Con A-activated Spleen Cells Research Articles

Antibacterial and antitumor activity of Bogorol B-JX isolated from Brevibacillus laterosporus JX-5.

Antimicrobial peptides are promising anti-infective agent candidates because they have a broad antimicrobial spectrum and bioactivity and are unlikely to elicit antibiotic resistance. The bogorols represent a new cationic antibiotic peptide and possess great therapeutic potential because of their bioactivity and precise mode of action. Here, we report that Bogorol B-JX (BBJX), a peptide previously isolated from Brevibacillus laterosporus JX-5 by us, has significant antibacterial and antitumor activities in vitro. BBJX was found to inhibit methicillin-resistant Staphylococcus aureus (MRSA) at 2.5µg/mL with distinct mechanisms of action from those against Bacillus bombyseptieus and Escherichia coli. It penetrates MRSA membrane with little visible destruction and binds to genomic DNA. BBJX could inhibit the proliferation of human histiocytic lymphoma cell line U-937 and ConA-activated spleen cells at 5µg/mL, but was not cytotoxic to the Jurkat cells, resting spleen cells or differentiated macrophage-like U-937 immunocytes. Moreover, BBJX caused apoptosis of U-937 cells by opening the mitochondrial permeability transition pore and stimulating the production of reactive oxygen species. Taken together, these studies provided basis for future medical application of the bogorols.

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4+T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4+CD25+FOXP3+Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg.This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4+, especially CD4+CD25+T cells. CD4+T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4+T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4+CD25+T cells' response to rIL-5 and expression of IL-5Rα was also assessed.rIL-5 was sufficient to promote transplant tolerance mediating CD4+T cells' survival in culture with specific-donor alloantigen. Tolerant CD4+T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4+T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4+CD25+T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4+CD25+T cells expressed IL-5Rα.This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4+CD25+T cells that mediate transplant tolerance.

The Sex Differences of Morphology and Immunology of SIRS of Newborn Wistar Rats.

The sex differences of infection and inflammatory diseases particularly appear at reproductive age and depend on the sex hormone level, varied between male and female. There are a few sets of data about the sex differences of infection and inflammatory diseases course, including systemic inflammatory response syndrome (SIRS) and sepsis, of newborns. The aim of our research was the estimation of morphological and immunological manifestation of SIRS of the newborn Wistar rats. Investigations were carried out on male and female two-day-old Wistar rats (10–12 g). SIRS was modeled by intraperitoneal injection of LPS (E. coli, O26: B6 strain, Sigma) in high dose—15 mg/kg. We did not find out any sex differences of the liver lesions severity between newborn males and females after LPS injection. The levels of endotoxin and estradiol in the serum, as the number of neutrophils in the intra-alveolar septa of the lungs, were higher in males than females with SIRS. Production of IL-2 and TNF-α by the spleen cells of males was higher than that in control group that reflects polarization predominantly on the Th1-type immune response. The secretion of IL-2, TNF-α, and IFN-γ by ConA activated spleen cells of females decreased that reflects the suppression of Th1-type immune response. We suppose that the LPS administration in the high dose causes the multidirectional reaction of the immune system of neonatal males and females Wistar rats.

Cerebral Gene Expression of Superantigen Encephalitis in the Lewis Rat Induced by Staphylococcal Enterotoxin A

Superantigens were suggested to play a role in the pathogenesis of different autoimmune diseases including multiple sclerosis (MS). Previously, it was demonstrated that local expression of the superantigen, staphylococcal enterotoxin A (SEA) in the brain of rats may lead to encephalitis which was amplified by using intravenous injection of concanavalin A (ConA)-activated splenocytes. In the present investigation, gene expression was studied in the rat brain 8 days after an injection of 50 mul of 1 mg/ml SEA or saline and 5 days after an intravenous injection of 1 x 10(7) ConA-activated spleen cells. Of 8800 genes investigated (Affymetrix, rat genome U34A), the expression of 106 genes was significantly and at least threefold increased with SEA, while the expression of 29 genes was decreased at least threefold. Increased gene expression was compatible with an intracerebral inflammatory response mediated by antigen-presenting cells and CD8+ T lymphocytes. Elevated chemokines comprised RANTES (CCL5), osteopontin, MCP-1 (CCL2) and CXCL10. Further, genes with increased expression were assigned to the extracellular matrix, microglia/macrophage cell elements, astrocytes (GFAP) and phagocytosis. There was considerable conformity between previously reported gene expression profiles for experimental autoimmune encephalomyelitis (EAE) or MS and the present findings. Our data are in line with the concept that T-cell superantigen locally expressed in the central nervous system induces an inflammatory response. Therefore, the study of gene expression profiles does not seem to allow clear conclusions with respect to the aetiology of central nervous system autoimmune diseases.

