Nude Mouse Spleen Cells Research Articles

Mitogenic Activity of CEL-I, anN-Acetylgalactosamine (GalNAc)-Specific C-Type Lectin, Isolated from the Marine InvertebrateCucumaria echinata(Holothuroidea)

An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.

Therapeutic efficiency of IL-2 gene transduced tumor vaccine for head and neck carcinoma

Transduction of the human interleukin-2 (IL-2) gene into tumor cells was carried out in order to develop a new immunotherapy for advanced head and neck carcinomas with a poor outcome. We transduced the IL-2 gene into KB cells, a head and neck squamous cell carcinoma cell line, using a defective herpes simplex viral (HSV) amplicon vector as a gene transfer vehicle. A high level of IL-2 was secreted by IL-2 gene-transduced KB cells (KB/IL-2). The IL-2 producibility of irradiated KB/IL-2 cells was almost the same as that of non-irradiated cells. In the tumor establishment model in nude mice, IL-2 and interferon-γ (IFN-γ) at high concentrations were detected in the sera of mice transplanted with KB/IL-2 cells. The spleen cells of nude mice transplanted with KB/IL-2 cells exhibited high cytotoxic activity compared to those from mice transplanted with KB cells and from untreated mice. Three of five mice transplanted with KB/IL-2 cells rejected tumors. In the treatment of established tumors, therapeutic effects due to irradiated KB/IL-2 were dose-dependent. The suppressive effects on tumor growth were blocked by anti-asialo GM1, anti-human IL-2 and anti-IFN-γ antibodies. Immunohistochemical observation revealed the presence of asialo GM1 + cells among the KB/IL-2 cells in tumors transplanted into nude mice.

Detection of thymopoietin-responsive proteins in nude mouse spleen cells by two-dimensional polyacrylamide gel electrophoresis and image processing.

Lymphoid cells isolated from the spleen of BALB/c nu/nu nude mice were treated with synthetic human thymopoietin, and newly synthesized proteins were labeled by [35S]methionine incorporation. In the control experiment, the same lot of spleen cells were incubated in the labeling medium without the addition of thymopoietin. Urea/detergent-soluble proteins were extracted from the cells after 3 h incubation to be separated by two-dimensional poly-acrylamide gel electrophoresis. Spots of [35S]methionine-labeled proteins were visualized by autoradiography and analyzed by image processing. The computer-aided spot matching screened out three major thymopoietin-responsive proteins, TRP-1, -2 and -3. [35S]Methionine incorporation into TRP-3, of which the isoelectric point and molecular mass were approximately pI 5 and 10 kDa, respectively, was decreased by the thymopoietin treatment. In contrast with the down regulation, TRP-1, which was slightly higher in pI and slightly larger in molecular mass, and TRP-2, which was slightly higher in pI and almost the same in molecular mass as TRP-3, were evidently induced by the treatment. However, TRPs could not be assigned to Thy-1 antigen on the difference in molecular mass. The specific induction by the thymopoietin treatment suggested that TRP-1 and -2 might be novel proteins related to the intracellular signal transduction.

Cytotoxicity induced by monoclonal antibody to alpha-fetoprotein coadministered with spleen cells of nude mice.

This paper describes an attempt to effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC) in nude mice. A monoclonal antibody against alpha-fetoprotein, 80G, coadministered with spleen cells from other nude mice bearing HuH-7N (xenograft of human hepatoma cell line, HuH-7) significantly suppressed the growth of HuH-7N as compared to treatment with 80G alone. 80G with spleen cells from normal nude mice also had some suppressive effect. In contrast, no effect was observed with each spleen cells alone as well as 80G alone. These results suggest that further supply of effector cells could enhance ADCC activity in nude mice.

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4.

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1+, Thy1-, and the majority of effector cells were aGM1+, Thy1+, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-alpha,beta antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-alpha,beta production was associated with LAK induction of NK lineage cells by IL-4.

