The Role and Regulation of mTOR in T-Lymphocyte Function

The conversion of naïve T cells into effector T cells is initiated by stimulation through the T-cell receptor (TCR). Upon activation, T cells undergo significant morphological and functional changes, putting new metabolic demands on the cell. Past research has identified the mammalian target of rapamycin (mTOR) as a critical regulator of cell metabolism, and the development of new genetic models has begun to reveal an important role for this pathway in the homeostasis and function of T lymphocytes. In this review, we focus on the most recent findings that demonstrate the ability of mTOR to regulate T-cell activation, CD8⁺ memory cell formation and function, and helper T lineage differentiation. Furthermore, we highlight the importance of tight control of mTOR signaling by tuberous sclerosis complex 1 for T-cell homeostasis, and the regulation of mTOR signaling by diacylglycerol kinases and the RasGRP1-Ras-Erk1/2 pathway in the context of TCR signaling.     

Interferon Production by Corynebacterium Parvum and BCG-Activated Murine Spleen Macrophages

Macrophages were identified to be a major source of interferon produced in murine spleen cell cultures after intravenous injection of Corynebacterium parvum (C. parvum), strain CN 6134 or Bacille Calmette Guérin (BCG). Another strain of C. parvum, CN 5888, which lacks RES stimulating activity and adjuvant activity in vivo, was not effective when injected intravenously. Protein synthesis was required for interferon activity to be produced and protein synthesis was also required for the antiviral state to be expressed. The antiviral activity was relatively stable to pH 2 and neutralized by an antiserum against virus-induced fibroblast interferon, thus exhibiting some properties of type I interferon. In vitro only CN 6134, the biologically active strain, could induce small amounts of interferon in spleen macrophage cultures. Macrophages from CN 6134 or BCG-infected athymic nu/nu mice produced similar interferon titers as their controls. It is concluded that infection with certain immunomodulators can activate splenic macrophages via a predominantly T-cell independent mechanism. Interferon in turn may operate locally as a mediator of immunoregulation.     

Anamnestic Chemiluminescence of Murine Spleen Cells

The oxidative response of murine spleen cells to secondary exposure to antigen was determined by luminol (5-amino-2,3-dihydro-1,4-pthalazinedione) amplified chemiluminescence, CL. BALB/cj and CBA/J mice were immunized with saline or an antigen solution of saline, luminol, and bovine serum albumin. Spleen cells were obtained from mice two and four days after immunization, and the CL response to in vitro antigenic exposure was measured for 35 minutes. At two days post-immunization, there was no difference in the CL of control and antigen-primed cells. By day four, the antigen-primed CL response differed significantly in both magnitude and time course from the primary antigen-stimulated response of the controls. This early development of differential CL response to antigenic challenge suggests a role for oxidative metabolic activity in the expression of the anamnestic immune response.     

Characterization and Functional Studies of the Murine T-Lymphocyte Response to Mycobacterium leprae Antigen

Mice were immunized with, Mycobacterium leprae in incomplete Freund's adjuvant, and sensitized lymphocytes were obtained from draining lymph nodes. The lymphocytes thus obtained proliferated specifically in vitro in the presence of M. leprae antigen, and this response was shown to be both T-ccll and macrophage dependent. T-cell blasts generated in vitro in response to M. leprae antigen were grown in the presence of interlcukin-2 (IL-2), The proliferative response of these blasts to M. leprae antigen was strictly dependent on the presence of syngeneic spleen cells as antigen-presenting cells. M. leprae-Immune F, blasts responding to the antigen in the context of either parental H-2 haplotype-hearing accessory cell could be obtained by positive selection from an F, hybrid-responding cell population. By means of flow microfluorometry the T-cell phenotype of the M. leprae-specific T-cell blasts was found to be Thy-1⁺ and to be composed of Lyt-1⁺ and Lyt-2⁺ subpopulations. Functionally, the blasts were shown to transfer delaycd-lype hypersensitivity locally to non-immunized recipients and to have cytolytic activity. Limiting dilution analysis showed the frequency of M. leprae -responding cells from blasts grown in IL-2 to be approximately 1/333.     

