Nylon Wool-nonadherent Spleen Cells Research Articles

Augmentation of natural killer cell activity in vitro against tumor cells by wild plants from Jordan

Thirteen aqueous extracts prepared from Jordanian plants, that are currently used in traditional medicine to treat various types of cancer, were tested in mice for their ability to augment natural killer (NK) cell function in vitro in generating cytotoxicity against YAC tumor targets. Lymphoid cells at a concentration of 5×10 6/ml were incubated in medium alone or in medium containing different dilutions of either plant extract or purified interferon α for 20 h and tested for NK activity. Maximum NK activity (62.3%) was obtained at 1:50 dilution of Nigella sativum fresh aqueous extract, 48.5% at 1:100 dilution for Allium sativum (and 38.3% at 1:50 dilution for Onopordum acanthium. Fresh aqueous plant extracts appeared to be more potent than old dried aqueous extract or ethanolic extracts. NK augmentation by plant extracts using nylon wool non-adherent spleen cells was slightly higher than the whole spleen cells.

Differential effects of B7-1 and B7-2 on the costimulation of mouse nonspecific cytotoxic T lymphocyte development in response to anti-CD3 antibody.

Despite extensive study, the relative contribution of B7-1 and B7-2 molecules to the costimulation of cytotoxic T lymphocyte (CTL) activation remains controversial. We used blocking mAbs to B7-1 and B7-2 molecules to determine the role of these B7 family members in the in vitro induction of mouse nonspecific CTL in response to soluble anti-CD3 mAb. Optimal induction of anti-CD3-activated killer-T (AK-T) cells was found to require interactions with B7-2 on residual accessory cells in nylon wool-nonadherent spleen cell preparations during the first 12 h of culture in the presence of anti-CD3 mAb. Because B7-1 is not expressed at high enough levels on residual accessory cells in primary T cell cultures to be an effective ligand for CD28, we used LPS-stimulated B cells, which express substantial B7-1, in addition to B7-2, to determine the contribution of B7-1 to AK-T cell development. Compared with B7-2, the contribution of B7-1 to the costimulation of AK-T cells in this system was modest because anti-B7-1 mAb had only a minimal inhibitory effect on the generation of cytotoxicity, whereas anti-B7-2 mAb strongly inhibited AK-T cell development. Anti-CD3-induced cytotoxicity of T cells from CD4 knockout mice and CD4-depleted nylon wool-nonadherent spleen cells from wild-type mice was inhibited by anti-B7-2 mAb, implying that B7-2 is able to bind directly to CD28 on CD8+ T cells and costimulate their activation. B7-1 blockade, on the other hand, did not affect the costimulation of CD8+ T cells. Blockade of B7-2/ CD28 interactions with anti-B7-2 mAb strongly inhibited granzyme B, but not perforin or Fas ligand gene expression, suggesting an explanation for the inhibitory effect of anti-B7-2 mAb on AK-T cell development. These data indicate that B7-2 is superior to B7-1 as a costimulator of mouse AK-T cell induction.

Cell-mediated immunity in host resistance against infection caused by Penicillium marneffei.

Penicillium marneffei is one of the most important opportunistic infectious pathogens in AIDS patients in Thailand and Southeast Asia. However, very little is known about the host defence mechanisms against P. marneffei infection. In the present study, we established the first experimental murine model of chronic pulmonary and disseminated infection using P. marneffei, and examined the immunological response to such infection in euthymic and athymic mice. In this model, micro-organisms inoculated intratracheally multiplied progressively in the lungs and disseminated to the liver and spleen. However, the number of organisms decreased gradually in these organs. In contrast, congenitally athymic mice developed severe pulmonary and disseminated systemic mycosis. Pulmonary penicilliosis marneffei was associated with a marked cellular inflammatory response as evident by histological abnormalities and increased intraparenchymal leucocyte count. To confirm the importance of cell-mediated immunity in host resistance to P. marneffei infection, we transferred nylon wool non-adherent spleen cells into the athymic mice. Such treatment significantly reduced the number of yeasts in the organs of athymic mice. Taken together, our results demonstrate that the cell-mediated immunity play a central role in a host defence mechanism against infection with P. marneffei, and suggest that our new model may be a useful approach for studying the pathogenesis of this fungal disease.

