Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Thy-1-negative and Ly-1-negative variants of T cells produce interleukin 2 in response to mitogens

IL 2 production by T cell variants, which lack the Thy-1 or Ly-1 surface glycoproteins, was studied. Cross-linking of the Thy-1 molecule resulted in IL 2 production by the EL4 thymoma and by a T cell hybridoma, suggesting that Thy-1 may play a role in T lymphocyte triggering. To further study the functional role of this molecule, Thy-1-negative variants were selected and analyzed for IL 2 production in response to phorbol-12-myristate-13-acetate (PMA) or to Con A. It was demonstrated that in spite of their failure to express Thy-1, the Thy-1-negative clones were capable of IL 2 production. These results indicated that although Thy-1 cross-linking triggers cell activation, a signal provided by Thy-1 is not indispensable for cell activation by mitogens. The T cell tumor line LBRM331A5 responds synergistically to IL 1 and PHA by releasing IL 2. It was demonstrated that anti Ly-1 monoclonal antibodies and PHA co-stimulated LBRM331A5 cells, as did IL 1 plus PHA. Thus, anti Ly-1 antibodies mimic the effect of IL 1, suggesting a role for Ly-1 antigen in T cell activation, perhaps by serving as an IL 1 receptor or as an associated molecule. To further study the functional role of Ly-1 and its relation to IL 1 receptor, Ly-1-negative variants of the LBRM331A5 cell line were selected and analyzed for IL 2 production in response to PHA plus IL 1. It was demonstrated that the Ly-1-negative clones were capable of IL 2 production as efficiently as Ly-1-positive clones. These results indicate that the Ly-1 and IL 1 receptor are distinct molecules, which are involved in different activation pathways.     

Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus

Inhibition of hepatitis B virus (HBV) replication and viral clearance from an infected host requires both the innate and adaptive immune responses. Expression of interferon (IFN)-inducible proteasome catalytic and regulatory subunits correlates with the IFN-alpha/beta- and IFN-gamma-mediated noncytopathic inhibition of HBV in transgenic mice and hepatocytes, as well as with clearance of the virus in acutely infected chimpanzees. The immunoproteasome catalytic subunits LMP2 and LMP7 alter proteasome specificity and influence the pool of peptides available for presentation by major histocompatibility complex class I molecules. We found that these subunits influenced both the magnitude and specificity of the CD8 T-cell response to the HBV polymerase and envelope proteins in immunized HLA-A2-transgenic mice. We also examined the role of LMP2 and LMP7 in the IFN-alpha/beta- and IFN-gamma-mediated inhibition of virus replication using HBV transgenic mice and found that they do not play a direct role in this process. These results demonstrate the ability of the IFN-induced proteasome catalytic subunits to shape the HBV-specific CD8 T-cell response and thus potentially influence the progression of infection to acute or chronic disease. In addition, these studies identify a potential key role for IFN in regulating the adaptive immune response to HBV through alterations in viral antigen processing.     

Molecular characterization, expression and mapping of porcine LMP2 and MECL-1 genes.

Low molecular mass polypeptides 2 (LMP2) and Multicatalytic endopeptidase complex subunit (MECL-1) are two of three catalytic beta-type subunits of the 20s proteasome and upon interferon gamma-induction. LMP2 is critical for the production of major histocompatibility complex (MHC) class I ligand and T-lymphocytes. The LMP2 gene is located in the MHC region, but MECL-1 is located outside the MHC region. They are involved in the antigen presentation and are important candidate genes for an initial exploration of relationships between the antigen processing genes and disease resistance. In this report, the porcine LMP2 and MECL-1 cDNA were cloned and A 5099 bp LMP2 genomic DNA structure was identified, then two single nucleotide polymorphisms were detected in the exon2 and exon5 of LMP2 gene in 367 individuals. The LMP2 and MECL-1 genes putative protein included 219,274 amino acids, respectively. Alignment and phylogenetic of predicted porcine LMP2 and MECL-1 amino acid sequence with their homologies were analyzed. Tissues expression of LMP2 and MECL-1 mRNA were observed by real time quantitative PCR (Q-PCR) method, the results revealed MECL-1 expressed widely in all tissues, but LMP2 was not detected in muscle. The porcine MECL-1 gene was mapped to chromosome 6, closely linked to microsatellite SW1108 (LOD = 4.09, 84cR) by radiation hybrid panel.     

Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera

The effects of Ly-5 alloantisera on the generation of cytotoxic T cells (CTL), on the effector phase of CTL killing, and on polyclonal mitogenesis were studied. Ly-5 antisera added at the beginning of mixed lymphocyte culture (MLC) suppressed the production of CTL in an allele-specific manner. Neither Ly-5.1 nor Ly-5.2 antisera inhibited the generation of cytotoxic effectors by Ly-5.1/Ly-5.2 heterozygous spleen cells; however, a combination of Ly-5.1 and Ly-5.2 antisera markedly suppressed the appearance of Ly-5 heterozygous CTL. Similarly, Ly-5 antisera inhibited the effector phase of CTL killing in an allele-specific manner. In addition, Ly-5 alloantisera specifically blocked concanavalin A and oxidative mitogenesis of splenocytes carrying the appropriate Ly-5 alloantigen. The results are discussed in light of a possible functional role of Ly-5 molecules in immune processes.     

Expression and regulation of interferon gamma-inducible proteasomal subunits LMP7 and LMP10 in the bovine corpus luteum

The proteasome is a large, polymeric protease complex responsible for intracellular protein degradation and generation of peptides that bind to class I major histocompatibility complex (MHC) molecules. Interferon gamma (INFgamma) induces expression of alternative proteasomal subunits that affect intracellular protein degradation, thereby changing the types of peptides that bind to class I MHC molecules. These alterations in class I MHC peptides can influence whether cells and tissues are tolerated by the immune system. Expression of two INFgamma-inducible proteasomal subunits, LMP7 and LMP10, in bovine luteal tissue was examined in this study. Northern analysis revealed the presence of mRNA encoding LMP7 and LMP10 in luteal tissue. Steady-state amounts of LMP7 mRNA did not change during the estrous cycle, but LMP10 mRNA was low in early corpus luteum (CL) and elevated in midcycle and late CL. Tumor necrosis factor alpha alone and in the presence of LH and/or prostaglandin F2alpha elevated steady-state amounts of LMP10 mRNA but did not affect LMP7 mRNA in cultured luteal cells. Immunohistochemistry revealed the presence of LMP10 primarily in small luteal cells. Numbers of LMP10-positive cells were lower in early CL than in midcycle and late CL. The finding that INFgamma-inducible proteasomal subunits are expressed in luteal tissue when the CL is fully functional was unexpected and suggests that proteasomes in luteal cells may generate peptides capable of stimulating a class I MHC-dependent inflammatory response.     

An altered T cell repertoire in MECL-1-deficient mice

Immunoproteasome subunits low-molecular mass polypeptide (LMP)2 and LMP7 affect Ag presentation by MHC class I molecules. In the present study, we investigated the function of the third immunosubunit LMP10/multicatalytic endopeptidase complex-like (MECL)-1 (beta2i) in MECL-1 gene-targeted mice. The number of CD8+ splenocytes in MECL-1-/- mice was 20% lower than in wild-type mice. Infection with lymphocytic choriomeningitis virus (LCMV) elicited a markedly reduced cytotoxic T cell (CTL) response to the LCMV epitopes GP276-286/Db and NP205-212/Kb in MECL-1-/- mice. The weak CTL response to GP276-286/Db was not due to an impaired generation of this epitope but was attributed to a decreased precursor frequency of GP276-286/Db-specific T cells. The expansion of TCR-Vbeta10+ T cells, which contain GP276-286/Db-specific cells, was reduced in LCMV-infected MECL-1-/- mice. Taken together, our data reveal an in vivo function of MECL-1 in codetermining the T cell repertoire for an antiviral CTL response.     