Characterization of the Murine T-Lymphocyte Response to Salmonella enterica Serovar Typhimurium Infection

Infection of mice with Salmonella enterica serotype Typhimurium induces a strong Th1 cell response that is central for the control of infection. We infected mice of a resistant background with a virulent strain of S. enterica serovar Typhimurium and analyzed the kinetics and magnitude of the T-cell response. After infection, the majority of CD4(+) and CD8(+) splenocytes acquired an activated phenotype, as indicated by expression levels of CD44 and CD62L. In addition, after 3 to 4 weeks of infection, more than 20% of the CD4(+) and more than 30% of the CD8(+) T cells produced gamma interferon (IFN-gamma) in response to short-term polyclonal stimulation. In contrast, we detected only a moderate (two- to threefold) expansion of both T-cell populations, and BrdU incorporation revealed that there was either no or only a limited increase in the in vivo proliferation of CD4(+) and CD8(+) T cells, respectively. Our results indicate that although an unexpectedly large population of both CD4(+) and CD8(+) T cells is activated and acquires the potential to secrete IFN-gamma, this activation is not paralleled by substantial expansion of these T-cell populations.     

Retention of Immune Complexes by Murine Lymph Node or Spleen Follicular Dendritic Cells

Using monoclonal anti-trinitrophenyl (TNP) antibodies complexed to TNP-myoglobin-coated gold particles, we analysed at the ultrastructural level the retention by follicular dendritic cells (FDC) of immune complexes containing various antibody isotypes. Gold-labelled immune complexes were injected subcutaneously or intravenously into naive mice and, after 24 h, germinal centres of draining lymph nodes or spleen were examined by electron microscopy. FDC generally retained complexes containing IgG2a and IgG2b better than those formed with IgG1 or IgG3. IgM was rarely retained. FDC isolated from lymph nodes or spleens were incubated in vitro with gold-labelled complexes in a serum-free medium. IgG2a and IgG2b complexes were also retained in vitro in large quantities by FDC; IgG1 and IgG3 complexes were retained in smaller quantities or in highly variable quantities compared with IgG2; IgM complexes were rarely seen on FDC. There was no difference between FDC isolated from lymph nodes or from spleen with respect to the Ig isotypes required for Fc-mediated retention of immune complexes.     

Mitogenic Responsiveness of Human T-Lymphocyte Subpopulations: Regulation by Suppressive Fc-Receptor-bearing T Cells and Influence of Fractionation Procedures

The proliferative response induced by leucoagglutinin (La) in different subpopulations of human T lymphocytes was studied. Subpopulations enriched in cells with either high- or low-avidity receptors for sheep erythrocytes (SRBC) (EH +, EL +) were prepared by sequential E-rosetting. In addition, T lymphocytes prepared by E-rosetting under optimal conditions (E + TOT) were fractionated on wheat germ agglutin (WGA)-Sepharose columns, rendering fractions enriched in lymphocytes with either low- or high-avidity receptors for WGA. The T lymphocytes were found to comprise at least three functionally distinct subpopulations, differing with respect to mitogen responsiveness. Cells characterized by high-avidity receptors for WGA and SRBC were highly responsive to La stimulation, regardless of the method used for purification. In contrast, cells with low-avidity receptors for WGA and probably also for SRBC but lacking Fc receptors for IgG responded only marginally but were conditioned to respond when subjected to E-rosetting under optimal conditions. This response was suppressed by lymphocytes with Fc receptors for IgG, which probably also had low-avidity receptors for WGA and SRBC. The lymphocytes with high-avidity receptors for WGA ans SRBC did not appear to be susceptible to suppression by Fc gamma + cells.     