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells.

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.

Trypanosoma cruzi: T-cell-dependent mechanisms of resistance during chronic infection

Effector mechanisms of resistance exerted by T cells from BALB/c mice chronically infected with Trypanosoma cruzi, Tulahuén strain, were studied. Spleen cells from chronically infected mice (Chro-SC) prestimulated with heat-killed trypomastigotes (HKT) and/or IL-2 destroyed PHA-labeled p-815 mastocytoma cells, HKT-pulsed macrophages, and normal peritoneal macrophages. However, HKT-stimulated Chro-SC did not affect the infectivity of free bloodstream forms of the parasite. Upon HKT stimulation, Chro-SC or their culture supernatant activated peritoneal macrophages for the destruction of intracellular amastigotes. The effect was abolished after Thy 1.2+ cell depletion. The addition of Cyclosporin A (CyA), which blocks T-cell activation, during HKT-stimulation of Chro-SC, diminished their ability to activate the trypanocidal activity of macrophages. CyA also inhibited the production of both macrophage-activating factors and interferon-γ by HKT-stimulated Chro-SC. CyA administration to recipients of nylon-wool nonadherent spleen cells from chronically infected mice inhibited their adoptively acquired resistance against T. cruzi, suggesting that the conferred resistance depended on the effect of specifically activated cells. When administered during the chronic stage of the infection, CyA abrogated the antigen-specific delayed type hypersensitivity response but increased the levels of anti- T. cruzi IgG antibodies. Neither parasitemia, tissular parasitism in myocardium or skeletal muscle, nor mortality were detected after CyA treatment, suggesting the presence of a CyA nonsensitive mechanism(s) in the control of T. cruzi during the chronic phase of the infection.

Protective immunity against Brugia malayi infective larvae in mice. I. Parameters of active and passive immunity.

Protective immunity against infective larvae of Brugia malayi was studied in different strains of mice using various sources of antigens. The following strains of mice were susceptible to infective larvae development for 2 weeks after primary ip challenge: BALB/c, C3H/HeJ, C3H/NeN, C3H/HeJms, C57BL/6Jms, and DDD. In comparison to gerbils, BALB/c mice developed stronger resistance to infective larvae after immunization with irradiation attenuated larvae or with killed microfilariae (mf). However, killed mf failed to enhance resistance in C3H/HeJ mice, although C3H/HeN mice were strongly protected and C3H/HeJms mice were protected to a lesser degree by this antigen. Extracts of mf with phosphate buffered saline and sodium dodecyl sulfate both induced high levels of resistance in BALB/c mice. Transfer of resistance from BALB/c mice immunized with attenuated infective larvae to naive mice was accomplished at a high level at protection with nylon wool nonadherent spleen cells (T cells) but not with adherent cells treated with anti-Thy 1.2 serum and complement. In contrast, sera from immunized mice were much less protective.

Successful in vitro graft-versus-tumor effect against an Ia-bearing tumor using cyclosporine-induced syngeneic graft-versus-host disease in the rat

Lethally irradiated LouM rats reconstituted with syngeneic bone marrow and then treated with cyclosporine (CsA) for 40 consecutive days following transplant developed a graft-v-host disease (GVHD)-like syndrome after CsA cessation. This model of GVHD was used to define and characterize a graft-v-tumor (GVT) effect against a syngeneic plasmacytoma CRL1662 cell line which expresses class II major histocompatibility (MHC) antigen (Ia). Nylon wool-nonadherent spleen cells from animals who developed syngeneic GVHD were capable of significant lysis against chromium-labeled tumor target cells in a four- hour chromium released cell mediated lympholysis assay; maximum lysis occurred five days following cessation of CsA when clinical signs first appeared. Cytolytic activity declined to baseline as GVHD symptoms resolved. Fractionation of splenocytes into lymphocyte subsets demonstrated that cytolytic lymphocytes (CTLs) of the OX8 phenotype (non-helper T) were capable of significant lysis against tumor target cells. Lysis of tumor cells was blocked by preincubation with monoclonal antibodies (MoAb) specific for the rat anti-class II MHC antigen but not with MoAb against class I. Incubation of tumor cells with gamma-interferon increased expression of tumor class II MHC antigens and significantly increased their susceptibility to lysis by nylon wool-nonadherent splenocytes from animals with syngeneic GVHD. These studies have demonstrated an in vitro GVT of syngeneic GVHD against an Ia-bearing tumor; the effector cell is a CTL of the OX8 phenotype specific for the class II MHC antigen.