Physalins B, F and G, seco-steroids purified from Physalis angulata L., inhibit lymphocyte function and allogeneic transplant rejection

Physalis angulata is a solanaceae widely used in folk medicine in various tropical countries in the world. We have previously described that seco-steroids (physalins) purified from P. angulata are potent inhibitors of macrophage activation, blocking the production of pro-inflammatory cytokines and LPS-induced lethality. Herein we investigated the immunomodulatory activities of these substances in lymphocyte proliferation and cytokine production and in transplantation. The addition of physalins B, F or G to concanavalin A-activated splenocyte cultures induced a concentration-dependent inhibition of proliferation. Physalin B also inhibited IL-2 production by Con A-activated spleen cells. The addition of 2 μg/ml physalin B to mixed lymphocyte reaction (MLR) caused a 100% inhibition of proliferation. More importantly, treatment of mice with physalin B, F or G prevented the rejection of allogeneic heterotopic heart transplant. Our results demonstrate the suppressive activity of physalins B, F and G in lymphocyte function and indicate the potential use of physalins as immunosuppressive agents for treatments of pathologies in which inhibition of immune responses is desired.

Aqueous extract from aerial parts of Artemisia vestita, a traditional Tibetan medicine, reduces contact sensitivity in mice by down-regulating the activation, adhesion and metalloproteinase production of T lymphocytes

In the present paper, the effect of the aqueous extract from aerial parts of Artemisia vestita (AV-ext), a traditional Tibetan medicine, on ear contact sensitivity was examined. AV-ext significantly reduced the ear swelling when administered during the induction phase of picryl-chloride (PCl)-induced ear contact sensitivity in mice. The extract also showed a dose-dependent inhibition on lymphocyte proliferation and IL-2 production in Con A-activated spleen cells. The proliferation inhibition was confirmed in the mixed lymphocytes reaction. Furthermore, the adhesion of the isolated spleen cells from PCl-sensitized mice to type IV collagen was significantly decreased in a dose-dependent manner by AV-ext. Such decrease was also seen in AV-ext-treated Jurkat T cells and the T cells purified from above spleen cells. The purified spleen T cells from PCl-sensitized mice produced more matrix metalloproteinase-9 (MMP-9) than naive T cells, and AV-ext remarkably reduced MMP-9 production both in vivo and in vitro. These results suggest that AV-ext may alleviate contact sensitivity through blocking the activation of T lymphocytes and decreasing their localization to the inflammatory sites via down-regulating the potential of cell adhesion and metalloproteinase production.

Expression and functional characterization of recombinant chicken interferon-gamma

A cDNA encoding chicken interferon-gamma (chIFN-γ) was cloned from a CD4 + T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli, COS- and CEC-32 fibroblast cell lines. In general, recombinant chicken IFN-γ (rchIFN-γ) expressed in the COS- and CEC-32 cell lines showed high bioactivity in vitro. The kinetics of IFN-γ gene expression were examined in concanavalin A (Con A)-activated spleen lymphocytes by Northern blot and RT-PCR. IFN-γ mRNA was detected as early as 30 min after Con A activation, reached peak expression at 2 h and then decreased starting at 4 h post Con A activation. A rabbit serum made to a synthetic peptide of IFN-γ immunoprecipitated a 60 kDa E. coli maltose-binding fusion protein of recombinant IFN-γ (MBP-IFN) and a 26–27 kDa secreted protein from COS cells and Con A-activated spleen cells. IFN-γ inhibited vesicular stomatitis virus (VSV) mediated cytotoxicity of chicken embryonic fibroblast (CEF) cells and upregulated the expression of many macrophage cell surface antigens, including class I and class II major histocompatibility complex (MHC) proteins. These results show that chicken IFN-γ possesses anti-viral activity and immunoregulates macrophage activities.