Influence of Intestinal Microflora on Murine Bone Marrow and Spleen Macrophage Precursors

To investigate the adjuvant effect of intestinal flora on macrophage–colony-stimulating factor-responsive macrophage progenitors from spleen and bone marrow, we compared progenitor numbers and phenotypic characteristics of in vitro matured macrophages in germ-free and flora-associated mice (conventional, Escherichia coli -monoassociated and conventionalized mice). The data obtained show that the flora affected differentially bone marrow and spleen progenitors. It increased the numbers of progenitors in the spleen but not in the bone marrow. It did not modify the expression of F4/80, Mac-1 and major histocompatibility complex (MHC) class II on bone-marrow-derived macrophages (BMDM), while it clearly up-regulated MHC class II expression on spleen-derived macrophages (SDM). This effect was more pronounced in flora-associated ex germ-free mice than in conventional mice and it was greatly enhanced in the absence of M-CSF. In vitro stimulation by lipopolysaccharide had no effect on marker expression of BMDM, while it decreased F4/80 and enhanced MHC class II molecules on SDM from germ-free and flora-associated mice. However, the expression of MHC class II remained lower in germ-free mice. Enhancement of MHC class II molecule expression on SDM may contribute to the protective role of flora, because successful immune responses are dependent on the expression of these molecules.     

Inhibition of Murine Splenic and Mucosal Lymphocyte Function by Enteric Bacterial Products

Previously we showed that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells. The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production. Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-γ) production by murine spleen cells, but IL-10 production was increased. The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor β (TGF-β). EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-γ production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer’s patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells. Lysates obtained from Shiga-like toxin-producing E. coli, E. coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells. In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-β.     

Flow Cytometric Analysis of in Vivo Activation of Murine Spleen Cells

The in vivo effects of two polyclonal immune stimulators were studied in several strains of mice by analyzing the percentages of cells in various phases of the cell cycle by flow cytometry. Both lipopolysaccharide (LPS) and polyriboinosinic, cytidylic acid (rI.rC) were capable of inducing an increase in the percentage of cells undergoing DNA synthesis (S phase) in the spleens of several mouse strains. The response to both LPS and rI.rC was maximal between days 3 and 5 following injection. Optimal in vivo responses to LPS occurred at 3-30 μg, and to rI.rC at 100 μg; however, responses were observed over a broad dose range. No similar increase in S-phase cells was observed following injection of non-mitogenic T-independent antigens. Specific antibody was also measured after in vivo administration of rI.rC. There was a dissociation between the ability of an injection to induce specific antibody and to induce proliferation. These studies extend our knowledge of in vivo lymphocyte activation, and provide a basis for a detailed anafisis of lymphocyte activation following a variety of immune modulators in vivo.     

Flow Cytometric Analysis of in Vivo Activation of Murine Spleen Cells

Abstract

The in vivo effects of two polyclonal immune stimulators were studied in several strains of mice by analyzing the percentages of cells in various phases of the cell cycle by flow cytometry. Both lipopolysaccharide (LPS) and polyriboinosinic, cytidylic acid (rI.rC) were capable of inducing an increase in the percentage of cells undergoing DNA synthesis (S phase) in the spleens of several mouse strains. The response to both LPS and rI.rC was maximal between days 3 and 5 following injection. Optimal in vivo responses to LPS occurred at 3–30 μg, and to rI.rC at 100 μg; however, responses were observed over a broad dose range. No similar increase in S-phase cells was observed following injection of non-mitogenic T-independent antigens. Specific antibody was also measured after in vivo administration of rI.rC. There was a dissociation between the ability of an injection to induce specific antibody and to induce proliferation. These studies extend our knowledge of in vivo lymphocyte activation, and provide a basis for a detailed anafisis of lymphocyte activation following a variety of immune modulators in vivo.     