T Cell Receptor‐independent Cell‐mediated Cytotoxicity by Nude Mouse Lymphokine‐activated Killer Cells

Lymphokine‐activated killer (LAK) cells, which can lyse a variety of tumor cells, can be induced from both normal and athymic nude mouse spleen cells by culture with high doses of recombinant interleukin 2 (rIL‐2). LAK cells generated from nude mouse spleen cells (Nude‐LAK cells) express just Thy 1.2 antigen, but not CD4 and CDS antigens. Nude‐LAK cells express neither T3 molecule, T cell receptor (TCR) αβ nor TCR γδ on their cell surface. The lack of TCR expression on Nude‐LAK cells was confirmed by the results of northern blot analysis. LAK cells generated from normal mouse spleen cells (Nor‐LAK) express TCR α, β transcripts, while Nude‐LAK cells express only sterile TCR β transcript, but not TCR α transcript. TCR γδ transcripts were scarcely detected in both Nor‐LAK cells and Nude‐LAK cells. Thus, it is strongly suggested that Nude‐LAK cells can recognize and lyse tumor cells by TCR‐independent mechanisms. Monoclonal antibody against lymphocyte function‐associated antigen (LFA‐1) molecule can block the Cytotoxicity of Nude‐LAK cells, indicating an important role of such accessory molecules in Nude‐LAK cell‐mediated Cytotoxicity.

A Newly Established Human Osteosarcoma Cell Line With Osteoblastic Properties

A human osteosarcoma cell line, HuO9, was established from a tumor that was heterotransplanted into athymic nude mice. Antiserum against nude mouse spleen cells was added to the early passage cultures to eliminate the host fibroblastic cells. The cell line retained a high activity of liver/bone/kidney-type alkaline phosphatase (ALP) and secreted osteocalcin, i.e., bone gamma-carboxyglutamic acid-containing protein (BGP), into the medium. The addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) increased the ALP activity as well as the level of BGP secreted into the medium. The ALP of 1,25(OH)2D3-treated cells has the same inhibition characteristics to heat and amino acids as that of untreated cells. Synthetic human parathyroid hormone stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) approximately 100-fold within five minutes. However, the stimulation was not observed with a synthetic human thyrocalcitonin. When HuO9 cells were transplanted into the back of a nude mouse, a tumor with an abundant osteoid formation and mineralization was produced. The results indicate that the HuO9 cell line expresses well-differentiated osteoblastic phenotypes. HuO9 is the first established human cell line to produce BGP, and it provides a useful model for the studies of osteoblasts and the regulatory mechanisms of BGP production.

Deletion of self-reactive T cells in nude mice grafted with neonatal allogeneic thymus.

The cellular basis of tolerance induction has been investigated in BALB/c(H-2d, thy 1.2, M1s1b2a) nude mice grafted with thymus of neonatal AKR/J mice(H-2k,Thy1.1,M1s1a2b). The spleen cells from nude mice grafted with AKR/J thymus showed a significantly decreased level of primary cytotoxic T cell response when stimulated with AKR/J cells, although these cells lysed well target cells of a third party C57BL/6 when stimulated with C57BL/6 cells. Consistent with CTL responses, T cells bearing V beta 6, that is important for recognizing M1s1a-encoded products of the thymic phenotype, were virtually abolished in the spleen and lymph node cells of nude mice 8 wk after grafting with AKR/J thymus. However, a substantial number of V beta 6-bearing T cells were detected in the peripheral organs of nude mice 23 wk after grafting with AKR/J thymus and in those of nude mice grafted with AKR/J fetal thymus depleted of macrophages/dendritic cells by incubating with 2'-deoxyguanosine in vitro before grafting. On the other hand, T cells bearing V beta 3, which are selectively related to M1s2a-encoded products of the host phenotype, were expressed neither on the peripheral T cells of nude mice grafted with AKR/J thymus at any stage after grafting nor on those of nude mice grafted with 2'-deoxyguanosine-treated AKR/J thymus. These data suggested that both V beta 6 and V beta 3 T cells were eliminated in the thymus of nude mice grafted with AKR/J thymus, presumably on the basis of interaction with both of graft-derived persisting and host-derived hemopoietic cells in the thymus and that thymic epithelium appears to have little capacity to eliminate T cells reactive to minor lymphocyte stimulating-encoded products.

Inhibition of Natural Killer Activity by Calcitonin Gene-Related Peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Balb/c mice and nude mice was studied. CGRP dose-dependently (10(-9) to 10(-7) M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10(-7) M CGRP was about 60%. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Balb/c spleen cells. Furthermore, when cells were treated with 10(-9) to 10(-7) M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM1 antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM1 completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM1 injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.

