IgG2b, IgG3 or IgA. In all lymphoid organs tested (spleen, bone marrow, mesenteric lymph nodes and thoracic duct), a considerable proportion of all B cells (5-20%) was induced by LPS to yield a clone of IgM-secreting cells. Frequency determinations of LPS-reactive cells giving rise to descendants secreting other Ig isotypes revealed that, on an average, and irrespective of the origin of the cells, 7% of all IgM-secreting clones switched to the synthesis of IgG1, 39% to IgG2, 41% to IgG3 and 1% to IgA. Roughly the same frequencies of B cells switching CH gene expression were found among spleen cells of athymic nude mice. No correlation was found between the clonal frequencies of CH gene expression in polyclonally activated B cells and the in vivo "background" Ig-secreting cells suggesting that the CH gene expression in B cells is influenced by the quality of stimulation and other regulating influences.

Polyclonal B cell activator associated with alpha-2-macroglobulin in the serum of patients with rheumatoid arthritis.

We investigated whether patients with rheumatoid arthritis have a polyclonal B cell activator (PBA) in their serum by using three methods: (1) the ability of any PBA to maintain the surface Ig of rabbit or human B cells in vitro; (2) the induction of blast transformation in human B lymphocyte cultures, and (3) stimulation of nude mouse spleen cells in vitro. All three methods indicated that a PBA is present in the serum of patients with rheumatoid arthritis but not in normal individuals or in patients with arthritis in which autoimmune phenomena have not been demonstrated. The entire PBA activity in rheumatoid arthritis patient serum was found associated with the macroglobulin fraction obtained by Sephadex G-200 chromatography and was precipitated by rabbit anti-human alpha 2-macroglobulin, but not by rabbit anti-Ig antibody. When alpha 2-macroglobulin was purified from patient serum the entire PBA activity was recovered in this fraction. Normal alpha 2-macroglobulin prepared by the same procedure had no PBA activity. Thus, the existence of a PBA associated with alpha 2-macroglobulin was demonstrated in serum of patients with rheumatoid arthritis.

Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes.

Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation of thymidine into DNA; the expected dilution of these two responses by nude spleen cells did not occur. However, if the nude splenocytes were added immediately prior to assay to the enriched T cells that had been precultured in presence of Con A, the expected dilution of the activated T-cell responses occurred; both 86Rb uptake and thymidine incorporation were reduced proportionally to the degree of dilution of the T cells by the nonresponding cells. These data indicate that during co-culture in presence of Con A there is interaction between the T cells, capable of responding to mitogens, and the nude spleen cells. Attempts to demonstrate a diffusible factor in the supernatants of stimulated T cells were unsuccessful. The measured interaction is sufficient to explain our previous paradoxical findings that enrichment of T cells as measured by membrane markers did not cause a corresponding enrichment for either cation transport or for thymidine incorporation, and that depletion of T cells in the B-enriched cultures did not cause a corresponding decrease in these two Con A induced responses.

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K, D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.

Enhancement of growth of a radiation-induced lymphoma by T cells from normal mice.

The effect of lymphocytes from normal mice on the growth of a syngeneic, radiation-induced, T-cell-derived lymphoma was investigated. Thymus and spleen cells enhanced the growth of admixed lymphoma cells in a reproducible manner. Growth enhancement was manifested by the earlier appearance and higher final incidence of tumours. Lymphocytes also enhanced the growth of radiation-damaged lymphoma cells. The enhancing activity of spleen cells was predominantly a property of T cells, since it was abolished by treatment with anti-theta serum plus complement and significantly less in spleen cells of nude mice. Tumour-enhancing thymocytes seem to belong to the immature thymic subpopulation, as indicated by their binding to peanut agglutinin.