Enhancement of nitric oxide production from activated macrophages by glycyrrhizin.

We studied the actions of glycyrrhizin on nitric oxide production from macrophages and a macrophage cell line RAW264-7. Although glycyrrhizin did not induce nitric oxide from resting macrophages, it enhanced the production of nitric oxide from IFN-gamma activated-macrophages or RAW cells. Glycyrrhizin also enhanced the production of nitric oxide from macrophages stimulated with the supernatant of con A-activated spleen cells. Further, glycyrrhizin enhanced tumor cell killing by macrophages activated with IFN-gamma. This tumor cell killing was mainly by nitric oxide.

Altered cytokine genes expression by ConA-activated spleen cells from mice infected by lymphocytic choriomeningitis virus

The intravenous injection of mice with lymphocytic choriomeningitis virus (LCMV) induces a rapid and long-lasting immunodeficiency. T lymphocytes from 7-day-infected mice do not proliferate in vitro in response to ConA stimulation, do not produce IL-2 but display high affinity IL-2 receptors on their membrane. The non-coordinated regulation of these genes suggested that other cytokine-encoding genes may also be affected in their regulation. We have thus analyzed the expression of the genes encoding different cytokines transcribed during spleen cell activation by ConA. The genes encoding T lymphocyte-derived cytokines can be classified in three groups: the genes expressed similarly by normal and LCMV-cells (the p55 and the p75 chains of the IL-2 receptor [1]), the genes under expressed in LCMV-cells (IL-2, IL-3, IL-4 and IL-5) and the genes over expressed by these cells (GM-CSF and IFN-γ). These results show that the viral infection has provoked a profound alteration of the overall regulation of the genetic program that follows T lymphocyte activation. Since T cell activation depends strictly on accessory cell-derived cytokines, we measured the level of transcription of IL-1, IL-6 and TNF-α; and our data show that the expression of these genes is equivalent in normal cells and in cells from LCMV-infected mice.

Macrophages suppress lectin-induced proliferation of lymphocytes from aged rats

Splenocytes from old rats exhibit a significant reduction in proliferative responses to Con A. This defect can be overcome by passing the cells over nylon wool, which suggests that nylon wool adherent cells contribute to the reduction in proliferation of mixed cell populations from aged rats. To examine the potential role of macrophages in this process, thioglycollate-elicited peritoneal macrophages were added to Con A-activated spleen cells from young and old rats. Addition of as few as 3% macrophages suppressed proliferation of Con-A-activated splenocytes and this inhibition was significantly greater if either the macrophages or spleen cells were derived from aged rats. Although prostaglandin E 2 (PG-E 2) inhibited the proliferation of splenocytes and nylon-wool enriched splenocytes from 3-month-old animals, its suppressive effects were much greater on Con A-activated lymphocytes derived from 24-month-old rats. Addition of catalase to deplete macrophaged-derived H 2O 2 resulted in greater augmentation of Con-A-induced proliferative responses of splenocytes from old rats than those from young rats. TGF-β2, but not TNF-α, also significantly suppressed the proliferation of Con A-activated splenocytes to a greater degree in aged than in young rats. Since macrophages are known to release PG-E 2, H 2O 2 and TGF-β2 after stimulation with Con A, these data support the idea that T cells within the splenocyte population become increasingly susceptible during aging to inhibitory products released by macrophages.

Abnormal activation and loss of suppressor T cells in the spontaneously hypertensive rat

Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.

Possible 110 kDa receptor for interleukin 2 in the chicken

Chicken lymphoblasts were generated from spleen cells which had been incubated with concanavalin A (Con A) for 48 h. Monoclonal antibodies (mAbs) were produced by immunizing mice with 48-h Con A lymphoblasts. These antibodies were used to identify chicken lymphokine receptors on Con A-activated lymphocytes. The cellular enzyme immunoassay (EIA), with lymphoblasts and spleen cells as the target cells, was used to select for specific mAbs. One mAb was found by EIA to have a strong response against the lymphoblasts but weak against resting spleen cells. By immunofluorescence staining, it reacted strongly with lymphoblasts and weakly with resting spleen, erythrocytes, and bursal cells. This mAb was also shown to inhibit the lymphokine activity present in conditioned medium that was collected from 24-h cultures of Con A-activated spleen cells. By immunoprecipitation, this mAb precipitated a lymphoblast surface antigen with a molecular weight of about 110 kDa. This receptor may be analogous to the putative interleukin 2 γ subunit recently described in human and mouse cell lines.