Immunoregulation of murine plasmacytoma. II. Target selectivity of anomalous killer cells and role of immune T cells for the induction.

Requirements for the induction of anomalous killer (AK) cells to syngeneic MOPC 104E-KI81 plasmacytoma, and their pattern of reaction were investigated. The AK cells are Thy-1.2 positive and were induced by cocultivation of normal BALB/c spleen cells for 5 days in vitro with mitomycin C-treated MOPC 104E-KI81 cells in RPMI 1640 medium supplemented with fetal calf serum and 2-mercaptoethanol. The pattern of reactivity was investigated by direct cytotoxicity and indirect cold target inhibition assays using a panel of target cells. The results from cold target inhibition show that anomalous killer cells react with various tumor targets in the same preferential order as the results shown in direct cytotoxicity assay, and indicates that the AK cell population may be detecting the same determinants expressed to different degrees on panels of target cells. Kinetic study showed that peak cytotoxic responses were observed on day 5 and day 4, for primary and secondary sensitizing cultures, respectively. Cytotoxicity was not generated when nude mouse spleen cells were used as responder cells, suggesting the essential role of T cells in the induction. Nylon wool-column-purified splenic T cells from mice primed in vivo with intraperitoneal injection of MMC-treated stimulator cells, but not from normal mice, were able to generate AK cells in vitro. These results indicate that generation of AK cells with NK-cell-like reactivity needs inductive factors provided by the immune reaction between sensitized T cells and stimulatory MOPC 104E-KI81 cells.

H-2 antigen expression and sensitivity of BL6 melanoma cells to natural killer cell cytotoxicity.

The sensitivity of H-2b-high and H-2b-low variants of BL6 melanoma to the cytotoxic action of NK and lymphokine-activated killer cells was investigated. BL6 mouse melanoma cells lack detectable H-2Kb and had low levels of expression of H-2Db Ag. The BL6T2 variant cells, obtained after treatment of BL6 cells with mutagen N-methyl-N-nitro-N'-nitro-soguanidine, had relatively high levels of expression of class I H-2b Ag. Poly(I:C)-stimulated spleen cells of nude mice were highly cytotoxic for BL6T2, whereas H-2b-low BL6 cells were less sensitive to NK activity in an 18-h 51Cr-release assay. Similar results were obtained after 4-h incubation of radio-labeled tumor cells with IL-2-activated effector cells. In contrast, both lines were equally sensitive to lysis by purified granules derived from rat large granular lymphocytes (LGL) or by macrophages. By using various clones selected from BL6 or BL6T2 cells, it was found that BL6 or BL6T2 clones with low H-2b Ag expression were less sensitive to lysis by NK cells than H-2b-high clones. After IFN treatment of either BL6 or BL6T2, the target cells became more resistant to lysis by either NK cells or by purified LGL granules. IFN-treated BL6 cells had substantially increased expression of H-2b Ag and in this respect became similar to untreated BL6T2. However, IFN-treated BL6 cells were more resistant than BL6T2 cells to lysis by NK cells and LGL granules, suggesting that augmentation of H-2b Ag expression and NK resistance could be two independent IFN-induced effects. With a cold target inhibition assay, it was found that BL6T2 or its H-2 positive clones were highly competitive and inhibited the cytotoxic activity of NK and lymphokine-activated killer cells against radiolabeled YAC-1 and BL6T2, whereas BL6 cells or H-2-negative clones of BL6T2 and BL6 lines showed poor competitive ability. Thus, our data indicate that the NK resistance of H-2-low BL6 cells may be due to a paucity of NK recognizable determinants. N-Methyl-N-nitro-N'-nitroguanidine treatment of BL6 melanoma cells was associated with an increase in class I H-2b Ag expression and NK sensitivity, suggesting the involvement of class I MHC Ag in the sensitivity of tumor cells to NK cell-mediated cytotoxicity.

Cellular control of abscess formation: role of T cells in the regulation of abscesses formed in response to Bacteroides fragilis.