Successful In Vitro Graft-Versus-Tumor Effect Against an Ia-Bearing Tumor Using Cyclosporine-Induced Syngeneic Graft-Versus-Host Disease in the Rat

Lethally irradiated LouM rats reconstituted with syngeneic bone marrow and then treated with cyclosporine (CsA) for 40 consecutive days following transplant developed a graft-v-host disease (GVHD)-like syndrome after CsA cessation. This model of GVHD was used to define and characterize a graft-v-tumor (GVT) effect against a syngeneic plasmacytoma CRL1662 cell line which expresses class II major histocompatibility (MHC) antigen (Ia). Nylon wool-nonadherent spleen cells from animals who developed syngeneic GVHD were capable of significant lysis against chromium-labeled tumor target cells in a four-hour chromium released cell mediated lympholysis assay; maximum lysis occurred five days following cessation of CsA when clinical signs first appeared. Cytolytic activity declined to baseline as GVHD symptoms resolved. Fractionation of splenocytes into lymphocyte subsets demonstrated that cytolytic lymphocytes (CTLs) of the OX8 phenotype (non-helper T) were capable of significant lysis against tumor target cells. Lysis of tumor cells was blocked by preincubation with monoclonal antibodies (MoAb) specific for the rat anti-class II MHC antigen but not with MoAb against class I. Incubation of tumor cells with gamma-interferon increased expression of tumor class II MHC antigens and significantly increased their susceptibility to lysis by nylon wool-nonadherent splenocytes from animals with syngeneic GVHD. These studies have demonstrated an in vitro GVT of syngeneic GVHD against an Ia-bearing tumor; the effector cell is a CTL of the OX8 phenotype specific for the class II MHC antigen.

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells.

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.

Induction of latent immunological memory in genetically nonresponsive mice.

C57BL/10 mice exhibit major histocompatibility complex linked nonresponsiveness to hen egg white lysozyme (HEL). When these animals are primed with HEL in Freund's complete adjuvant (FCA), their secondary splenic plaque forming cell responses to aqueous HEL challenge are minimal to nonexistent. This notwithstanding, we show here that concomitant priming with both HEL and keyhole limpet hemocyanin (KLH) leads to an enhanced response to the HEL component following secondary challenge with an HEL-KLH conjugate. This enhancing effect can be transferred by nylon wool nonadherent spleen cells from HEL/FCA primed animals. Adoptive transfer studies with fractionated spleen cell populations suggest also that B cells are primed in these animals. Thus, animals which are incapable of mounting a secondary response to this antigen nevertheless appear to be primed at both the T-cell and B-cell levels following exposure to the antigen in FCA. The implications of this finding are discussed.

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction: IV. Effect of delayed-type hypersensitivity augmentation factor on in vitro induction of DTH

We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR), or in the culture supernatant of the mixture of sensitized T cells and specific antigens. This factor (DTH augmentation factor; DAF) was confirmed to augment DTH in transferred recipients. In this paper, such an activity of DAF was further investigated using the system with in vitro induction and local transfer of DTH. DAF also augmented the primary in vitro induction of DTH, when spleen cells from mice transferred with the DAF-containing serum 12 hr previously or spleen cells incubated with the DAF-containing serum on ice for 2 hr were cultured with heterologous erythrocytes. DAF acted on the induction phase of DTH and augmented a typical DTH which was dependent on Thy-1-positive T cells. DAF showed antigen specificity, but was not assigned to conventional immunoglobulin. The activity of DAF was detected when nylon-wool nonadherent cells were incubated with DAF prior to the culture of those cells and antigens, but not detected when only nylon-wool adherent cells were incubated with DAF. Thus, DAF exerted its effect through binding to acceptor cells which were included in nylon-wool nonadherent spleen cells from normal mice.