Prevention of Adoptive Transfer in BB Rats by Prophylactic Insulin Treatment

Prophylactic insulin can prevent diabetes in the BB rat. We evaluated its use to prevent adoptive transfer of diabetes by activated splenocytes. ConA-activated spleen cells from acutely diabetic BB rats were divided into two equal aliquots and injected intravenously in paired diabetes-prone BB rat littermates. At the time of cell injection, subcutaneous insulin injections (15 U.kg-1.day-1) were started in one of each pair (n = 21) of littermates, and the control littermates (n = 21) were injected with saline. The incidence of diabetes, observed 35 days after cell injection, was 95% in control rats compared with 29% in insulin-treated rats (P less than 0.05). To confirm the absence of diabetes, insulin was stopped in all nondiabetic insulin-treated rats at 63 days of age. An OGTT was performed at 65 days of age: 4 rats were glucose intolerant. All rats received comparable numbers of ConA-activated splenocytes. At the time the rats were killed, 3 insulin-treated rats had a completely normal morphology and a normal glucose tolerance. All control rats had insulitis whether they were diabetic or not. No significant difference in any mononuclear subset was observed in relation to insulin treatment. We conclude that prophylactic insulin can prevent adoptive transfer of diabetes in the BB rat without inducing changes in the mononuclear cell subsets.

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.

Studies on Autoimmunity For Initiation of β-Cell Destruction: V. Decrease of Macrophage-Dependent T Lymphocytes and Natural Killer Cytotoxicity in Silica-Treated BB Rats

Administration of silica, which is selectively toxic to macrophages, to young BB rats resulted in the prevention of insulitis and diabetes. However, the mechanism leading to the prevention of an autoimmune process in silica-treated BB rats is not known. This study was undertaken to investigate the mechanism involved in prevention of insulitis and diabetes. Neonates of diabetes-prone BB (DPBB) rats injected with concanavalin A (ConA)-activated spleen cells from silica-treated DPBB (STDPBB) rats did not develop insulitis or diabetes, whereas DPBB neonates injected with ConA-activated spleen cells from the untreated DPBB rats developed both insulitis and diabetes. Not only was there a decrease of natural killer (NK) cell activity in splenocytes from STDPBB rats, but there was also a significant reduction in the number of immunocytes such as T lymphocytes (helper/inducer and cytotoxic/suppressor) and NK cells. The number of macrophages in both spleen and peripheral blood was significantly decreased in STDP rats compared with untreated DP rats. In contrast to the changes in T lymphocytes and NK cell activity, there was no change in target beta-cells in STDPBB rats with regard to the susceptibility to adoptive transfer of insulitis. It is concluded that the prevention of insulitis and diabetes in STDPBB rats is due to a decrease in macrophage-dependent T lymphocytes and NK cell cytotoxicity.

Cell division-associated expression of an epitope, KH17, on early developing thymocytes

A newly established monoclonal antibody, KH17, detects a unique epitope temporarily expressed on early developing CD3-thymocytes confined to a cycling stage. KH17 is detectable on a part of CD4-CD8-,CD4-CD8+, and CD4+CD8+ cells, but not on CD4+CD8- thymocytes. By four-color flow cytometry analysis using KH17, we were able to define the heterogeneity of immature CD4-CD8- thymocytes by the expression of KH17 and IL-2R. In Thy-1-congeneic bone marrow chimeras, the appearance of KH17-IL-2R+ thymocytes preceded the increase of KH17+IL-2R- cells. The antibody could also divide CD3-CD4-CD8+ cells into two subpopulations, KH17+ and KH17-, which showed a continuum. In the fetal thymus there was a rapid and dramatic increase of KH17-CD4+CD8+ thymocytes concomitant with a decrease of KH17+CD4-CD8+ thymocytes in later gestation days. KH17 is not expressed on resting peripheral T cells, but is expressed on a large proportion of Con A-activated blastic spleen cells. The KH17 molecules precipitated from Con A-activated spleen cells were 55 and 75 kd polypeptides, but different from IL-2R subunits.

Immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with Vaccinia virus (neurotropin™)—II. Restorative effect on immune responses through the recovery of IL-2 production in aging mice

To examine the immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with Vaccinia virus (Neurotropin), its effect on the immune responses in aging BALB/c mice was examined. Neurotropin clearly restored the decreasing T-cell-dependent immune responses such as delayed-type hypersensitivity (DTH) response and plaque-forming cells (PFC) response to sheep red blood cells (SRBC) when administered i.p. from 13 months old (mo) to 16 mo. However, Neurotropin administration from 2 to 5 mo had no effect on the immune responses of young animals. Neurotropin administration from 13 to 16 mo restored not only the T-cell proliferation of spleen cells induced by concanavalin A (Con A) and phytohemagglutinin (PHA), but also the interleukin-2 (IL-2) production by spleen cells activated with Con A. However, Neurotropin did not affect the responsiveness of Con A-activated spleen cells to exogenous recombinant IL-2. An absence of suppressor cells capable of inhibiting the IL-2 production in the spleens was confirmed in the 16 mo mice. Neurotropin administration also restored IL-1 production by peritoneal macrophages stimulated with lipopolysaccharide (LPS). These results suggest that long-term administration of Neurotropin restores the decreasing T-cell-dependent immune responses through the recovery of IL-2 and in part IL-1 production, but not the responsiveness to IL-2 in aging BALB/c mice.

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F 1 mice

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F 1 (B/W F 1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F 1 mice as compared with young B/W F 1 mice. Neurotropin, when administered i.p. to aged B/W F 1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F 1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F 1 mice. Neurotropin, administered to the aged B/W F 1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F 1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F 1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F 1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F 1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory activity in B/W F 1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F 1 mice by the recovery of IL-2 production.

Studies of the MHC class I molecules of veto-active and veto-inactive lectin-stimulated spleen cell populations.

Mouse spleen cells activated for three days with ConA, but not with PHA and LPS, were found to possess strong veto activity. Thus, ConA-activated spleen cells were capable of preventing a cytotoxic T cell response generated in MLC against the H-2 haplotype of the ConA-activated cells. Radiolabelling and chemical cross-linking of veto-active and veto-inactive cells, followed by immunoprecipitation of H-2, class I antigens, and analyses of the precipitates by one and two dimensional SDS PAGE were performed. By these procedures we were unable to demonstrate qualitative and quantitative differences between precipitates of veto-active and veto-inactive cell populations.

Suppressive and Enhancing Effect of T-2 Toxin on Murine Lymphocyte Activation and Interleukin 2 Production

T-2 toxin, a fungal metabolite shown previously to exert potent immunosuppressive effects, was examined for its effects on activation and interleukin 2 (IL 2) production by murine and rat splenocytes. Splenocytes (1 X 10(6) cells/well) were incubated with 1 microgram Concanavalin A (Con A) for 48 h at which time cellular protein and DNA synthesis by these cells were ascertained using radiolabeled precursors. IL 2 synthesis was determined from the cell supernatant using the IL 2-requiring cell line CTLL. Spleen cells from mice treated for 4 consecutive days with 2 mg/kg toxin exhibited a 50% reduction in in vitro Con A activation but the supernatant IL 2 levels from these cells was 4-fold higher than cells from control mice. In vitro exposure of Con A-activated normal spleen cells to various toxin doses for 48 h resulted in diminished protein and DNA synthesis at 0.4 ng toxin with maximum inhibition at 1 ng (50% inhibition (TCID50) = 0.5 ng). Enhanced synthesis of both products was observed at lower toxin concentrations. IL 2 production by these cells followed a similar toxin dose response. Rat splenocytes were slightly more resistant and CTLL cells were slightly more sensitive to T-2 toxin than mouse splenocytes. These results indicate the variable effects a cytotoxic agent can have on lymphoid cells and that dosage is an important parameter for these effects.