Although abscesses are a major sequela of infection, little is known about which cellular events initiate and which prevent this pathologic response. These studies are the first to indicate a role for T cells in the important pathogenic process of abscess development and also in immunity to abscesses induced by Bacteroides fragilis. We have shown that T cells initiate the formation of abscesses in mice after i.p. challenge with B. fragilis. These T cells bear both Ly-1 and Ly-2 surface markers. Nude mice (which have been shown by others to have T cell or T cell precursors) are also able to form abscesses. Cyclophosphamide-treated mice (with depressed T cell function) were not capable of developing abscesses. Reconstitution with normal or nude mouse spleen cells restored this ability. However, reconstitution with anti-Thy-1.2-treated, anti-Ly-1, or anti-Ly-2-treated spleen cells (or a mixture of the two cell populations) failed to allow abscess formation after bacterial challenge. Immunity to abscesses caused by B. fragilis requires two T cells. The first Ly-1-2+ T cell has an IJ surface marker and has been shown to release a small m.w. soluble factor (ITF) that is antigen specific. Immunity to abscesses, however, depends on the interaction of ITF with a second Ly-1-2+ T cell, demonstrated in reconstitution experiments with nude mice. The data presented document a critical role for T cells in abscess induction and suggest the existence of a suppressor-like T cell circuit in immunity to abscesses.

Activation of murine B lymphocytes by suramin

Suramin stimulated DNA synthesis in spleen cell cultures of all inbred strains of mice tested, including, for example, CBA, DBA/2, C57BL/6, and the lipopolysaccharide (LPS)-nonresponsive strain C3H/HeJ. The cells responding to the drugs were removed by passage through nylon wool columns, but they were not eliminated by in vivo treatment of the mice with anti-Thy 1.2 antibody. Spleen cells of homozygous nude mice (C57BL/6 or BALB/c background) were as reactive as those of their heterozygous littermates. Collectively the data show that suramin is a B-cell mitogen in the mouse.

Recombinant interleukin 2 allows the differentiation of Thy 1.2 + LAK cells from nude mouse spleen cells

Culture of nude mouse spleen cells with recombinant human interleukin 2 (r-IL 2) resulted in the proliferation and generation of lymphokine-activated killer (LAK) cells which could lyse a variety of tumor cells. Flow cytometry study indicated that nude mouse spleen cells contained almost no Thy 1.2 + cells at the initial times of the culture, whereas LAK cells obtained from nude mouse spleen cells by culture with r-IL 2 (nude-LAK cells) expressed high intensity of Thy 1.2 antigen and lymphokine-activated cell-associated (LAA) antigen. The cytotoxic activity of nude-LAK cells was greatly reduced by treatment with anti-Thy 1.2 antibody plus complement but not with anti-Ly 1.2 or Ly 2.2 antibody plus complement treatment. Moreover, nude-LAK cells were resistant to the treatment with antiasialo GM1 antibody plus complement, in contrast to resident nude natural killer (NK) cells. These data strongly suggested that r-IL 2 allowed the nude mouse spleen cells to differentiate into Thy 1.2 +, Ly 1 −,2 −, asialo GM1 − LAK cells which were distinct from Thy 1.2 +, Ly 2 +, asialo GM1 − LAK cells induced from normal mouse spleen cells.

Analysis of splenic lymphocytes with high electrophoretic mobility in adult and aged nude mice

Electrophoretic mobility (EPM) and surface markers of splenic lymphocytes in adult (8 weeks old) and aged (over 1 year old) nude mice were investigated. Splenic lymphocytes in nude mice showed a bimodal pattern consisting of low mobility lymphocytes (LML) corresponding to B cells and high mobility lymphocytes (HML). The HML of nude mice showed the following immunological characteristics: (1) surface Ig − cells; (2) asialo GM 1 + cells; (3) an increase in natural killer (NK) activity after depletion of B cells; (4) abrogation of the HML peak and NK activity after treatment with anti-asialo GM 1 and complement. These findings suggested that HML in nude mice were NK cells. The mobility of NK cells was slightly lower than of T cells in normal mice, although their histograms greatly overlapped each other. In the spleen cells of nude mice, there was a significant increase in the numbers of Thy-1 + cells and a decrease in the intensity of asialo GM 1 antigen as a function of age. The surface markers of HML were Thy-1 +− asialo GM 1 ++ in adult nude mice, but were Thy-1 + asialo GM 1 + in aged nude mice. However, although HML in aged nude mice became Thy-1 +, these had almost the same EPM as those in adult nude mice.