Variants of 3T3 cells lacking mitogenic response to the tumor promoter tetradecanoyl-phorbol-acetate

The potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) can stimulate quiescent, nonproliferating 3T3 cells to reenter the cell cycle and divide. We have previously used a slection technique developed in our laboratory to isolate variant cell lines which no longer divide in response to epidermal growth factor. We have now utilized the same selection procedure to isolate, from 3T3 cells, two variant cell lines, TNR-2 and TNR-9, which retain growth control and divide in response to elevated serum or fibroblast growth factor, but which do not respond to TPA. The variants do not incorporate precursors into DNA in response to TPA, demonstrating that the cells do not enter the S phase of the cell cycle. The TPA nonresponsive variant TNR-2 cannot respond to epidermal growth factor; TNR-9 responds to this mitogen. TNR-2 variant cells, which do not respond to EGF, do not bind ¹²⁵I-EGF. TPA can modulate ¹²⁵I-EGF binding to TNR-9 cells in a manner similar to its action on parental 3T3 cells. This TPA-induced alteration of EGF binding indicates that TNR-9 cells still interact with TPA, despite their inability to mount a mitogenic response.     

MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells

Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this was abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 was barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation was similar, indicating that the MSK1 'knockout' did not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells.     

T mitogens trigger LPS responsiveness in mouse thymus cells.

Mouse thymus cells are essentially unresponsive to lipopolysaccharide (LPS) stimulation. However, when cultured with minimally mitogenic levels of concanavalin A or submitogenic ratios of mitomycin-treated allogeneic spleen cells, in combination with LPS, they demonstrate levels of DNA synthesis de novo or greater than those induced by the T cell mitogen alone. Dose-response kinetics were characteristic of LPS. The subpopulation containing the LPS responsive cells was of low net buoyant density. Neither phytohemagglutinin nor pokeweed mitogen acted synergistically with LPS in this model to trigger thymus cells. The data suggest that LPS triggering may involve interaction with a T cell subpopulation.     

Specific binding of coupling factor 1 lacking the delta and epsilon subunits to thylakoids

An improved procedure for the preparation of chloroplast coupling factor 1 (CF1) lacking the delta subunit is described. In addition, CF1 deficient in the epsilon subunit was isolated by a new method and CF1 lacking both of the smaller subunits was prepared. The ability of the subunit-deficient forms and of CF1, either heated or incubated with dithiothreitol to activate its ATPase activity, to bind to thylakoids from which CF1 had been removed was studied. All CF1 preparations bound in a cation-dependent manner to similar extents. CF1 lacking the delta subunit required higher cation concentrations for maximal binding. All preparations competed similarly with control CF1 for binding sites on the depleted membranes. The alpha subunit of all forms of CF1 in solution was rapidly cleaved by trypsin. After reconstitution, however, the alpha subunit of CF1, as well as of the subunit-deficient and the activated forms, was resistant to attack by trypsin. Moreover, treatment of the membranes with either trypsin or N,N'-dicyclohexylcarbodiimide inhibited the binding of all CF1 forms. These results suggest that the binding of the subunit-deficient and activated forms of CF1 is specific. CF1 lacking the epsilon subunit restored neither proton uptake nor ATP synthesis to the depleted membranes. In contrast to our previous results, CF1 lacking the delta subunit was partially effective. Previously, we used a suboptimal Mg2+ concentration for binding the delta-deficient enzyme which we show here was partially deficient in the epsilon subunit. These results show that the delta and epsilon subunits are not required for binding CF1 to the membranes and that the delta subunit is not an absolute requirement for ATP synthesis.     