Differential Regulation of DC Function by Siphonodiol

Siphonodiol is a polyacetylene diol isolated from marine sponges Callyspongia sp. We demonstrate that the effect of Siphonodiol on the phenotypic and functional maturation of human monocyte derived DC in vitro. Human monocytes were exposed to Siphonodiol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on LPS-primed DC were partially enhanced by Siphonodiol. Siphonodiol augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC. Siphonodiol dose-dependently enhanced the production of IL-12p70 by LPS-primed DC and this cytokine production was inhibited by anti-TLR4 mAb. IFN-γ secretion from naïve T cells co-cultured with DC differentiated with LPS was augmented by Siphonodiol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by Siphonodiol depends on TLR4 and via the activation of IL-12p70.     

Functional Analysis of Pokeweed Mitogen-Dependent Cell Interactions in Murine Spleen Cells

The nature of lymphocyte responses on addition of pokeweed mitogen (PWM) to normal murine spleen cells was studied in low cell density cultures. PWM, over a wide range of concentrations, stimulated proliferation in a set of cells roughly 10-fold smaller than the lymphocyte populations responding to either concanavalin A or lipopolysaccharide. PWM also induced a relatively small number of B lymphocytes in these cultures to mature to Ig-secreting plaque-forming cells (PFC). Proliferative and PFC responses were completely abrogated by T-cell removal from normal spleen cell cultures. Moreover, cell mixture and irradiation experiments demonstrated that B lymphocytes do not proliferate in response to PWM, even in the presence of an excess of normal T cells, suggesting that PFC development results from terminal maturation without proliferation. Finally, parallel titrations of cloned helper cells, normal splenic T cells or T-cell blasts induced by PWM showed that the poor B-lymphocyte responses in normal spleen cell cultures is due to the very low frequency of competent helper cells in these populations. PWM, however, was competent to activate and expand this set of helper lymphocytes in primary cultures.     

Malformations in the Murine Kidney Caused by Loss of CENP-F Function

Centromere-binding protein F (CENP-F) is a large and complex protein shown to play critical roles in mitosis and various other interphase functions. Previous studies have shown that the disruption of CENP-F function leads to detrimental effects on human development. Still, it is important to note the lack of studies focusing on the effects that the loss of this essential protein may have on specific adult organs. In the current study, we used a novel global knockout murine model to analyze the potential consequences deletion of CENP-F has on adult kidney structure and function. We discovered several structural abnormalities including loss of ciliary structure, tubule dilation, and disruption of the glomerulus. Along with these structural irregularities, renal dysfunction was also detected suggesting hydronephrosis and acute kidney injury in these knockout organs. Importantly, this is the first study linking CENP-F to kidney disease and hopefully these data will serve as a platform to further investigate the molecular mechanisms disrupted in the kidney by the loss of CENP-F. Anat Rec, 302:163–170, 2019. © 2018 Wiley Periodicals, Inc.     

Tim-3中和抗体对哮喘小鼠T淋巴细胞功能的影响

目的 探讨Tim-3中和抗体对支气管哮喘(简称哮喘)小鼠外周血及支气管肺泡灌洗液(BALF)中T淋巴细胞体外增殖功能和细胞因子合成功能的影响.方法 分别采集实验组和对照组外周血和BALF,并分离出T淋巴细胞进行体外分组培养,各组分别以不同浓度的Tim-3中和抗体进行干预,并在不同的时间进行检测.用MTT法分别测定各组细胞的增殖情况,用酶联免疫吸附试验(ELISA)测定T淋巴细胞白细胞介素4(IL-4)、干扰素γ(IFN-γ)的水平.结果 与对照组比较,Tim-3中和抗体对体外培养的实验组小鼠外周血和BALFT淋巴细胞的增殖均有抑制作用,其作用强度(在一定范围内)随剂量的增加和时间的延长而加强(均P<0.05);而且对实验组小鼠的抑制作用明显强于对照组小鼠(P<0.05);同时实验组小鼠外周血和BALFT淋巴细胞经抗体干预后,IL-4的表达水平较正常组明显下降,IFN-γ明显增加(P<0.05).结论 Tim-3中和抗体能抑制实验组小鼠T淋巴细胞的增殖和IL-4的合成,增加IFN-γ的分泌.Tim-3可能作为哮喘治疗新的分子靶点.