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1 +, Lyt 1 +2 + and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.

Comparison of the biological properties of purified natural and recombinant human interleukin-2

We compared the biological properties of the purified recombinant human IL-2 derived from E. coli with those of purified natural IL-2. Both had almost the same specific in vitro activities on a weight basis to (i) support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; (ii) induce killer cells in normal mouse spleen cells; and (iii) induce antibody forming cells in nude mouse spleen cells. No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography.

Effects of Cimetidine on Tumor Growth and Immune Function in Nude Mice Bearing Human Ovarian Carcinoma<xref ref-type="fn" rid="FN2">2</xref>

The effects of cimetidine on the growth of a human ovarian cancer cell line inoculated into BALB/c nude mice were examined. The cell line, designated "KK," was derived from a cystadenocarcinoma of the ovary. The passage number was about 40, and its tumorigenicity in nude mice was 100% even when 10(5) cells were inoculated. About 2 weeks after inoculation, the KK cells formed palpable tumors, and the tumor volume reached 2.29 cm3 on day 36. Conversely, the nude mice given cimetidine (100 mg/kg/day) orally with drinking water had about one-third the tumor volume (0.81 cm3) of that in untreated nude mice on day 36. The natural killer activity against the YAC-1 (a T-cell lymphoma) cell line in spleen cells of the nude mice challenged with human xenogeneic tumor (KK cell line) was not affected by treatment with cimetidine while inhibiting the tumor growth. The capacity to lyse the KK cells did not exist in the spleen cells of nude mice challenged with the KK cell xenograft and not treated with cimetidine. The cimetidine-treated spleen cells acquired the capacity to lyse the KK cells on day 14. Thereafter, the capacity was maintained at the same level as long as cimetidine was administered, whereas that in untreated nude mice remained undetectable.

Follicular Dendritic Cells in the Regulation and Maintenance of Immune Responses

The objectives of this report are to explore known and potential regulatory roles of follicular dendritic cells (FDCs) in immune responses. FDCs are found in B-cell areas of lymphatic tissue and are especially rich around germinal centers where B-cell proliferation is occurring. Recent advances in isolating FDCs in our laboratories made it possible to begin investigating interactions of FDCs with other cells in vitro. In a series of experiments, we sought to determine whether a low concentration of FDC-enriched cells (2-10%) could enhance B-cell and/or T-cell mitogenesis. The results showed that an enriched population of FDCs markedly enhanced lipopolysaccharide-induced stimulation of nude mouse spleen cells, while an equivalent number of resident peritoneal cells or thymocytes had no effect. Depletion of Thy 1.2 positive cells or an exposure to 2400 rads of gamma radiation did not abrogate the enhancing capacity of FDC-enriched cells. FDC-enriched cells also increased phytohemagglutinininduced proliferation. Scanning electron microscopy of FDC preparations showed that antigen-bearing dendritic processes of Type 2 FDCs have a «beaded» appearance. It was proposed that these immune complex coated microspheres may represent an important mechanism for exposing persisting antigen to the immune system. The gradual shedding of these microspheres may play an important role in regulating and maintaining immune responses within lymphoid tissues.

Functional clonal deletion of class I-specific cytotoxic T lymphocytes by veto cells that express antigen.

Intravenous injection of class I incompatible spleen cells into mice results in a drastic reduction of the recipient's cytotoxic T lymphocyte (CTL) response against the injected, but not against third party, class I antigens when measured in bulk cultures initiated 5 to 6 days after the injection. This specific suppressive effect is partly due to T cells but can also be seen when high numbers of anti-Thy-1 and complement-treated spleen cells of nude mice are injected. Such cells suppressing CTL responses against self histocompatibility antigens are called "veto cells." The precursor frequency of CTL specific for the injected class I antigen is found to be reduced greater than 200-fold at days 5 to 6 after the injection, whereas the frequencies of CTL specific for third party class I antigens are not significantly changed. These results indicate that there is a functional clonal deletion of the CTL recognizing class I incompatible veto cells in vivo. The role of such a veto phenomenon in the induction and maintenance of self tolerance and allograft tolerance is discussed.