Apparent desensitization of Swiss 3T3 cells to the mitogens FGF and vasopressin

The growth factors FGF and vasopressin were found to have only a transient effect on confluent quiescent monolayers of Swiss 3T3 cells. Whether measured as cumulative entry into S-phase by autoradiography, or as cell division by time-lapse filming, the elevated rate of cell proliferation was maintained only over 10-15 hr. Several trivial or artifactual explanations for this transience were ruled out, including toxicity of 3H-thymidine; exhaustion or degradation of medium components, nutrients or growth factors (although some medium depletion was observed); and the generation during quiescence of cells incapable of division. We have also eliminated heritable variation in the capacity to respond to individual growth factors. However, unstable phenotypic heterogeneity in growth factor requirements between cells may play some part, as found elsewhere for the response to low concentrations of serum (Brooks et al, 1984). Cell populations that had ceased to respond to vasopressin recovered their sensitivity after 2-3 days' incubation in conditioned medium lacking vasopressin. The phenomenon thus resembles the mitogen-induced desensitization described by Collins and Rosengurt (1982, 1983). However, in our case, the loss of sensitivity was not selective for vasopressin but applied also to epidermal growth factor (EGF) and to prostaglandin F2 alpha. Furthermore, changes in responsiveness to vasopressin with time were associated with changes in cell density. Although some element of selective desensitization has not been ruled out, the transient response in our experiments can be accounted for in terms of unstable heterogeneity in growth factor requirements and/or in terms of density-dependent regulation of growth.     

CD40L启动CD4^＋T细胞缺乏小鼠免疫反应的研究

随着HIV感染者及各类医疗措施导致的免疫受损者的增多,探讨一种适合免疫缺陷人群的预防机会感染的策略越来越受到重视。研究表明,CD4^＋T细胞是抵抗肺孢子菌等机会感染的最主要因素,但不是唯一的因素。其中CD40配体（CD40L）被认为是一种可以启动B细胞和CD8^＋T细胞反应的关键因子。为探讨CD4^＋L是否能在缺乏CD4^＋T细胞的小鼠体内启动免疫反应,本文研究了用卵白蛋白（OVA）作为模型抗原,联合应用CD40L引起的免疫反应。结果显示,同时应用OVA和CD40L,可使CD4^＋T细胞耗竭小鼠体内抗OVA IgG抗体和抗原特异性IFN明显增多,提示在CD4^＋T细胞缺乏的宿主体内,CIMOL可以启动B细胞和CD8^＋T细胞类免疫反应。该结果为抗肺孢子菌等机会性感染的免疫预防研究提供可贵的资料。     

Lymphocyte receptors for polyclonal T mitogens: I. Concanavalin A binding to lymphocytes inhibits mitogenesis induced by galactose oxidase

Mitogenic stimulation of mouse lymphocytes by the enzyme galactose oxidase (GO) is inhibited if Con A is present during the enzymatic oxidation. The mechanism of this inhibition appears to involve steric hindrance of GO action at cell surface sites which bind Con A because (a) similar pulse exposure of unoxidized cells to Con A does not affect their subsequent ability to respond to GO stimulation; (b) Con A binding to fetuin interferes with GO oxidation of that glycoprotein substrate; and (c) sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cells labeled by $\text{GO}\text{NaB}{}^3\text{H}{}_4$ in the presence of Con A shows a selective inhibition of labeling of some high-molecular-weight glycoproteins compared to controls labeled in the absence of Con A.     

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.     

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening–induced replicative senescence is dependent on the ATM‐p53‐p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53‐responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti‐proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum‐dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late‐passage cells, and ectopic Pin1 expression rescues cells from BTG2‐induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.     

T cells in G1 provide a memory-like response to secondary stimulation

The commitment of naive T cells to proliferate is a function of the strength and duration of stimuli mediated by the TCR and coreceptors. Ranges of 2-20 h of stimulation have been reported as necessary in vitro. Whether T cells actually experience uninterrupted stimulation for such long periods under physiological conditions is controversial. Here we ask whether commitment to proliferate requires continuous stimulation, or can T cells integrate intermittent periods of stimulation. T cells were stimulated for two short-term (subthreshold) periods (5-7 h) either sequentially or separated by an interval of rest. Naive lymph node T cells were able to integrate interrupted stimulation, even when the duration of rest was as long as 2 days. Furthermore, when short-term-stimulated T cells were separated by density, three populations were observed: low density blasts, intermediate density G(1) cells, and high density G(0) cells. Low density cells progressed to division without further stimulation, whereas G(0) and G(1) cells remained undivided. However, after a period of rest, a second subthreshold stimulation caused the G(1) but not the G(0) fraction to quickly proceed through the cell cycle. We conclude that noncycling T cells in the G(1) phase of the cell cycle remain in a state of readiness for prolonged periods of time, and may represent a population of memory-like effectors capable of responding rapidly to antigenic challenge.