Capacity of different cell types to stimulate cytotoxic T lymphocyte precursor cells in the presence of interleukin 2.

Plastic-adherent cells enriched for dendritic cells (AC) were found to be among the most potent stimulator cells for the activation of cytotoxic T lymphocytes (CTL) in vitro in the presence of interleukin 2 (IL 2) and a constant second set of allogeneic stimulator cells. Concanavalin A-activated nylon wool-nonadherent spleen cells ( CNWT ), concanavalin A-activated unfractionated spleen cells ( Cspl ), and some variants of the ESb T lymphoma line were equally effective as stimulator cells, however, and provoked a substantial cytotoxic response at concentrations of 10(4) cells per culture or less. In contrast, nonactivated nylon wool-nonadherent spleen cells ( NWT ) or unfractionated spleen cells (Spl) and cells of the P815 mastocytoma, the Meth A fibrosarcoma, and the T cell lymphomas Ly 5178 Eb and ESb did not stimulate cytotoxic responses at these cell concentrations. The strong stimulatory potential of the Cspl preparation was reduced by treatment with anti-Thy-1 antibody plus complement, whereas the stimulatory activity of the AC preparation was resistant to this treatment. All cell types tested expressed class I major histocompatibility antigens. Nonactivated NWT cells, in contrast to the CNWT preparation, showed no detectable staining with anti-I-E or anti-I-A antibodies and also a slightly weaker staining with class I antisera. Experiments with the tumor cell lines revealed, however, that there was no strict correlation between stimulatory potential and density of class I alloantigens or the expression of I-E determinants. Experiments on primary cytotoxic responses in vivo gave similar results. Experiments in cultures with a single set of stimulator cells and I region-compatible responder cells indicated that AC and Cspl or CNWT also have a markedly stronger capacity than NWT to induce IL 2-dependent DNA synthesis.

Ia-dependent interleukin 2 production in syngeneic cellular interactions.

In the murine syngeneic mixed lymphocyte response (SMLR), the co-culture of nylon wool-nonadherent spleen cells or young (4 to 6-day-old) thymocytes with syngeneic nylon wool-adherent adult spleen cells, results in proliferation and Interleukin 2 (IL 2) production. In both the thymus and spleen SMLR, proliferation and IL 2 production are the function of Lyt-1+2-3- (Lyt-1) responder cells and require the expression of IA (IB) and/or IE cell surface antigens on the stimulator cell. IL 2 production in both spleen and thymus SMLR is independent of heterologous serum factors (i.e., fetal calf serum), which, however, influences the magnitude of the in vitro proliferation. The IL 2-producing cell in spleen is capable of self-renewal in the absence of the thymus; it is detectable 6 to 7 mo after adult thymectomy. These results suggest the in vitro SMLR may measure a cellular interaction between the Lyt-1 subset of T cells and self Ia antigens on macrophage-like cells that produces IL 2 in an antigen-independent fashion. This interaction may have physiologic significance in the expression and regulation of T cell function.

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes.

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.

In vitro effects of prealbumin on T-cell-dependent immune responses of the mouse

The immunological activities of human prealbumin preparation were investigated in vitro. Prealbumin was effective at inducing precursors in nylon wool-nonadherent spleen cells of nu/ nu mice to express Thy-1 antigens. At 100–250 μg/ml a four- to sixfold enhancement of cytotoxic index was observed. In the presence of prealbumin, mitogenic responses of spleen cells from normal C57B1/6J mice to phytohemagglutinin (PHA) or concanavalin A (Con A) but not lipopolysaccharide (LPS) were increased. The most significant effect of prealbumin at the optimal concentration (250 μg/ml) on PHA-induced proliferation of spleen cells was found in mice 12 months of age. An in vitro plaque-forming cell (PFC) assay showed prealbumin to exert a stimulating effect on PFC formation in spleen cells from normal C57B1/6J mice at different ages. No such activity was exhibited by human serum albumin preparation. In comparison with prealbumin, synthetic facteur thymique serique (FTS) was tested in mitogenic responses and the PFC assay. FTS showed no influence on responses of mouse spleen cells to PHA, Con A, or LPS. At concentrations of 100–200 μg/ml, however, FTS enhanced PFC formation from normal spleen cells. The findings indicate that it is unlikely that FTS is responsible for the activities of the prealbumin preparation.

Dendritic cells are accessory cells for the development of anti-trinitrophenyl cytotoxic T lymphocytes.

This study establishes that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro. We developed a model in which nylon wool-nonadherent spleen cells were used both as the responding and stimulating cells, the latter having been TNP-modified and x-irradiated. Thy-1-bearing CTL developed in C57BL/6, B6D2F1, and CBA mice only when small numbers of DC were added. Maximal responses in 5-d cultures were achieved with 0.5-1 DC/100 responding T cells. The DC did not have to be TNP modified directly. Anti-Ia and complement inactivated accessory cells, whereas similar treatment of the responders had no effect. DC exposed to ultraviolet radiation were ineffective, but x-irradiated DC were fully active. Culture media from DC, or from DC-nylon wool-passed spleen T cell cocultures that contained abundant CTL, would not substitute for viable DC. Enriched preparations of macrophages (M phi) were obtained from blood, peritoneal cavity, and spleens of BCG-immune and unprimed mice. M phi added at doses of 0.2-4% were weak or inactive as accessory cells. The level of Ia antigens on test M phi populations was quantitated and visualized by binding of a radioiodinated monoclonal anti-I-Ab,d antibody, clone B-21. M phi that bore substantial amounts of Ia from all organs were weak accessory cells. Addition of M phi to DC-T cell cocultures produced inhibitory effects, usually at a dose of 2% M phi. In contrast, 0.5% Ia-bearing M phi from BCG-immune boosted mice inhibited > 80% of the DC-mediated CTL response. Addition of indomethacin reversed M phi inhibition, and 10(-9) M prostaglandin E2 in turn blocked the indomethacin effect. Indomethacin also restored a low level of accessory cell function in immune-boosted adherent peritoneal cells, but not in preparations of monocytes and spleen M phi. Small numbers of DC were identified in preparations of immune-boosted peritoneal cells and may have accounted for the observed accessory activity. We conclude that the development of anti-TNP CTL is an immune response in which (a) DC are the critical accessory cells; (b) Ia-bearing M phi are weak or inactive; and (c) M phi can inhibit DC-mediated response by an indomethacin-sensitive mechanism.

Activation of helper T cells by immune complexes

Spleen cells from mice primed with dinitrophenylated human γ-globulin (DNP-HGG) did not mount a secondary anti-DNP response in diffusion chamber cultures upon stimulation with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH). The same cells, however, responded to stimulation with DNP-KLH complexed with anti-KLH antibody of rabbit or mouse origin. There is an optimal antigen:antibody ratio at which the immune complexes (IC) must be formed for maximal activity. T cells are required for the immunogenic activity of IC, since T-cell-depleted cultures did not respond. It was found that IC made with carrier and anticarrier antibody stimulated the development of carrier-specific helper T cells in cultures of spleen cells, thymocytes, and nylon wool nonadherent spleen cells from nonimmune mice. In contrast, free carrier did not elicit helper T cells. IC made with carrier and the F(ab′) 2 fragment of anticarrier antibody were immunogenic, but those made with carrier and the Fab′ fragment of anticarrier antibody were not, suggesting that helper T-cell activation is triggered by crosslinking of antigen-specific surface receptors.

Adoptive transfer of immunity and suppression by cells and serum in early Mycobacterium lepraemurium infections of mice

The ability of lymphocytes and serum from mice infected with 10(7) Mycobacterium lepraemurium to adoptively transfer resistance to irradiated (600 rad 60 cobalt) syngeneic mice was studied. Two strains of mice were used: BALB/c mice, which are highly susceptible to moderate cutaneous infections with M. lepraemurium, and C57Bl mice, which are comparatively resistant. In both strains of mice recipients of nylon wool non-adherent spleen cells, and thus T lymphocyte recipients, demonstrated increased resistance to subcutaneous infection with 10(7) M. lepraemurium. Significantly increased susceptibility to infection was noted in the BALB/c recipients of nylon wool adherent spleen cells or serum. No suppressor activity could be demonstrated in the resistant C57Bl strain.